Some of these mutants appear to have acquired novel functions beyond merely losing wild-type functions. To investigate these gain-of-function effects in vivo, we generated mice of three different genotypes: MMTV-+/+Hras/p53, MMTV--/-Hras/p53, and MMTV-R172H/R172HHras/p53. Salivary tumors from these mice were characterized with regard to age of tumor onset, tumor growth rates, cell cycle distribution, apoptotic levels, tumor histopathology, as well as response to doxorubicin treatment. Microarray analysis was also performed to profile gene expression. The MMTV--/-Hras/p53 and MMTV-Hras/R172H/R172Hp53 mice displayed similar properties with regard to age of tumor onset, tumor growth rates, tumor histopathology, and response to doxorubicin, while both groups were clearly distinct from the MMTV-+/+Hras/p53 mice by these measurements. In addition, the gene expression profiles of the MMTV--/-Hras/p53 and MMTV-Hras/R172H/R172Hp53 tumors were tightly clustered, and clearly distinct from the profiles of the MMTV-+/+Hras/p53 tumors. Only a small group of genes showing differential expression between the MMTV-Hras/-/-p53 and MMTV-Hras/R172H/R172Hp53 tumors, that did not appear to be regulated by wild-type p53, were identified. Taken together, these results indicate that in this MMTV-Hras-driven salivary tumor model, the major effect of the p53 R172H mutant is due to the loss of wild-type p53 function, with little or no gain-of-function effect on tumorigenesis, which may be explained by the tissue- and tumor type-specific properties of this gain-of-function mutant of p53. The vast majority of these missense mutations are clustered in the central DNA-binding domain of the p53 protein, either affecting residues directly contacting DNA (DNA contact mutations), or those important for maintaining the conformation of the DNA-binding domain . Both classes of mutations disrupt the sequence-specific DNA-binding function of the p53 protein, thus preventing p53 from acting as a transcription factor [in vitro [Most p53 mutations arise somatically during tumor development and progression. However, p53 mutations can also be transmitted in the germ-line and give rise to cancer predisposition conditions called Li-Fraumeni Syndrome (LFS) and Li-Fraumeni-like Syndrome (LFL) –10. Unlin factor ,12. In an factor ,14. Furtin vitro –21, incrin vitro ,20,22–24in vitro –30, exacin vitro ,31–33, ein vitro , disruptin vitro , and incin vitro ,36,37. Ain vitro . Interacin vitro ,38–40. Tin vivo. For example, when p53 R172H, which is equivalent to the hot-spot human p53 R175H mutant , was expressed in the epidermis [Although the majority of studies characterizing the gain-of-function properties of mutant p53 have been conducted using cell culture systems, a variety of genetically engineered mouse tumor models have also been developed for examining the effects of mutant p53 pidermis  or mammapidermis  of transpidermis ,46. Althpidermis ,44.ras (MMTV-Hras) transgenic mouse mammary/salivary tumor model [Hras/p53+/+ mice develop both mammary and salivary tumors, salivary tumorigenesis was greatly accelerated in the MMTV-Hras/p53-/- mice. The MMTV-Hras/p53-/- salivary tumors had higher histopathological grades, growth rates, S-phase fractions, and genomic instability than the MMTV-Hras/p53+/+ tumors [Hras/p53+/+ tumors responded better to doxorubicin treatment than the MMTV-Hras/p53-/- tumors, due to a decrease in S phase fraction and an increase in G1 phase fraction, effects which were absent in the treated MMTV-Hras/p53-/- tumors [p53 status and treatment, likely due to the anti-apoptotic effects of the activated Ras signaling pathways driving tumorigenesis in this model.Although the mutant p53 knock-in mice allowed for the comparison of tumorigenesis and tumor properties in mice carrying p53 null and p53 gain-of-function alleles, direct comparisons within a particular tumor type were complicated by the fact that mice in these models develop multiple tumor types. We therefore sought to create animal models that would allow for a head-to-head comparison of the effects of p53 null and gain-of-function mutations within the context of a single tumor type. Our lab previously utilized the MMTV-v-Ha-or model  crossed + tumors . In addi- tumors . No signMMTV-Hras mice to p53 R172H knock-in mice [Hras/p53+/+ tumors differed significantly from both the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors in these measurements, p53 null and R172H tumors exhibited very similar properties. We also performed gene expression profiling on salivary tumors of the three genotypes and identified the differentially regulated genes among the three classes. Again, the p53+/+ tumors clustered separately from the other two classes, but there were relatively few gene expression differences between the p53 null and R172H tumors. These results indicate that, within the context of activated Ha-ras expression in the mouse salivary gland, the primary effect of p53 R172H mutation is the loss of wild-type p53 function, with little discernable gain-of-function effect on tumorigenesis.In the current study, we have included p53 R172H as the third p53 status in this tumor model by crossing the -in mice , and hav3 or displaying indications of morbidity.All animal studies and care were performed under the guidelines of the Virginia Commonwealth University (VCU) Institutional Animal Care and Use Committee (IACUC), in accordance with the principles and procedures outlined in the National Research Council “Guide for the Care and Use of Laboratory Animals” under Assurance Number A3281-01. Specific approval for these studies was granted by the VCU IACUC under protocol #AM10313, entitled “Molecular Genetics of Tumorigenesis in Transgenic Mice”. Pain and distress resulting from tumor development was minimized by euthanizing any mouse with a palpable tumor of 2500mmHras and MMTV-Hras/p53-/- mice were generated as described in [p53R172H/+ mice were generously provided by Dr. Gigi Lozano (The University of Texas MD Anderson Cancer Center) [Hras/p53R172H/R172H mice, p53R172H/+ mice on a C57BL/6 background were initially mated to MMTV-Hras/p53+/- mice on a CB6 background. The colony was subsequently maintained on the C57BL/6 x BALB/c mixed background by interbreeding of mice from this colony. Mice were genotyped by PCR for determination of ras transgene and p53 status [MMTV-ribed in , and hav Center) . To gene3 status ,46.2)/2, where L is the length and W is the width of the tumor in mm. Tumor-bearing mice were sacrificed when the weight of the tumor approached 2500 mg  and the tumor was dissected out. The tumor mass was then cut into multiple sections, one of which was placed in 10% formalin and the rest into 1.2 ml cryovials which were then flash frozen in liquid nitrogen and stored at -80°.Mice were monitored visually or by palpation 2–3 times weekly for the presence of tumors arising from the salivary glands . Once a tumor was detected, tumor size was measured  by caliper measurement of the width and length of the tumor. Tumor weight was estimated using the formula: tumor weight (mg) = . For immunohistochemistry, tumor tissues fixed in 10% phosphate-buffered formalin were transferred to 70% ethanol after 48 hours to preserve antigens. Fixed tumor tissues were paraffin embedded and cut into 5μm sections for staining. Primary antibodies and dilution used for immunoblotting include: p53  at 1:1000; p19ARF  at 1:1000; p16  at 1:1000; p15  at 1:1000 and Actin  at 1:1000. HRP-conjugated Secondary antibodies (Jackson ImmunoResearch Laboratories) were used at 1:2000 dilution. An enhanced chemiluminescence (ECL) assay (PerkinElmer) was used and documented either on Kodak X-Omat Blue XB-1 film or by an AlphaEase digital imaging system (Alpha Innotech). Primary antibodies and dilution used for immunohistochemistry include: Ki-67  at 1:200; cleaved caspase 3  at 1:100; p53  at 1:100.For immunoblotting, tumor protein was extracted from frozen tissue using PIERCE RIPA buffer, supplemented with additional 1X protease inhibitor cocktail containing 50mM NaF, 1mM NaA subset of salivary tumor-bearing mice in each genotypic group were treated with doxorubicin. When the weight of the tumor reached 500–700 mg, the mouse was subjected to a 9-day treatment schedule at a dose of 2 mg/kg doxorubicin/day, injected IP. During the treatment period, tumor dimensions were measured daily and tumor weight calculated the same way as in the tumor growth analysis. Only tumors with initial palpable size <800 mg were used to calculate mean tumor weights for plotting tumor growth curves.For RNA extraction, 20–40 10 μm frozen sections from each candidate tumor were collected. RNA was extracted using TRIZOL reagent (Invitrogen Life Technologies), according to the manufacture’s protocols. Total RNA isolated from the tumor sections was subjected to a cleanup step using the RNeasy Mini Kit (Qiagen). Microarray analysis was performed using the Affymetrix GeneChip platform and the Mouse 430A 2.0 array according to the Affymetrix standard protocol. Multiple levels of data analysis were performed in the BRB-ArrayTools software, version 3.5.0 . The probe-level microarray data (in .CEL files) were collated in BRB-ArrayTools using the RMA (Robust Multi-chip Average) method. Microarray data used in this study are deposited in the Gene Expression Ominibus (GEO) database with accession # GSE59452.3 μg of total RNA from each tumor sample was loaded in each RT reaction, using the SuperScript III First-strand Synthesis system (Invitrogen Inc.) according to the manufacture’s protocol, and oligo(dT) was selected to prime the RT reactions. Primers for the real-time PCR assays were designed using the Primer Express Version 3.0 software (Applied Biosystems) using the standard melting temperature (60°C) and at least one of the two primers for each gene was selected to span an exon-intron boundary, to avoid amplifying genomic DNA contaminants. The real-time PCR assay was performed on the Applied Biosystems 7500 Real-time PCR System (Applied Biosystems). The Relative Standard Curves method was used as the study design for the quantitative PCR assay . β-actin was used as the internal control. The raw data were analyzed in the Sequence Detection Software Version 1.3.1 (Applied Biosystems) according to the manufacture’s protocol. Sequences of the primers used in the quantitative PCR assays are listed in Hras male transgenic mice), we generated cohorts of mice of the following genotypes: MMTV-Hras/p53+/+, MMTV-Hras/p53-/-, and MMTV-Hras/p53R172H/R172H. Although female MMTV-Hras/p53+/+ mice develop primarily mammary tumors, male mice of this genotype develop primarily salivary tumors (at a later average age of tumor onset). However, we have previously shown that when bred to p53 knock-out mice, both males and females of the MMTV-Hras/p53-/- genotype develop primarily salivary tumors due to an acceleration of salivary but not mammary tumorigenesis in this model [Hras/p53R172H/R172H mice. Therefore, in order to compare tumors of a single cell type of origin arising in mice of a single gender, we restricted our analysis to salivary tumorigenesis in male mice of each of the three genotypes. To determine the potential differential effects of p53 loss-of-function and gain-of-function mutations on tumor phenotype within the context of a single tissue of origin  without salivary tumors were sacrificed due to development of Harderian gland tumors, consistent with previous reports [Hras/p53-/- and MMTV-Hras/p53R172H/R172H mice were much earlier than in the MMTV-Hras/p53+/+ mice and the overall salivary tumor incidence was much higher than in the MMTV-Hras/p53+/+ mice. As shown in Hras/p53+/+ mice, 68 days for the MMTV-Hras/p53-/- mice and 109 days for the MMTV-Hras/p53R172H/R172H mice. We observed a statistically significant difference between the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H mice in age of tumor onset , with a slightly delayed tumor onset in the MMTV-Hras/p53R172H/R172H mice. However, both the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H mice developed salivary tumors much more rapidly than the MMTV-Hras/p53+/+ mice .ransgene ,44, whiccterized ,44,48, w reports . AnimalsHras/p53+/+ mice had acquired somatic mutations in the p53 gene which might confound our subsequent analyses, exons 5–9 of the p53 locus of 4 MMTV-Hras/p53+/+ salivary tumors  were sequenced, and no p53 mutations were detected (data not shown).To exclude the possibility that tumors from the MMTV-Hras/p53-/- mice have growth rates that are nearly double those of salivary tumors arising in MMTV-Hras/p53+/+ mice [Hras/p53+/+, MMTV-Hras/p53-/-, and MMTV-Hras/p53R172H/R172H) was compared , 34 MMTV-Hras/p53-/- tumors , and 39 MMTV-Hras/p53R172H/R172H tumors  was obtained. The mean tumor growth curves for each genotype are shown in Hras/p53+/+ tumors grew significantly slower than the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors, as was previously reported for the comparison of MMTV-Hras/p53+/+ and Hras/p53-/- tumors [Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors.We have previously shown that salivary tumors arising in MMTV-+/+ mice . To detecompared ; S1 Fig.- tumors . HoweverHras/p53R172H/R172H tumors, and to characterize the expression patterns of mutant p53 protein in the tumor tissues, western blotting and immunohistochemical analysis were performed on tumors of each genotype, using an antibody that detects both wild-type and mutant p53. Western blot analysis showed that mutant p53 was present in very high levels in all of the MMTV-Hras/p53R172H/R172H tumors tested, whereas p53 was barely detectable in the MMTV-Hras/p53+/+ tumors and completely absent in the MMTV-Hras/p53-/- tumors , which is the activated version of a key apoptosis mediator. As for the Ki-67 staining, there was also significant variation in the staining patterns for Cc3, either among different tumors, or among different regions of the same tumor. Cc3 immunostaining revealed low indices of apoptosis in all three genotypic groups of tumors (data not shown).Previous studies also demonstrated that apoptosis levels were constitutively low in the tumors with the MMTV-ated Ras . To inveHras/p53-/- mice are significantly impaired in their response to doxorubicin compared to those arising in MMTV-Hras/p53+/+ mice [Hras/p53+/+ tumors plateaued following the first day of treatment, with some of the tumors actually regressing during the 9-day treatment period. In contrast, both the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors continued to grow with unaltered rates for 3–4 days, after which the growth of most of the tumors began to plateau. Although the average size of the MMTV-Hras/p53R172H/R172H tumors at the initiation of treatment was larger than that of the MMTV-Hras/p53-/- tumors, the overall shape of the tumor response curves was nearly superimposable. Interestingly, 3 of the 18 MMTV-Hras/p53R172H/R172H tumors in the treatment study exhibited no plateau phase and continued to grow at an unaltered rate during the 9-day treatment period . The expression levels and potential meaning of the 6 clusters of genes are summarized in iii), which represent the differentially expressed genes between the wild-type p53 (WT) and p53-null status in this analysis. Of these 184 genes, only a few were previously reported as regulated by wild-type p53, including 3 well-characterized p53 target genes Cdkn1a (p21), Mdm2, and Ccng1. These genes are listed in As our major interest was to identify differentially expressed genes that may indicate gain-of-function effects, we compared the expression profiles of the three groups of tumors, and tumors with wild-type p53 were used to control for residual wild-type functions of mutant p53. To identify the genes differentially expressed between the three groups, we performed class comparison analysis using Significance Analysis of Microarray (SAM). The multi-class comparison function of SAM gave rise to the identification of 188 genes with a low 1% False Discovery Rate (FDR). The 188 genes were then subjected to hierarchical clustering to identify co-expressed gene clusters, the result of which is shown in iii and v define the genes with higher expression levels in the mutant p53-expressing samples than in the wild-type p53-expressing and p53-null samples (iv), or intermediate levels (Cluster i and vi) for a significant fraction of the remaining genes. This indicates the presence of residual wild-type functions with the mutant p53 in regulating gene expression. However, when examining the overall expression profiles based on these 188 genes, the two groups showing the greatest similarity were the mutant p53-expressing tumors and p53-null tumors, while both showed significant differences from the tumors with wild-type p53, in agreement with the results from the unsupervised clustering of the samples (t-test with Benjamini-Hochberg correction). From this analysis we found that despite the different scales of significant genes identified by different mathematical and statistical approaches, there are consistently very few genes standing out from the comparisons between MT-p53 and p53-null samples, in sharp contrast to those between wild-type p53 and p53-null samples  displayed no significant difference in survival time of the mice when compared to p53-null mice [p53R248Q allele, which significantly reduced lifespan when compared to p53-null mice [p53R172H/R172H and p53-/- mice, while in the third study [p53R172H/- mice developed carcinomas and hemangiosarcomas with increased incidence compared to p53-/- mice. The discrepancy between these studies may be due to the fact that mice with different genetic backgrounds were used. The common gain-of-function effect observed in all three studies was that tumors in the p53R172H/+ mice metastasized with a high incidence, a feature the p53+/- tumors lacked.Interestingly, the data from this study demonstrated that MMTV-e tumors . Comparep53 loss . In anotl glands . These oull mice ,44,51. Oull mice . In two rd study , p53R17Hras/p53-/- tumors displayed more malignant properties than the MMTV-Hras/p53+/+ tumors, consistent with our previous findings [Hras/p53R172H/R172H tumors closely resemble the MMTV-Hras/p53-/- tumors with regard to most of these parameters, suggesting that loss of the sequence-specific DNA binding function of wild-type p53 is the primary cause of the difference in tumor histology between tumors with mutant and wild-type p53.In this study, with a set of well-defined histopathologic parameters, the MMTV-findings . The MMTHras/p53-/- tumors displayed a delayed response upon doxorubicin treatment, as compared to the MMTV-Hras/p53+/+ tumors. Apoptotic levels were shown to be minimal in both types of tumors, either with or without treatment. Instead, doxorubicin treatment caused significant changes in cell cycle distribution in these tumor cells. In the present study, the MMTV-Hras/p53R172H/R172H tumors demonstrated indistinguishable response patterns compared to the MMTV-Hras/p53-/- tumors. Immunohistochemical analysis of cleaved caspase 3 (Cc3) revealed similarly low levels of apoptosis in tumors of all three genotypes (data not shown). Thus, the results demonstrated again that apoptosis does not play a major role in tumor properties or response to chemotherapeutic treatments in this model, likely due to the anti-apoptotic effects of the activated Ras signaling driving tumorigenesis in this model. Thus, it is important to note that, with the low level of both spontaneous and drug-inducible apoptosis in this tumor model, it may be difficult to detect any influence of mutant p53 on apoptosis, which is an important aspect of the gain-of-function activities identified in in vitro cell culture systems.In a previous study from our lab , the MMTR172H [R172H was found to be stabilized in all the tumors tested by both Western blotting and immunohistochemistry. It is reasonable to speculate that activated Ras signaling is providing the stabilizing signal for mutant p53 in this tumor model.One possible explanation for the failure to detect a p53 gain-of-function phenotype distinct from the knock-out phenotype could potentially be a lack of stabilization and accumulation of the mutant p53 protein. The Lozano group demonstrated that in the R172H knock-in mice, mutant p53 was not stabilized in either normal or tumor tissues, due to the basal level of MDM2’s inhibitory activity on mutant p53 [R172H . Howeverin vivo analyses we performed, it is not surprising that the gene expression profiles of the different groups of tumors closely reflect this relationship. We have tested three prevalent probe-level data summarization algorithms for Affymetrix microarray data, and two False Discovery Rate-based class comparison approaches, and none of the combinations could detect reasonable numbers of significant genes from the comparison between the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors, while substantial numbers of genes were identified between the MMTV-Hras/p53+/+ tumors and either the MMTV-Hras/p53-/- or the MMTV-Hras/p53R172H/R172H tumors.Given the similarity between the p53 R172H-expressing and p53-null tumors, but the significant difference between both groups and tumors with wild-type p53 in the in vivo setting or under physiological conditions. Rather, most of the observations have been made by over-expressing mutant p53 in p53-null cells, and in many cases, in the presence of stress signals. Additionally, almost all of the previous studies only utilized a comparison between cells without p53 and those expressing mutant p53, a study design lacking the ability to detect any residual wild-type function of the mutants.Several mechanisms have been proposed for the gain-of-function activities of mutant p53 . One of Hras/p53R172H/R172H tumors  in human) was previously identified by another group as induced by p53R 175H overexpressed in p53-null H1299 non-small cell lung carcinoma cells. BCL2L1 is a member of the BCL2 family of anti-apoptotic proteins [BCL2L1 was dependent on mutant p53 in SKBR3 mammary adenocarcinoma cells expressing p53R 175H, and in HT29 human colorectal adenocarcinoma cells expressing p53 R273H, another p53 mutant with gain-of-function activities [Hras/p53R172H/R172H tumors, Csnk2a1, which encodes casein kinase 2, alpha 1 polypeptide, has also been shown to have an anti-apoptotic function, but regulation of its expression hasn’t been associated with mutant p53[The class comparison analysis in this study identified a small subset of genes with the highest expression levels in the MMTV-in vitro ,61–63 ore models . One of proteins . In addiutant p53–68).in vitro cell migration as well as in vivo tumor metastasis [Hras/p53R172H/R172H tumors. Indeed, a few genes have been reported to promote cell motility and/or metastasis, including Etv4/E1A-F [Mia1 [Cap1 [EFEMP2 (EGF-containing fibulin-like extracellular matrix protein 2) was recognized as a binding partner of mutant human p53 proteins, with the highest specificity to structural mutants like human p53R175H. Even more intriguingly, co-expression of EFEMP2 and p53 R175H had synergistic effects in promoting neoplastic transformation and tumor cell growth [Because increased metastatic potential has also been commonly observed with gain-of-function mutants of p53 ,44, and v4/E1A-F ,70, Mia1-F [Mia1 , and Capl growth . The facHras/p53+/+ and MMTV-Hras/p53R172H/R172H tumors as compared to the MMTV-Hras/p53-/- tumors  on in vivo tumor development and test the gain-of-function effects of a p53 mutant in an in vivo setting. Overall, the MMTV-Hras/p53R172H/R172H mice closely resembled the MMTV-Hras/p53-/- mice with regard to age of tumor onset, tumor growth rates, tumor histopathological properties, tumor responses to doxorubicin, and gene expression profiles, while both groups were clearly distinct from the MMTV-Hras/p53+/+ mice. These results indicate that in this MMTV-Hras-driven mouse salivary tumor model, the primary effect of p53 R172H mutation is the loss of wild-type p53 function, with little or no gain-of-function effect on tumorigenesis, underscoring the tissue- and tumor type-specific properties of the gain-of-function mutants of p53.In conclusion, this study has led to the generation of MMTV-S1 Fig(A-C) Once a tumor was detected, growth was monitored by daily caliper measurements, and calculated tumor weights (mg) of the three groups of tumors were plotted over time. Each line represents the growth of an individual tumor.(TIF)Click here for additional data file.S2 FigTumor histopathology was graded on a 3-point scale taking into account (A) N/C (nuclear to cytoplasmic) ratio, degree of nuclear pleomorphism, and overall tumor architecture; percentage of tumor cells showing (B) spindle cell morphology, (C) “giant cells”, (D) apoptosis, (E) mitotic figures, and (F) necrosis.(TIF)Click here for additional data file.S3 FigHras/p53+/+; middle: MMTV-Hras/p53-/-; bottom: MMTV-Hras/p53R172H/R172H; left: representative tumors of each genotype with high levels of Ki-67 staining; right: representative tumors of each genotype with low levels of Ki-67 staining. (B) An example of an MMTV-Hras/p53+/+ tumor in which different regions show markedly different levels of Ki-67 staining.(A) Heterogeneity of Ki-67 staining in different tumors from the same genotypic group. Top: MMTV-(TIF)Click here for additional data file.S4 Fig. (A-C) Calculated weights (mg) of the three groups of tumors during the treatment period are plotted over time (days). Each line represents the growth of an individual tumor.(TIF)Click here for additional data file.S5 FigGenes up-regulated in wild-type p53 tumors compared to p53-null tumors are colored in red and those down-regulated are colored in green.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.


In this paper, a putative E subunit of the V-type H+-ATPase gene, W36 was isolated from stress-induced wheat de novo transcriptome sequencing combining with 5'-RACE and RT-PCR methods. The full-length of W36 gene was 1097 bp, which contained a 681 bp open reading frame (ORF) and encoded 227 amino acids. Southern blot analysis indicated that W36 was a single-copy gene. The quantitative real-time PCR (qRT-PCR) analysis revealed that the expression level of W36 could be upregulated by drought, cold, salt, and exogenous ABA treatment. A subcellular localization assay showed that the W36 protein accumulated in the cytoplasm. Isolation of the W36 promoter revealed some cis-acting elements responding to abiotic stresses. Transgenic Arabidopsis plants overexpressing W36 were enhanced salt and mannitol tolerance. These results indicate that W36 is involved in the plant response to osmotic stress.The vacuolar type H Plants have three major methods to remit Na+ infections, including restricting Na+ absorption, expediting Na+ exocytosis, and energizing Na+ segmentation in the vacuole [+/H+ antiporters could take advantage of the proton gradient formed by these pumps to exchange Na+ for H+, many evidences suggest that tonoplast Na+/H+ antiporter which drives Na+ from cytosol into vacuole play a major role in Na+ compartmentalization in plant leaves [+-gradient generated by primary-active H+ pumps located at the tonoplast, such as vacuolar type H+-ATPase (V-type H+-ATPase or V-H+-ATPase) and V-type H+-PPase to active secondary transport of Na+ from the cytosol into the vacuole via tonoplast Na+/H+ antiporter to eliminate Na+ toxicity, and consequently enhance salt resistance [Salt stress is one of the main environmental factors that cause osmotic stress and reduction in plant growth and crop productivity ,2. Salt  in vivo . To ensu vacuole ,5. ATPast leaves ,7. Theresistance ,9.Arabidopsis, P-type H+-ATPase are encoded by a 12-member gene family (AHA1 to AHA12) [AHA1 produce a constitutive protein activity and reduce sensitivity to ABA-mediated stomatal closure [+-ATPase enhanced plant salt tolerance [+-ATPases account for about 6%–8% of the total tonoplast proteins and even reach to 30%. The molecular weight of V-H+-ATPases is approximately 400–650 kD, which are composed of 7–10 subunits and divided into hydrophilic V1 subunit group (made up of 8 kinds of subunits (A-H)) and hydrophobic V0 ). The optimal pH of this enzyme is about 7.2. It can be activated by anions, for instance Cl−. V-type H+-ATPases not only maintain the dynamic balance of cytoplasm ion and cell metabolism as a kind of “dominate enzyme”, but also respond to environmental factors through appropriately changing subunits expression and modulating enzyme activity. The structure of V-type H+-ATPases appears to be conserved across eukaryotes [+-ATPases subunits are encoded by small multigene families, which have been detected in many plant genomes [Arabidopsis, V1 subunits B, E and G are each encoded by three different VHA genes, and multiple isoforms exist for each of the five V0 subunits [Arabidopsis VHA-c1 and c3 subunit isoforms have been knocked down by RNAi, each resulting in reduced root length and decreased tolerance to moderate salt stress [+-ATPases subunits may respond to specific developmental or environmental cues, which allow each subunit to be amplified or suppressed as required [+ transmembrane transshipment and participates in ATP synthesis process. Both F- and V-type H+-ATPases are composed of multiple subunit complexes. Homology sequence analyses of the catalytic and noncatalytic subunits of the F- and V-type H+-ATPases have suggested that these genes are derived from a common ancestral gene [ATPases can catalyze the degradation of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and a free phosphate ion. There are three types of ATPases on plant cell membrane . The firo AHA12) ,15. Muta closure . Additioolerance . The seckaryotes . However genomes . In Arabsubunits . Arabidot stress . The prerequired . The thiral gene .W36 (ABC70183.1), a V-type H+-ATPase subunit E gene, was isolated from stress-induced de novo transcriptome sequencing of wheat. Molecular characteristics and functions of W36 were analyzed to clarify the molecular mechanism of V-type H+-ATPase, which will provide theory basis for further realizing stress physiology process and completing signal-transporting systems in plants.Wheat is an important food crop and occupies a momentous position in the national economy. Salt stress caused by soil salt and alkali seriously affects the yield and quality of wheat. In this study W36 was isolated by analysis of the de novo transcriptome sequencing. Combining with 5'-RACE and RT-PCR methods, W36 full-length sequence was obtained. The full-length of W36 cDNA was 1097 bp, which contained a 681 bp open reading frame (ORF) and encoded 227 amino acids. The 5'-non-coding and 3'-non-coding region was 144 and 251 bp, respectively. The predicted molecular weight and pI value was 19.7 kD and 6.87, respectively. A vATP-synt-E  conserved domain of W36 was discovered in GenBank which was a typical characteristic of V-type H+-ATPase E subunit family.A 833 bp cDNA fragment named W36 and maize VHA-E, Glycine max VHA-E, three rice VHA-E  and three Arabidopsis genes, AtVHA-E1 and AtVHA-E2, and AtVHA-E3. W36 shares an 87% identity with ZmVHA-E and showed an 86%, 75%, 73%, 63%, 72%, 65%, and 69% identity with OsVHA-Ec, GmVHA-E, AtVHA-E1, AtVHA-E2, AtVHA-E3, OsVHA-Ea, and OsVHA-Eb, respectively  results suggested that nditions . Under dW36 cDNA sequence was fused to the C-terminus of the hGFP reporter gene and subcloned into an expression vector under the control of the CaMV 35S promoter. This construct was transferred into wheat protoplast cells by PEG (Polyethylene glycol) to investigate intra-cellular localization. Relative to the hGFP reporter, the W36::hGFP fusion protein was observed to be mainly localized in cytoplasm  a hW36 could improve abiotic stress tolerance, we tested the high salinity, mannitol, drought, and cold tolerance of W36 overexpressing plant lines .In addition, transgenic plants and control plants did not differ significantly in terms of survival rates and phenotypic changes under drought and cold treatments although T. aestivum cv. Xiao Baimai) seedlings were grew on seedbed at 25 °C under a 16 h light/8 h dark photoperiod for 10 days and then subjected to various stresses. Seedlings were placed in a 4 °C chamber for cold stress, and exposed to air on filter paper under 60% humidity conditions for rapid drought treatment. To mimic salinity, H2O2 and ABA treatments, seedlings were transferred into solutions containing 2% NaCl, 10 mM H2O2 and 100 µM ABA, respectively. Materials were collected at 0.5, 1, 2, 6, 12, 24, and 48 h after stress treatment.Wheat  in the two samples were investigated.To investigate the transcriptome response to stress in wheat plant, the Make multiple sequences comparison and homologous analysis by GenBank and DNAMAN software. Multiple alignments of the amino acid sequences were performed using ClustalX. Phylogenetic tree was constructed using MEGA 5.1 with neighbor-joining method.T. aestivum cv. Xiao Baimai) genome DNA (25–30 µg) was extracted according to SDS-phenol-chloroform method, digested individually with AluI, Sau3AI, BamHI, EcoRV or EcoRI at 37 °C for one night, fractionated on 0.8% agarose gel, and then blotted onto a nylon memebrane. Southern blotting was completed using 3'-termi of W36 labeled with α-32P-dCTP as a probe, washed with 2 × SSC/0.1% SDS at 65 °C, and then X-ray-plate was performed.Wheat , 5 μL of the 1:5 diluted cDNA, 0.5 μL each of 20 µM forward and reverse primers and 4 μL of PCR-grade water, in an ABI7300 real-time PCR machine . The PCR program consisted of 60 °C for 45 s, 94 °C for 3 min, followed by 40 cycles of 94 °C for 15 s, and 68 °C for 31 s. W36RTF: 5'-GGAAAGAGAAGCAGGTTGATGTTC-3', W36RTR: 5'-TCATAGAAACAGCTGTACCATCACC-3' Wheat actin gene was used as an internal standard [Total RNAs of the control and the treated seedlings were extracted with Trizol  kit following the manufacturer’s instructions and then treated with DNAase. First-strand cDNA was produced using 1 μg of total RNA as a template with 100 U of M-MLV reverse transcriptase  at 42 °C for 90 min with 1 μL Olig(dT)standard ,25.W36 were cloned into the SalI and XbaI sites of hGFP. The blank GFP vector (hGFP) was used as the control. The recombinant W36-hGFP fusion plasmids were transformed to wheat by the PEG-mediated method [For generation of the GFP fusion protein, the coding sequences of d method . Expressd method .TaqI, purified using ethanol precipitation, ligated using T4 DNA ligase and then used as template. Based on the cloned sequence obtained above, the 5'-flanking region of W36 was obtained through inverse PCR. PLACE [cis-acting regulatory DNA elements were used to analyze the 5'-flanking region.To amplify the 5'-flanking region, the genomic DNA was completely digested by restriction endonuclease R. PLACE , a databArabidopsis, the full-length W36 cDNA was ligated into the modified vector pBI121 under the control of the CaMV35S promoter. Columbia (Col-0) ecotype Arabidopsis plants were transformed using the Agrobacterium-mediated method [−1 kanamycin. T3 generation plants were used for further analysis.To construct an expression vector for d method . TransfoArabidopsis were placed on MS agar medium containing 150 mM NaCl and 300 mM mannitol for certain durations, respectively. Seed germination and growth were under long-day conditions (15 h light/9 h dark) at 22 °C. The percentage of germinated seeds was calculated based on the number of seedlings that reached the cotyledon stage at 2 weeks [In one set of experiments, seeds of wild-type and the transgenic  2 weeks .For drought treatment, plants were grown at normal growing conditions without watering for 2 weeks; and then rewatered under normal growing conditions. Cold treatments were performed by transferring the plants in a pre-cooled medium to growth chambers at −6 °C for 3 h, and then rewatered under normal growing conditions. Survival rates were assessed after two weeks .+-ATPase conducted proton motive force (PMF) and pumped H+ from cytoplasma into vacuole using the energy produced by hydrolyzing cytoplasma ATP. Thereby, V-type H+-ATPase could motive solute transportation between cytoplasma and vacuole, maintain solute relatively constant, and guarantee normal life activities. In this present study, the wheat W36 mainly localized in cytoplasm  in plant. Intersection of ABA and H2O2 signaling has been documented in previous studies. Evidence for ABA in triggering the production of H2O2 [2O2 up-regulating the ABA catabolism genes [2O2 [W36 contributed to V-type H+-ATPase in response to environmental stimuli.ABA, one of the “classical” plant hormones, regulates many aspects of plant growth and development, including seed dormancy and germination ,47,48. H of H2O2  and H2O2sm genes  has beennes [2O2 . ConsideW36, a single-copy gene, encoded the E subunit of the V-type H+-ATPase that accumulated in the cytoplasm in wheat. qRT-PCR analysis revealed that W36 could be induced by drought, cold, salt, and exogenous ABA treatment, which corresponds to the containment of stress-responsive elements in W36 promoter. Overexpression of W36 enhanced salt and mannitol tolerance in transgenic Arabidopsis plants. Therefore, W36 is involved in the plant response to osmotic stress.


To the Editor: Infection with group A rotavirus is among the leading causes of gastroenteritis in children, especially in sub-Saharan Africa (www.who.int/nuvi/surveillance/RV_Case_Defs.pdf). After informed consent and epidemiologic and clinical data had been obtained, a fecal specimen was collected from each child. Samples were transported to the Institut Pasteur de Bangui, where they were tested for rotavirus-A antigen by using the VIKIA Rota-Adeno test, . Results were immediately reported  to the referring physician.Rotavirus-A G-type (virus protein [VP] 7) and P-type (VP4) genotyping were performed by using previously described 2-step amplification methods . Prevalence was expressed in percentages. The χ−6). Because data were collected for only 8 months, annual rotavirus-A prevalence might have been underestimated or overestimated, a possible limitation of the study.A total of 250 infants and young children with diarrhea  were enrolled in this study. Results obtained by the VIKIA Rota-Adeno test revealed that 100 (40%) of these children were infected with rotavirus-A, mostly male children .The proportions of rotavirus-A infection in children <9 months of age and those ≥10 months of age were 37.3% (62/166) and 45.2% (38/84), respectively, (p = 0.2). Rotavirus-A infections were more prevalent during February–March  than during April–September  ; among P genotypes, type P[8] predominated , followed by P[6] . The predominant genotypic combination was G1P[8]  and G1P[6]  .–Despite the limitations of a short study period and low number of genotyped strains, this study reports useful information. It reveals that 40% of children hospitalized with acute diarrhea at Complexe Pédiatrique, Bangui, were infected with rotavirus-A during the study period, which included the end of the rotavirus-A gastroenteritis season. Most patients were <9 months of age. These results are similar to those found in the 1980s study at Complexe Pédiatrique, Bangui (Our study provides relevant data about the genotypes of rotavirus-A from children in the Central African Republic, 25 years after the most recent study (


Jatropha curcus is a good candidate plant for biodiesel production in tropical and subtropical regions. However, J. curcus is susceptible to the geminivirus Indian cassava mosaic virus (ICMV), and frequent viral disease outbreaks severely limit productivity. Therefore the development of J. curcus to carry on durable virus resistance remains crucial and poses a major biotechnological challenge.J. curcus plants expressing a hairpin, double-stranded (ds) RNA with sequences homologous to five key genes of ICMV-Dha strain DNA-A, which silences sequence-related viral genes thereby conferring ICMV resistance. Two rounds of virus inoculation were conducted via vacuum infiltration of ICMV-Dha. The durability and heritability of resistance conferred by the dsRNA was further tested to ascertain that T1 progeny transgenic plants were resistant to the ICMV-SG strain, which shared 94.5% nucleotides identity with the ICMV-Dha strain. Quantitative PCR analysis showed that resistant transgenic lines had no detectable virus.We generated transgenic J. curcus plants to include a resistance to prevailing geminiviruses in Asia. These virus-resistant transgenic J. curcus plants can be used in various Jatropha breeding programs.In this study we developed transgenic The online version of this article (doi:10.1186/s13068-014-0149-z) contains supplementary material, which is available to authorized users. Jatropha curcas, a small woody plant belonging to the Euphorbiaceae family, is a non-food oil seed crop mainly grown in the tropical and subtropical regions. This plant possesses several traits render this woody plant suitable for biodiesel feedstock production. It is easy to propagate and grows rapidly. It has a short gestation period, low seed cost and high oil content. Moreover, the ability of J. curcus to thrive on degraded soil and its wide adaptability to different growth conditions allows the use of marginal or non-arable wasteland for the application of this plant on an industrial scale. However, the productivity of J. curcus in the field is limited by the occurrence of Jatropha curcas mosaic disease (JcMD) [e (JcMD) -3. The de (JcMD)  and up te (JcMD) .Jatropha plants in Africa and South Asia [Jatropha plantations prompted us to investigate the biology of the virus in more detail.We have previously reported the first full-length genome sequence of one geminivirus, a strain of Indian cassava mosaic virus (ICMV-Dha), as the causative pathogen of JcMD found in Southern India . Followiuth Asia -6. Recenuth Asia . The recGeminiviridae can be classified into four genera: Begomovirus, Mastrevirus, Curtovirus and Topocuvirus. So far, all five Jatropha viral pathogens belong to one genera: Begomovirus. Most of these viruses contain two genomic components termed DNA-A and DNA-B (approximately 2.7 to 3.0 kb) and they are all exclusively transmitted via the whitefly, Bemisia tabaci. The virus DNA-A-positive strand encodes the coat protein (CP/AV1) involved in the encapsidation of viral DNA, virus movement and viral transmission by B. tabaci). Among other encoded proteins, the replication associated-protein (Rep) AC1 is absolutely required for the replication of both genomic components. The transcriptional activator protein (TrAP) AC2 is needed for transcriptional activation of viral gene transcription and plant host gene expression. The replication enhancer protein (Ren) AC3 greatly enhances viral DNA accumulation by interacting with Rep/AC1. Another viral protein, AC4, acts as a gene-silencing suppressor to compromise the host defense system. All these five genes are essential for the virus life cycle and pathogenesis [Geminiviruses, which are single-stranded DNA viruses infecting a range of economically important crop species  in tropical and subtropical regions, have become a major threat to world agriculture in the past decade . Based oogenesis .Jatropha germplasms. This strategy has the advantage that segregation patterns can be clearly observed between resistant and susceptible lines [J. curcus. Recently, we and other groups have established transformation platforms which facilitate the transfer of foreign genes into the J. curcus genome [J. curcus.Because of the capacity of geminiviruses to evolve rapidly by mutation, recombination and pseudo-recombination, the development of plants with durable virus resistance continues to be a major challenge. One strategy involves genetic crossing of resistant and susceptible le lines . Howeverle lines . Therefos genome -13, and J. curcus lines by expressing a hairpin double-stranded (ds) RNA targeting five key geminivirus DNA-A genes. Some of the transgenic lines displayed broad resistance to related geminiviruses, with 94.5% nucleotide identity in the transgene sequences.A major strategy to produce transgenic plants with virus resistance is based on the concept of pathogen-derived resistance (PDR) in which the transgene is derived from viral sequences. The mechanism of PDR includes protein-mediated resistance and RNA interference (RNAi). Both mechanisms have been shown to confer geminivirus resistance in transgenic tomato, common bean, cassava and others -18. HereAC5 gene, fragment 2 (250 bp) targets genes for TrAP/AC2 and Ren/AC3 and fragment 3 (609 bp) targets genes for Rep/AC1 and AC4  gene  35S promoter with double enhancers and named it pX9-hpICMV RNAi. Upon chemical induction with 17β-estradiol, promoter . The PCRAgrobacterium and a total of 133 T0 transgenic plants were generated. We performed PCR analysis on genomic DNA using two pairs of primers  value difference between symptomless resistant transgenic plants and uninfected control plants. In total, 15 independent transgenic lines presented with the symptomless phenotype and no virus could be detected in these resistant transgenic plants. However, we detected very high virus titers in susceptible transgenic plants, which were similar to those found in infected WT plants Figure . Six monNext, we performed Southern blotting to examine the transgene copy number in resistant lines. As there are two EcoRV sites around the two loxP sites, the genomic DNAs were digested with EcoRV Figure A. SoutheJ. curcus plants with between three and four true leaves were inoculated with ICMV-Dha. Two months after the virus challenge, WT plants and transgenic line 82–3 showed very obvious virus disease symptoms, whereas transgenic plants 82–1 and 82–2 grew normally (like uninfected control plants) -PCR sequencing) showed a consistent virus resistance trait Figure C. T1 see) Figure A. qPCR a) Figure B. To ana) Figure C. Southe) Figure D. OveralJatropha tree , which shares 94.5% nt homology with the ICMV-Dha strain [We also tested whether the ICMV resistance conferred by hpRNAi has broad virus resistance. We recently reported a second ICMV isolate from the Singaporean a strain . Before J. curcus.We reasoned these ICMV-resistant plants should be also resistant to multiple ICMV strains within a similar evolution clade. Multiple sequence alignment of full-length DNA-A was carried out using the ClustalV program Vector NTI®  with default parameters. Phylogenetic trees were constructed from multiple alignments using the neighbor-joining method in the MEGA4 program , and a bAC1 gene , the coat protein gene and AC4 gene have been shown to confer geminivirus resistance in tomato, bean and tobacco plants [Chickpea chlorotic dwarf Pakistan virus (CpCDPKV), a member of Mastrevirus  a multi-target approach was adopted. The RNAi trigger sequences include those encoding the N-terminus of the Rep gene, the large intergenic  and the N-terminus of the movement protein gene [Phaseolus vulgaris) showed that the begomovirus Bean golden mosaic virus (BGMV) can also be suppressed by the expression of a hpRNA transgene derived from AC1 [During the last decade geminiviruses have emerged as one of the major causative pathogens of economically important crops such as cassava, cotton and tomato. As resistance genes are not always available in the relevant crop germplasm, biotechnological approaches have been used to generate transgenic plants to confer virus resistance. Several different strategies have been reported to confer virus resistance to transgenic model plants by expression of: (1) trunked viral proteins, (2) artificial zinc finger nuclease, (3) peptide aptamers capable of binding to viral proteins, (4) non-pathogen-derived antiviral agents and so on ,22-25. Ho plants -16,30-32from AC1 .J. curcus plants with high resistance to multiple virus infection. After two rounds of virus challenge, resistant plants were free of virus symptoms and no virus could be detected , thereby allowing us to ascertain resistance to related viruses. There is 95.3% nt identity between the ICMV-Dha and ICMV-SG strains if we include sequences that we used for the hpRNAi construction. Two other n Figure . In viewn Figure  and 7. LJ. curcus plants with resistance to ICMV via expression of a hairpin dsRNA with sequences homologous to five key ICMV DNA-A genes. Virus resistance was confirmed through two rounds of inoculation with J. curcus-isolated strains of ICMV, ICMV-Dha and ICMV-SG. The T1 progeny transgenic plants were virus resistant, confirming that the resistance phenotype was heritable. Transgenic plants generated from this study can be used in various Jatropha breeding programs.We generated transgenic F1-5: 5’-AGCGCCTCGAGCGTTTGAATCTAGACACGATGTGCTC-3’,F1-3: 5’-TGCAGTGATGAGTTCCCCTGTGCGTGAACATGTTAAACACCTCACCGAAATCCT-3’,F2-5: 5’-GATTTCGGTGAGGTGTTTAACATGTTCACGCACAGGGGAACTCATCACTGCAGC-3’,F2-3: 5’-GGAATGTTCCCCATTCCAAGGTATCGAATACCCTCAAGAAACGCCAGGTCTGAG-3’,F3-5: 5’-CAGACCTGGCGTTTCTTGAGGGTATTCGATACCTTGGAATGGGGAACATTCCAG-3’,F3-3: 5’-AGCGCAAGCTTTAGCTGGAATTGGGCCCTGGATTGCAGA-3’.We replaced the G10-90 promoter in pX7-GFP  with a sJ. curcus .The PCR fragment was inserted in the sense orientation into the t vector  to generJatropha curcas (JcMD) elite plants  [Seeds were obtained from ngapore) . Cotyledngapore) .Genomic DNAs were prepared with DNeasy plant mini kits  according to the manufacturer’s instructions. Approximately 50 ng of genomic DNA were used for PCR. The reactions were subjected to 94 C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes for 40 cycles. Primers P1 (5’-ATCTCCACTGACGTAAGGGATGAC-3’) and P2 (5’-GTTTAAGATCTACTTACGTAATCAAGC-3’) were used to check the occurrence of marker excision events. DNA fragments containing the hygromycin resistance gene were amplified by either P3-1 (5’-GAGGGCGAAGAATCTCGTGCTTTC-3’) and P4-1 (5’-TACTTCTACACAGCCATCGGTCCA-3’) or P3-2 (5’-GAAGAATCTCGTGCTTTCAG-3’) and P4-2 (5’-CAACCAAGCTCTGATAGAGT-3’). The product amplified by primers P3-1 and P4-1 was 885 bp, whereas the product amplified by primers P3-2 and P4-2 was 745 bp  and ran in ABI7900HT. Forward primer 5’-CTGCACAATGTGGGACCCTTTG-3’ and reverse primer 5’-CTTCGCCCTGATGACAGAGATC-3’ were used for the amplification of viral DNA-A of either ICMV-Dha (128–290 nt) or ICMV-SG (127–289 nt). All samples were run in triplicate and the data was analyzed with RQ manager at a preset threshold cycle value . The o et al. , the rel+ membrane  following standard procedures [Genomic DNA was digested with restriction enzymes and separated on 0.8% agarose gels. The gels were processed and resolved DNA bands transferred to a nylon Hybond-Nocedures . Membran


C677T and A1298C are the most common allelic variants of Methylenetetrahydrofolate Reductase (MTHFR) gene. The association between MTHFR polymorphisms and the occurrence of non-alcoholic fatty liver disease (NAFLD) remains controversial. This study was thus performed to examine whether MTHFR mutations are associated with the susceptibility to NAFLD.st, 2016), were searched for eligible case-control studies. The odd radio (OR), 95% confidence interval (CI) and P value were calculated through Mantel-Haenszel statistics under random- or fixed-effect model.A first meta-analysis on the association between the MTHFR polymorphisms and NAFLD risks was carried out via Review Manager 5.0 and Stata/SE 12.0 software. The on-line databases, such as PubMed, EMBASE, CENTRAL, WOS, Scopus and EBSCOhost (updated to April 1P = 0.04) and recessive model , but not the other genetics models, compared with control group. For A1298C, significantly increased NAFLD risks were detected in allele model , homozygote model , dominant model  and recessive model , but not heterozygote model.Eight articles (785 cases and 1188 controls) contributed data to the current meta-analysis. For C677T, increased NAFLD risks were observed in case group under homozygote model (T/T vs C/C, OR = 1.49, 95% CI = 1.03~2.15, T/T genotype of MTHFR C677T polymorphism and C/C genotype of MTHFR A1298C are more likely to be associated with the susceptibility to NAFLD. Human Methylenetetrahydrofolate Reductase (MTHFR) gene is located at chromosome 1p36.3 and contains 11 exons . As a kiNon-alcoholic fatty liver disease (NAFLD), the most common chronic liver disease, is the hepatic manifestation of the metabolic syndrome without a history of excess alcohol consumption –14. The To our knowledge, no meta-analysis on the association of MTHFR genetic variants and overall NAFLD risks has been reported. Hence, we first carried out a meta-analysis to investigate the relationship between MTHFR polymorphisms (C677T and A1298C) and susceptibility to NAFLD. Our finding showed that both C677T and A1298C polymorphisms of MTHFR gene might positively correlate to the risks of NAFLD.st, 2016. There was no language or region restriction. The combinations of following keywords were used: “Methylenetetrahydrofolate Reductase (NADPH)” or “Methylene-THF Reductase (NADPH)” or “MTHFR” or “Methylenetetra hydrofolate Reductase”; “Non-alcoholic Fatty Liver Disease” or “NAFLD” or “Fatty Liver, Nonalcoholic” or “Livers, Nonalcoholic Fatty” or “Nonalcoholic Fatty Livers” or “Nonalcoholic Steatohepatitis” or “Nonalcoholic Steatohepatitides” or “Steatohepatitides, Nonalcoholic” or “Steatohepatitis, Nonalcoholic”; “Polymorphism, Genetic” or “Genetic Polymorphisms” or “Genetic Polymorphism” or “Polymorphism (Genetics)” or “Polymorphisms, Genetic”. The full details of databases searching terms were also provided , Cochrane Central Register of Controlled Trials (CENTRAL), Web of Science (WOS), China National Knowledge Infrastructure (CNKI)/WANFANG, Scopus and EBSCOhost in April 1provided .The eligible case-control studies were identified according to the following inclusion and exclusion criteria. Inclusion criteria: 1) The data on the association between MTHFR polymorphisms and susceptibility to NAFLD was provided; 2) The individual genotype frequencies for MTHFR polymorphisms could be extracted. Exclusion criteria: 1) duplicated studies; 2) reviews or books; 3) non-clinical data; 4) other genes; 5) non-NAFLD diseases; 6) case, trial, or non-polymorphism; 7) meeting/conference abstracts; 8) unavailable data.2 and P value of Hardy-Weinberg Equilibrium (HWE) test in control group. HWE value was calculated by chi-squared test and P value less than 0.05 was considered a departure from HWE.Data was extracted from qualified articles independently by the authors (MYS LZ SLS JNL) using the same reporting form. The controversial evaluations were resolved through discussion. If the data was unavailable, an attempt was made to contact corresponding author to request missing data via E-mail. The following information was extracted: mutation site, first author, year of publication, country, ethnicity, sample sizes in case and control group, source of control, genotyping method, gender and age in case group, disease group, allele and genotype frequencies in each group, The ΧP value, odd radio (OR) and corresponding 95% confidence interval (CI) were calculated by Mantel-Haenszel statistics under the allele, homozygote, heterozygote, dominant or recessive models. P value <0.05 was considered statistically significant association between C677T and A1298C polymorphisms of MTHFR and NAFLD risks. Χ2-based Q statistic and I2 test were applied to analyze the overall heterogeneities. When I2 values < 25% or P value of heterogeneity >0.10, a fixed-effect model was selected for Mantel-Haenszel statistics. Otherwise, a random-effect model was used [The was used –35. Whenwas used . Statistst, 2016), to obtain the eligible case-control studies. Flow chart of studies selection in meta-analysis was shown in We searched the on-line electronic databases, including PubMed, EMBASE, CENTRAL, WOS, CNKI/WANFANG, Scopus and EBSCOhost , EMBASE (n = 29), CENTRAL (n = 0), WOS (n = 24), CNKI/WANFANG (n = 3), Scopus (n = 144) and EBSCOhost (n = 11). After 50 duplicated articles were removed, the 153 articles were excluded by screening the title and abstract: 59 articles are reviews or books; 11 articles do not provide the clinical data; 16 articles are related to the other genes; 46 articles focus on non-NAFLD diseases; 21 articles are case, trial or fail to contain the data of gene polymorphism. 18 potentially articles were then assessed for eligibility. The data was extracted from all these full-text articles. As shown in 2 = 56% and P = 0.004) revealed that high heterogeneity among studies was detected for C677T polymorphism. Random-effect model was thus applied for meta-analysis. The data on the association between C677T allele frequency of MTHFR and susceptibility to NAFLD was obtained . In addition, the potential publication bias was evaluated by Begg’s funnel plot with pseudo 95% confidence limits. The result of P = 0.893) excludes the presence of large publication bias.Next, the genetic association between MTHFR C677T polymorphism and susceptibility to NAFLD was measured. As shown in 2 = 47% and P = 0.02), C/T+T/T vs C/C (I2 = 45% and P = 0.03) and T/T vs C/C+C/T (I2 = 26% and P = 0.16) data indicated the existence of the moderate degree of heterogeneity across studies (2 = 21% and P = 0.21). Pooled analysis for the association between C677T genotype frequencies and the risks of NAFLD was shown in P = 0.04) and recessive model , but not the other genetics models . In addition, the results of HWE test . The subgroup analyses under all genetic models were also performed based on ethnicity or HWE  and HWE P>0.05 subgroup . In order to evaluate the influence of each study on the overall OR under all genetic models, the sensitivity meta-analyses, in which one study is omitted at a time, were also performed. As shown in The contrast of the homozygote model (T/T vs C/C), heterozygote model (C/T vs C/C), dominant model (C/T+T/T vs C/C) and recessive model (T/T vs C/C+C/T) was then detected respectively, through the meta-analysis, in that the data on genotype frequencies of MTHFR C677T polymorphism was available. Genotype distribution and characteristics of MTHFR C677T polymorphism in different case-control studies were shown in  studies . A randoHWE test  in contry or HWE  via StatP>0.05). In addition, all the case-control studies were performed in Caucasian population. We then first performed the meta-analysis between the allele frequency of MTHFR A1298C and the susceptibility to NAFLD under C vs A model. As shown in 2 = 66% and P = 0.001) for meta-analysis. The data  was obtained in C vs A comparison of MTHFR A1298C. The basically symmetric plot  did not provide the statistical evidence for publication bias , A/C vs A/A model (I2 = 53% and P = 0.02), A/C+C/C vs A/A model (I2 = 63% and P = 0.003) was obtained and random-effect model was used. For the C/C vs A/A+A/C model, fixed-effect model was used (I2 = 14% and P = 0.31). A significantly increased NAFLD risks was observed in homozygote model , dominant model  and recessive models , but not heterozygote model . Moreover, similar results were obtained in the sensitivity meta-analyses under all genetic models  and susceptibility to many clinical diseases, such as gastric cancer, hepatocellular carcinoma, NAFLD, neural tube defects, acute lymphoblastic leukemia and renal/heart failure , 38–42. The positive correlation between NAFLD susceptibility and two MTHFR variants (C677T and A1298C) was observed in our statistical evidence. For C677T polymorphism, an increased NAFLD risk was observed under homozygote model (T/T vs C/C) and recessive model (T/T vs C/C+C/T), but not T vs C, C/T vs C/C and C/T+T/T vs C/C models, suggesting that T/T genotype of MTHFR C677T polymorphism might have the increased risks of NAFLD in general population. Moreover, we found that a significantly increased NAFLD risk was detected in Asian population under the comparison of T vs C, T/T vs C/C, C/T vs C/C; C/T+T/T vs C/C. Similarly, the meta-analysis of A1298C polymorphism based on 11 case-control studies in Caucasian population provided the evidence that a significantly increased NAFLD risk was observed under allele model (C vs A), homozygote model (C/C vs A/A), dominant model (A/C+ C/C vs A/C) and recessive model (C/C vs A/A+A/C), but not heterozygote model (A/C vs A/A), suggesting that C/C genotype of MTHFR A1298C polymorphism might be linked to the susceptibility to NAFLD in Caucasian population.The C677T polymorphism means the substitution of C (cytosine) to T (thymine) at nucleotide position 677, which results in the transition from alanine to valine, while A1298C polymorphism refers to the transition of A (adenine) to C (cytosine) at position 1298, which leads to an amino acid substitution from glutamic acid to alanine . Folate There are some shortages or limitations in this meta-analysis, which should be pointed out. For example, no large sample size was included in the case/control groups of meta-analysis. It is still possible that other unpublished or undetected studies are present, although we selected the eligible studies independently according to the inclusion and exclusion criteria. The potential selection bias still may affect the reliability of our findings. Different degree of heterogeneity and departure from HWE was also detected in some comparisons or case-control studies. Furthermore, it was reported that C677C/C1298C compound genotype confers increased risks of NASH in Turkish women patients . UnfortuVery complicated natural history of NAFLD was existed, and multiple genetic or environmental factors contribute to the occurrence and progression of the NAFLD –53. NAFLAll in all, this is the first meta-analysis to provide evidence that C677T and A1298C mutations of MTHFR are significantly associated with an increased risk of NAFLD. The homozygous T/T genotype of MTHFR C677T and C/C genotype of MTHFR A1298C polymorphism seem to be more susceptible to NAFLD. More case-control studies are warranted to validate the conclusion.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


L-phenylalanine, 4-methoxy-L-phenylalanine, 4-phenyl-L-phenyalanine, O-tert-butyl-L-tyrosine) inverts the enantiotope preference of the enzyme toward 2-chloro-1-phenylethanone with close side chain correlation. Analyses of enzyme activity, substrate affinity and ternary structure of the mutants revealed that substitution at Trp222 causes a notable change in the overall enzyme structure, and specifically in the entrance tunnel to the active center. The size of residue 222 in DKR is vital to its enantiotope preference. Trp222 serves as a “gate keeper” to control the direction of substrate entry into the active center. Consequently, opposite substrate-binding orientations produce respective alcohol enantiomers.Trp222 of diketoreductase (DKR), an enzyme responsible for reducing a variety of ketones to chiral alcohols, is located at the hydrophobic dimeric interface of the C-terminus. Single substitutions at DKR Trp222 with either canonical  or unnatural amino acids (UAAs) (4-cyano- Enantioselectivity is an intriguing property that allows certain enzymes to be exploited to yield enantiomerically pure chemicals for use in diagnostics, materials and pharmaceuticals Directed evolution is the most promising approach to fine-tune enzyme enantioselectivity to a desired level R-preference. In screening different mutants against the mono-ketone substrate of 2-chloro-1-phenylethanone, enantioselectivity of the W222F variant changed from an Re- to Si-preference (Diketoreductase (DKR), a homodimeric protein containing 283 amino acids in each subunit, is a useful biocatalyst that stereoselectively reduces β,δ-diketo esters to corresponding dihydroxy products for biosynthesis of statin side chains eference . This reAccording to a recently solved crystal structure and elucidated catalytic mechanism of DKR Escherichia coli strains DH5α and BL21 (DE3) were obtained from Tiangen Biotech Co., Ltd. . The AxyPrep Plasmid Miniprep Kit was from Axygen Biotech Ltd. (USA). The pEVOL-ONBYRS plasmid was a gift from Professor Peter. G. Schultz at Scripps Research Institute . The UAAs 4-cyano-l-phenylalanine, 4-methoxy-l-phenylalanine, 4-phenyl-L-phenyalanine and O-tert-butyl-l-tyrosine were purchased from Adamas Reagent Co., Ltd. (Switzerland). Isopropyl-β-d-thiogalactopyranoside (IPTG), NADH, and acetoacetyl-CoA were purchased from Sigma Chemical Co. .Primers used in site-directed mutagenesis were synthesized by Invitrogen Inc. . Mutations were confirmed by DNA sequencing with an ABI Genetic Analyzer 3730 . The pET22b(+)-DKR construct containing plasmid pET22b and full-length WT-DKR was used as a template for mutagenesis. Site-directed mutagenesis was performed with the QuikChange Lightning site-directed mutagenesis kit  according to the manufacturer's protocol. Two overlapping complementary primers containing the desired nucleotide changes were designed for each mutation reaction. The plasmid pET22b-DKR222TAG with Trp222 mutated to TAG and a stop codon (TGA) was used to incorporate UAAs.p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNFRS) BglII & SacI sites were synthesized by Invitrogen Inc. . The primer MGU110 (5′- CATATGTAACGCCGTTATACGTTGTT -3′) and MGU111 (5′- GACGTCAAAAGCACGCAAACTCAATA -3′) were used to amplify the pCNFRS gene containing NdeI and PstI sites at the N-terminus and C-terminus, respectively. These two genes were then digested with restriction enzymes NdeI & PstI, and BglII & SalI, respectively, and then ligated into the plasmid pEVOL in which the ONBYRS gene was deleted after digestion with NdeI and PstI. The resulting plasmid contained pCNFRSII-tRNA with two copies of pCNFRS (expressed under the control of an araBAD promoter) and a copy of suppressor tRNA (expressed under the control of an Ipp promoter). ONBYRS and CNFRS are largely homologous, with differences at only nine amino acid residues , and thus recognize the same cognate tRNA.The et.al.E. coli BL21 (DE3) cells were co-transformed with pCNFRSII-tRNA and pET-DKR222TAG. The transformed cells were recovered in 1 ml of LB medium shaken for 1 h at 37°C before plating on a LB agar plate containing chloramphenicol  and ampicillin . An abridged method for expressing UAA mutants was adapted from a previous report −1 Cm and 100 µg·ml−1 Amp. Cells were grown at 37°C with shaking at 220 r.p.m. When the OD600 reached 0.5, expression was induced by addition of 1 mM IPTG and 0.2% arabinose for 30 h.WT-DKR was expressed as described by Wu The procedures for purification of DKR and DKR mutants were described previously −1. Mobile phases A and B consisted of 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile, respectively. Elution was achieved with a gradient of 25–30% B in 25 min, and kept at 30% B for an additional 5 min. The retention times of (R)-1-hydroxy-2-chloro-phenylethane and (S)-1-hydroxy-2-chloro-phenylethane were 10.4 min and 12.2 min, respectively.Chiral HPLC was performed on a Chiralcel OD-RH column  at 25°C with an injection volume of 10 µl and a flow rate of 0.5 ml·min−1 cm−1), as described by Huang et al.The enzyme activity of wild type and mutant DKR was determined on a UV-1700 array spectrophotometer  by monitoring the decrease in absorbance of NADH  or different NADH quantities  were prepared in the standard assay conditions. d) for substrate and NADH were determined by fluorescence quenching experiments in 96-well plates. DKR was titrated with increasing amounts of substrate/NADH and the resulting decrease in protein fluorescence intensity was monitored in a volume of 200 µl/well containing 0.1M Tris-HCl (pH 6.5), 6.3–7.0 µM enzyme and different final concentrations of the substrate (0–1000 µM)/NADH(0–300 µM). Kd values were calculated by fitting the data to the quadratic equation F), and [P0] (µM) is the enzyme concentration.Fluorescent spectra were recorded at room temperature on a Tecan Flurospectrometer  with a Safire microplate reader in combination with XFLUOR4 software, version 4.5. The excitation and emission monochromators were set at 5 nm slit widths. After excitation at 290 nm for selective tryptophan excitement, sample emission was measured from 300 to 400 nm. The average of three separate scans was analyzed, and background due to buffer was subtracted. Because the decrease in fluorescence was attributed to the binding between substrate or NADH and DKR, dissociation constants  + complex was minimized with an energy minimization algorithm that uses the Amber 99 force field. This energy-minimized structure was used as a template for virtual screenings. The binding site was defined as a sphere encompassing protein residues within 4.5 Å of S122-H143-N146-N194, and was followed by restoration through London dG. Ligand placements were refined again by the Amber 99 force field. Default values were applied for other parameters, and 20 genetic algorithm runs were performed for each docking. Finally, a three-dimensional protein-substrate binding model was generated using MOE-2009. Ligand conformation was evaluated based on the S score, which measures interactions. Compounds showed different binding modes, and those with the lowest S scores were chosen for evaluation.WT and nine mutants of DKR were chosen as targets for docking-based virtual screening. Receptor files, ligands and docking parameter files were prepared using MOE. The X-ray crystal structure of WT-DKR (PDB: 4E13) containing two NAD molecules was used. All hydrogen atoms and partial charges were added to the protein using Protonate 3D. The energy of the DKR-NADRe face preference, while a Phe substitution at Trp222 inverted the enantiotope selectivity from Re- to Si-  values that were comparable to W222F, W222Y showed an e.e. that was increased by 6.2-fold compared with W222F. Due to the small difference in molecular volume between Phe and Tyr, this significant and unusual increase of enantiopreference might be the result of increased polarity and additional H-bonding between the hydroxyl group of Tyr and the residue(s) in active center. Nevertheless, compared to WT-DKR, the results confirm that the smaller size of the side chain did affect the enantiotope preference and thus there could indeed be a correlation between the side chain and enantiopreference.WT-DKR reduces 2-chloro-1-phenylethanone to its corresponding alcohol 1-hydroxy-2-chloro-phenylethane with an - to Si- . This realcohols . AlthougRe- to Si-, if the molecular volume of the side chain is a major factor, substitution of Trp222 with bulky residues should result in a preference for the Re-face and production of R-alcohol, which is similar to WT, and the e.e would thus increase with side chain size. Trp has the largest side chain among the twenty canonical amino acids, so we genetically incorporated UAAs to increase the side chain size using an expanded genetic code. In this system, UAAs can be genetically incorporated into proteins using engineered orthogonal tRNA/aminoacyl-tRNA synthetase pairs. Briefly, an orthognal tRNA/synthetase pair evolved to be specific for the UAA should be expressed in the target cell together with the gene of interest. An amber stop codon UAG is introduced at the desired site for UAA insertion in the target gene. The orthogonal tRNA synthetase charges the UAA onto the cognate tRNA, which recognizes the UAG codon and then incorporates the UAA during translation p-cyanophenylalanine-specific aminoacyl-tRNA synthetase (pCNFRS), together with its cognate amber nonsense suppressor tRNA, can incorporate multiple UAAs with bulky side chains and the polyspecificity of pCNFRS allowed substitutions with multiple UAAs l-phenylalanine (MeOF); 4-phenyl-l-phenyalanine (BiF); and O-tert-butyl-l-tyrosine (BuOF) (dkr gene in the pET22b(+) vector for UAA incorporation. Then the plasmid pCNFRSII-tRNA (E. coli BL21(DE3) cells with plasmid pCNFRSII-tRNA and pET22b(+)-DKR222TAG, four variants incorporating UAAs were expressed after IPTG induction. Mutant proteins were purified through two chromatographic steps as described previously Since smaller amino acids at residue 222 in DKR caused preferential enantiotope inversion from e (BuOF) . We firsSII-tRNA , which hRe-preference by the three mutants containing larger amino acids at residue 222. DKR Trp222 replacement by CNF, which contains a side chain that has a lower molecular volume than Trp, retained the Si-preference . Unlike smaller amino acid substitutions, increases in Re-preference correlated with side chain size increases when Trp222 was replaced by bulky residues, which supports the thinking that increased side chain bulk results in a more marked Re-preference.Similar to WT-DKR, 2-chloro-1-phenylethanone was reduced with eference . As indimK. Mutant binding affinity for the substrate increased with bulkier UAAs. With the exception of the BiF substitution, the catk values of all other variants decreased by varying degrees, with W222V, which has the smallest side chain, being only 20% that of WT-DKR. Meanwhile, the BiF mutant, with a biphenyl group at residue 222, showed a slight increase in catk (0.34 to 0.50), which may result from a stronger BiF-dependent aromatic-aromatic interaction in the protein structure that enhances transition state stabilization. Overall, the catalytic efficiency (catk/mK) of these mutants was largely affected by mK rather than catk.We compared kinetic behaviors of mutants with that of WT-DKR . MutantsKd) values was roughly consistent with catk. The differences in Kd values were very minor and similar to WT values , an overlay between the structures of nine mutants and WT-DKR revealed a notable change in backbone architecture (RMSD>1.0 Å) . The RMSEven though a marked change in α-helix 12 was observed in the mutants, this helix does not directly interact with the active center. A structural overlay between WT-DKR and the nine mutants was used to determine how the mutations might affect enzymatic enantiopreference . RemarkaS)-configuration binding mode were lower for V, L, M, F, Y and CNF substitutions, whereas WT and substitutions of MeoF, BiF and BuOF favored the pro-(R)-configuration binding mode. However, the docking score differences between each mode were not obvious, indicating that the enantiotope preference depends strongly on Trp222 substitution instead of the orientational preference for the active site itself.In an attempt to rationalize these observations, we used computational docking to investigate the effects of these mutations on the binding of 2-chloro-1-phenylethanone to DKR variants. When WT-DKR and nine mutants were docked with the substrate, two opposing binding modes that produced respective alcohol enantiomers were found . Active Si-preference mutants, substrate orientation was asymmetric and disordered  and a copy of a suppressor tRNA (expressed under the control of an Ipp promoter).(DOC)Click here for additional data file.Figure S2MALDI-TOF/MS analysis of purified mutants with UAAs at residue 222 after tryptic digestion. (A) CNF substitution; (B) MeOF substitution; (C) BiF substitution; (D) BuOF substitution.(DOC)Click here for additional data file.Figure S3RMSD between WT-DKR and nine DKR mutants. The RMSD by residue plot shows the residue-by-residue quality of superposition. For each alignment column used during the superposition, the RMSD value is represented by a vertical bar. Poor RMSD values are highlighted by dotted red horizontal lines with a 2.0 Å cutoff. Residue pairs above this line indicate obvious change. Residues marked by the pink line are located in α-helix 12. Residues located in α-helix 14 are marked by a blue line.(DOC)Click here for additional data file.Figure S4Comparison of models for enzymatic catalysis. A hydride attack from two opposite orientations produces respective alcohol enantiomers. Substrate 2-chloro-1-phenylethanone is shown as a teal sphere. Residues that serve as proton donors are highlighted in magenta. (A1) Pro-(S)-configurations in W222V; (A2) Pro-(R)-configurations in W222V; (B1) Pro-(S)-configurations in W222L; (B2) Pro-(R)-configurations in W222L; (C1) Pro-(S)-configurations in W222M; (C2) Pro-(R)-configurations in W222M; (D1) Pro-(S)-configurations in W222F; (D2) Pro-(R)-configurations in W222F; (E1) Pro-(S)-configurations in W222Y; (E2) Pro-(R)-configurations in W222Y; (F1) Pro-(S)-configurations in CNF; (F2) Pro-(R)-configurations in CNF; (G1) Pro-(R)-configurations in WT; (G2) Pro-(S)-configurations in WT; (H1) Pro-(R)-configurations in MeOF; (H2) Pro-(S)-configurations in WeOF; (I1) Pro-(R)-configurations in BiF; (I2) Pro-(S)-configurations in BiF; (J1) Pro-(R)-configurations in BuOF; (J2) Pro-(S)-configurations in BuOF.(DOC)Click here for additional data file.Figure S5Binding interactions of 2-chloro-1-phenylethanone with WT-DKR and DKR mutants. (A1) Substrate was attached from the Si face in W222V; (A2) Substrate was attached from the Re face in W222V; (B1) Substrate was attached from the Si face in W222L; (B2) Substrate was attached from the Re face in W222L; (C1) Substrate was attached from the Si face in W222M; (C2) Substrate was attached from the Re face in W222M; (D1) Substrate was attached from the Si face in W222F; (D2) Substrate was attached from the Re face in W222F; (E1) Substrate was attached from the Si face in W222Y; (E2) Substrate was attached from the Re face in W222Y; (F1) Substrate was attached from the Si face in CNF; (F2) Substrate was attached from the Re face in CNF; (G1) Substrate was attached from the Re face in WT; (G2) Substrate was attached from the Si face in WT; (H1) Substrate was attached from the Re face in MeOF; (H2) Substrate was attached from the Si face in MeOF; (I1) Substrate was attached from the Re face in BiF; (I2) Substrate was attached from the Si face in BiF; (J1)Substrate was attached from the Re face in BuOF; (J2) Substrate was attached from the Si face in BuOF.(DOC)Click here for additional data file.Table S1Stereochemical quality and model evaluation of WT-DKR and DKR mutants.(DOC)Click here for additional data file.Table S2a.Root mean square deviations  of WT-DKR and mutants(DOC)Click here for additional data file.


HOXB1 gene plays a critical role as an oncogene in diverse tumors. However, the functional role of HOXB1 and the mechanism regulating HOXB1 expression in glioma are not fully understood. A preliminary bioinformatics analysis showed that HOXB1 is ectopically expressed in glioma, and that HOXB1 is a possible target of miR-3175. In this study, we investigated the function of HOXB1 and the relationship between HOXB1 and miR-3175 in glioma. We show that HOXB1 expression is significantly downregulated in glioma tissues and cell lines, and that its expression may be closely associated with the degree of malignancy. Reduced HOXB1 expression promoted the proliferation and invasion of glioma cells, and inhibited their apoptosis in vitro, and the downregulation of HOXB1 was also associated with worse survival in glioma patients. More importantly, HOXB1 was shown experimentally to be a direct target of miR-3175 in this study. The downregulated expression of miR-3175 inhibited cell proliferation and invasion, and promoted apoptosis in glioma. The oncogenicity induced by low HOXB1 expression was prevented by an miR-3175 inhibitor in glioma cells. Our results suggest that HOXB1 functions as a tumor suppressor, regulated by miR-3175 in glioma. These results clarify the pathogenesis of glioma and offer a potential target for its treatment.The  Glioma is the most frequent primary malignant tumor of the adult central nervous system (CNS), and is characterized by high morbidity and poor survival –3. DespiThe HOX genes encode a highly conserved family of transcription factors, containing a 60-amino-acid, helix-turn-helix DNA-binding domain, that play important roles in development, regulating numerous processes, including apoptosis, receptor signaling, differentiation, motility, and angiogenesis . HOXB1 iHOXB1 3′-untranslated region (3′-UTR), and the miRNA they all predicted was miR-3175. In this study, we suggest that HOXB1 functions as a tumor suppressor gene in glioma and that the expression of HOXB1 is regulated by miR-3175. These results extend our understanding of the molecular mechanism of the tumorigenesis of glioma and offer a potential target for glioma therapies.The transcription of the HOX genes is regulated by several proteins and RNAs, including the trithorax group proteins, polycomb repressor complex 2 (PRC2), HOTAIR, and microRNAs (miRNAs) . It is whttp://www.cgga.org.cn/) and 180 gliomas from the Gene Expression Omnibus website .Human glioma specimens and normal tissues were obtained from the Department of Neurosurgery, China-Japan Union Hospital of Jinlin University. This study obtained Institutional Review Board (IRB) approval regarding the use of human samples for experimental studies from the Ethics Committees of the China-Japan Union Hospital of Jilin University and written informed consent was obtained from all the patients or their families. We also obtained written informed consent from guardians on behalf of the minors enrolled in the study. Forty glioma tissue specimens were taken from patients who underwent surgery between September 2010 and October 2012. All glioma specimens were histologically classified and graded according to the World Health Organization Classification of Tumors of the Central Nervous System guideline. Complete clinical data were acquired at the initial diagnosis and follow-ups. To maintain the integrity of the data, we adhered to the principles expressed in the Declaration of Helsinki. Eight nonneoplastic brain tissue specimens, for use as controls, were obtained from patients with severe traumatic brain injury, who had required partial resection of their brain tissue. All human tissue specimens were freshly resected at surgery and immediately frozen in liquid nitrogen until analysis. Overall survival (OS) and progression-free survival (PFS) were assessed and calculated from the follow-up data of the glioma patients. OS was defined as the period from the date of the pathological diagnosis to death; PFS was defined as the period from the date of the pathological diagnosis to glioma progression, presenting as new neurological symptoms. We downloaded mRNA expression microarray data for 212 gliomas from the Chinese Glioma Genome Atlas (CGGA) data portal , were purchased from the Type Culture Collection of the Chinese Academy of Sciences . The cells were cultured in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum , 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% COHOXB1 and miR-3175, respectively. qRT-PCR was performed with the StepOnePlus™ Real-Time PCR System  using the UltraSYBR Mixture . HOXB1 expression was normalized to that of the endogenous control housekeeping gene ACTB, encoding β-actin, and miR-3175 expression was normalized to that of U6. The PCR cycling parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s. The qRT-PCR results were evaluated with the 2–ΔΔCt method. The following primers were used in the PCR reactions: HOXB1 forward: 5′-GGTATGCTCCTGCCGCCTGCA-3′ and HOXB1 reverse: 5′-ATCAGCATAGGCCGGTGCAA-3′; miR-3175 stem-loop primer 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACGTCACT-3′, miR-3175 forward: 5′-GATACTCACGGGGAGAGAACGCAG-3′, miR-3175 reverse: 5′-GTGCAGGGTCCGAGGT-3′; β-actin forward: 5′-GGCACCACACCTTCTACAAT-3′ and β-actin reverse: 5′-GTGGTGGTGAAGCTGTAGCC-3′; and U6 forward: 5′-CTCGCTTCGGCAGCACA-3′ and U6 reverse: 5′-AACGCTTCACGAATTTGCGT-3′.Total RNA was extracted from the human tissue specimens and glioma cells with TRIzol Reagent , according to the manufacturer’s instructions. The first-strand complementary DNAs (cDNAs) were synthesized with the HiFi-MMLV cDNA Kit , according to the manufacturer’s instructions. The traditional oligo(dT) primer method and the stem-loop-specific primer method were used to verify the expression levels of Total proteins were extracted from all the cultured cells with RIPA buffer  containing 1 mM phenylmethanesulfonyl fluoride, according to the manufacturer’s protocol. The protein concentrations were determined with a BCA Protein Assay Kit , and the proteins were then separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked overnight at 4°C with 5% bovine serum albumin . After the membranes were washed with Tris-buffered saline-Tween 20 at room temperature, they were incubated sequentially with diluted (1:500) primary antibodies directed against HOXB1  and other proteins, including procaspase-3, p53, and cytochrome C . The membranes were washed again and incubated with a horseradish-peroxidase-conjugated secondary antibody . β-Actin was used as the endogenous reference control.2O2) and nonspecific antigens were blocked with 5% BSA. The slides were then incubated overnight at 4°C with a primary antibody directed against HOXB1 . After the slides were washed three times with phosphate-buffered saline (PBS) for 5 min each at room temperature, a biotinylated secondary antibody (1:1000) was added and the samples incubated for 1 h. After the slides were rinsed three times with PBS, the streptavidin-biotin–peroxidase complex (1:100) was applied to them. The reaction products were visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB), according to the manufacturer’s instructions. All tissue sections were viewed with a Nikon Eclipse 80i microscope  fitted with a camera. The images were analyzed with the ImagePro Plus 6.0 software.Immunohistochemical staining was performed to determine the expression and subcellular localization of the HOXB1 protein in the human glioma specimens and nonneoplastic brain tissues. The tissues were embedded in paraffin and cut into 4 μm thick sections, which were deparaffinized and rehydrated, and then retrieved with heat-induced epitope retrieval. Endogenous peroxidase was inhibited with 3% hydrogen peroxide (HHOXB1 was performed using the programs miRanda (http://www.microrna.org/), DIANA TOOLS (http://diana.imis.athena-innovation.gr), and TargetScan (http://www.targetscan.org/).A predictive search for miRNAs targeting HOXB1 was synthesized, annealed, and inserted into the XbaI and AhaIII sites of the pmirGLO luciferase reporter vector , downstream from the stop codon of the luciferase gene. The sequence of wild-type (WT) HOXB1 complementary to the binding site of miR-3175 in the 3′-UTR (TCTCCCC) was replaced with a mutated (MUT) HOXB1 sequence (TGAGGCC). The constructs were confirmed with DNA sequencing. Glioma U87 cells were cotransfected with pmirGLO-WT-HOXB1 or pmirGLO-MUT-HOXB1, the pRL-TK Renilla plasmid (Promega), and equal amounts of the negative control, miR-3175 mimic or miR-3175 mimic. The pRL-TK Renilla luciferase reporter vector was used as the internal control. After the cells were incubated for 48 h, the luciferase activity was measured with the Dual Luciferase Reporter Assay Kit .The 3′-UTR of HOXB1 (si-HOXB1) and an siRNA negative control (si-NC) were designed and synthesized by GenePharma, Si-HOXB1-1 forward: 5′-GATCCCCTTCTTAGAGTACCCACTTTCAAGAGAAGTGGGTACTCTAAGAAGGTTTTTTA-3′, si-HOXB1-1 reverse: 5′-AGCTTAAAAAACCTTCTTAGAGTACCCACTTCTCTTGAAAGTGGGTACTCTAAGAAGGG-3′; si-HOXB1-2 forward: 5′-GATCCCTTCTCAGTACTACCCTCTTTCAAGAGAAGAGGGTAGTACTGAGAAGTTTTTTA-3′, si-HOXB1-2 reverse: 5′-AGCTTAAAAAACTTCTCAGTACTACCCTCTTCTCTTGAAAGAGGGTAGTACTGAGAAGG-3′; si-HOXB1-3 forward: 5′-GATCCCTCAATGAAACACAGGTCATTCAAGAGATGACCTGTGTTTCATTGAGTTTTTTA-3′, si-HOXB1-3 reverse: 5′-AGCTTAAAAAACTCAATGAAACACAGGTCATCTCTTGAATGACCTGTGTTTCATTGAGG-3′; si-NC forward 5′-AATGGGTAAACCGTTAAACAGTCCCTAAGAGATAAATGGCCTCGAATCCTATC-3′, si-NC reverse: 5′-CCCTGAAGGAAGAAAGGATTCGCAAACGTATCAAGGGATCGGTTAACTTAACC-3′. One day before transfection, the glioma cells were plated at a density of 3–5 × 105 cells/well in six-well plates so that the cells were grown to 70%-80% confluence at the time of transfection. The cells were transfected with the oligonucleotides described above using Lipofectamine 2000 , according to the manufacturer’s protocol. The glioma cells were harvested 48–72 h after the initial transfection for further analysis.The miR-3175 mimic, the miR-3175 inhibitor, and the corresponding negative controls (NC) were synthesized and purified by GenePharma, . The sequences were: miR-3175 mimic: 5′-ACGUCACUGCGUUCUCUCCCCG-3′; miR-3175 inhibitor: 5′-CGGGGAGAGAACGCAGUGACGU-3′; miR-3175 mimic NC: 5′-UACUTCGAACCAACCAGCGUUACT-3′; and miR-3175 inhibitor NC: 5′-CATUCCUUGTUGUGUAAUCCUUTT-3′. Three small interfering RNAs (siRNAs) targeting 3/well). The cells were incubated for another 48 h in medium containing 10% FBS. MTT solution  was added to each well and the plate was incubated for 4 h at 37°C in an incubator. After the medium was removed, the purple formazan crystals were dissolved in 150 μl/well dimethyl sulfoxide  for 30 min. The absorbance of the formazan was measured at 490 nm on a SpectraMax M3 microplate reader .The viability of the glioma cells transfected with specific oligonucleotides was determined with the 3--2,5-diphenyltetrazolium bromide  uptake method. Briefly, after the cells were incubated for 24 h, they were transfected with specific oligonucleotides, and seeded in a 96-well plate . The upper chamber was loaded with 200 μl of serum-free medium and the lower chamber was loaded with 700 μl of medium containing 10% FBS. After incubation for 18 h in an incubator at 37°C, the upper-chamber medium was cautiously removed, the glioma cells were fixed in 4% paraformaldehyde for 15 min and stained with Giemsa for 20 min, and the noninvasive cells were removed with a cotton swab. Images of the glioma cells that had migrated to the other side of the membrane were captured at 100× magnification with a Nikon Eclipse Ti-S microscope (Nikon) with the NIS-Element S.F. software. The numbers of cells were counted in three nonoverlapping, randomly selected fields. In the scratch wound assay, the glioma cells transfected with the appropriate oligonucleotides were incubated for 48 h. A linear wound was made across the cell layer with a 200 μl pipette tip, and three different reference points were marked. The cells were incubated in medium supplemented with 1% FBS for 24 h. After 0 h and 24 h, the wound widths at the same reference points were photographed and measured under a Nikon Eclipse Ti-S microscope  with the NIS-Element S.F. software.The Transwell assay and scratch wound assay were used to determine the invasion capacity of the transfected cells in vitro. In the Transwell assay, glioma cells transfected with the appropriate oligonucleotides were incubated for 48 h, and 3 × 102 at 37°C in six-well plates. The cells were harvested with pancreatic enzymes without EDTA and washed twice with precooled PBS. The cells were resuspended at a concentration of 1 × 105 cells/ml in 500 μl of binding buffer containing 5 μl of annexin V-fluorescein isothiocyanate (FITC) and 5 μl of propidium iodide (PI), according to the instructions of the manufacturer of the Annexin V-FITC Apoptosis Detection Kit . After incubation for 15 min at room temperature in the dark, all the samples were analyzed within 1 h with a guava easyCyte 8 flow cytometer . The data were analyzed with the De Novo software.Glioma cells were transfected with the appropriate oligonucleotides and incubated for 72 h in an incubator under 5% COHOXB1 and miR-3175 expression was analyzed with Spearman’s rank correlation. Prognostic factors were examined with univariate and multivariate Cox regression models. The data are expressed as means ± SD. Pairwise comparisons were made with t tests. P < 0.05 was considered statistically significant.All experiments were performed in triplicate. SPSS version 18.0  and GraphPad Prism version 5.0  were used for the statistical analyses. Survival curves were constructed with the Kaplan-Meier life table method. The correlation between HOXB1 expression in whole-gene profiles of human glioma tissues from two independent gene expression data sets, CGGA and GEO. We found that HOXB1 expression differed significantly between the glioma tissues and nontumor brain tissues, in that HOXB1 expression was obviously lower in the gliomas  than in low-grade glioma tissues  , but the immunohistochemical analyses showed that HOXB1 protein expression levels were clearly downregulated in HGG. HOXB1 expression was also examined in glioma cell lines . As shown in HOXB1 functions as a tumor suppressor gene in glioma and that the downregulated expression of HOXB1 may be related with the degree of malignancy.We analyzed and summarized the GSE4290; . We alsoI) CGGA; . To furtI) CGGA; . The resP = 0.004, 95% confidence interval [95% CI] 0.008–0.394) was associated with worse survival in this prospective cohort of glioma patients , and HOXB1 expression  were associated with the progression of glioma. A multivariate analysis showed that HOXB1 expression  and glioma grade  were associated with shorter survival  and HOXB1 expression  were associated with PFS. In the Cox regression analysis, the low expression level of HOXB1 was an independent predictor of poor prognosis in glioma patients. These data support the notion that low HOXB1 expression is associated with worse survival in glioma patients and may act as a tumor biomarker in malignant glioma.We evaluated the prognostic value of HOXB1 expression for patient OS and PFS based on the follow-up data of glioma patients. OS and PFS were stratified by HOXB1 expression using the Kaplan-Meier life table method and Cox regression models. In this study, we found that high HOXB1 expression predicted longer OS and PFS . Univaripatients . Howeversurvival . The varHOXB1 expression in glioma cell lines  for the targeted knockdown of ll lines . A cell l growth . The effl growth . In the l growth . A cell poptosis . These rP = 0.12). The expression of miR-3175 was elevated in the three glioma cell lines compared with that in the control cells. Because miR-3175 expression is upregulated in glioma, we investigated the biological functions of miR-3175 in glioma. After U87 cells were transfected with the miR-3175 mimic, miR-3175 inhibitor, miR-3175 mimic NC, or miR-3175 inhibitor NC, qRT-PCR was used to determine the expression of miR-3175  homeodomain factors that act synergistically with HOX , and it c cancer , and micc cancer . HoweverHOXB1 expression. In this study, HOXB1 was shown experimentally to be a novel target of miR-3175. A luciferase reporter assay confirmed that miR-3175 directly recognizes the 3′-UTR of HOXB1 transcripts. An miR-3175 mimic downregulated HOXB1 expression and an miR-3175 inhibitor upregulated it, demonstrating that the expression of both HOXB1 mRNA and HOXB1 protein are significantly regulated by miR-3175 in glioma cell lines. The inverse correlation between HOXB1 expression and miR-3175 expression was confirmed in clinical specimens with Spearman’s rank correlation. A cotransfection analysis was performed to verify that the tumorigenesis induced by the reduced expression of HOXB1 in glioma is regulated by miR-3175. The oncogenicity induced in glioma cells by low HOXB1 expression was not only reversed by an miR-3175 inhibitor, evident as reduced cell proliferation and increased cell apoptosis, but the expression of several apoptosis-related proteins was affected by low HOXB1, including that of procaspase-3, p53, and cytochrome c. Procaspase-3 expression was enhanced and cytochrome c expression was reduced in cells in which HOXB1 expression was downregulated. Therefore, we suggest that HOXB1 triggers both the mitochondrial and caspase-dependent apoptotic pathways [This finding supports a role for miR-3175 as a tumor promoter in glioma and raises the possibility that miR-3175 regulates pathways .CBP) [MDM2), is also involved in p53-induced apoptosis [The expression of HSP70 interacting protein (ST13), c-Jun N-terminal kinase 2 (JNK2), and programmed cell death 10 (PDCD10) is HOXB1-dependently upregulated, and these proteins are involved in mitochondrial permeabilization and the induction of apoptosis [CBP) , and CBPCBP) . The HOXpoptosis –39, a TApoptosis . More impoptosis . In thisHOXB1 is a tumor suppressor gene in glioma. The downregulation of HOXB1 expression promotes glioma cell proliferation and invasion and inhibits cell apoptosis. More importantly, HOXB1 was shown experimentally in this study to be a direct target of miR-3175. Although much remains to be investigated regarding the role of HOXB1 in the pathogenesis of glioma, as a novel biomarker, HOXB1 represents a potential target for the diagnosis and treatment of glioma.In conclusion, our data suggest that S1 Dataset(A) The whole western blot images show HOXB1 protein expression in glioma cell lines A172, U251, and U87. (B) The whole western blot images show HOXB1 protein expression in U87 cells after transfection with three siRNA targeting different sites of HOXB1 and a siRNA negative control at 48 h. (C) The whole western blot images show HOXB1 protein expression levels in glioma cell lines after transfection with miR-3175 mimics, miR-3175 inhibitor and the corresponding NCs at 72 h. (D) The whole western blot images show several apoptosis-related proteins  expression in U87 cells after transfection with si-NC + inhibitor NC, si-HOXB1 + miR-3175 inhibitor and si-HOXB1 + inhibitor NC.(RAR)Click here for additional data file.