However, no differences were demonstrated in either viral replication or oncogenicity between the two rescued viruses in chickens. These results indicated that the 19-nt insertion contributed to ALV-J replication in vitro but was not related to its pathogenicity in vivo.Subgroup J avian leukosis virus (ALV-J) was first isolated from meat-type chickens that had developed myeloid leukosis and since 2008, ALV-J infections in chickens have become widespread in China. A comparison of the sequence of ALV-J epidemic isolates with HPRS-103, the ALV-J prototype virus, revealed several distinct features, one of which is a 19-nucleotide (nt) insertion in the leader sequence. To determine the role of the 19-nt insertion in ALV-J pathogenicity, a pair of viruses were constructed and rescued. The first virus was an ALV-J Chinese isolate (designated rSD1009) containing the 19-nt insertion in its leader sequence. The second virus was a clone, in which the leader sequence had a deleted 19-nt sequence (designated rSD1009△19). Compared with rSD1009△19, rSD1009 displayed a moderate growth advantage  Retroviridae, subfamily Orthoretrovirinae, and genus Alpharetrovirus. ALVs that infect chickens can be classified into six subgroups  based on their host ranges, viral cross-neutralization patterns, and viral envelope interference Avian leukosis viruses (ALVs) constitute a species belonging to the family gag-pol-env sequence followed by a 3′ LTR gag, pol, and env genes encode Gag (a group-specific antigen), a reverse transcriptase integrase, and the envelope glycoproteins, respectively ALV-J has an overall structure of a typical slowly transforming replication-competent ALV: a 5′ long terminal repeat (LTR) leader in vitro and in vivo.In recent years, comparisons of the sequences of ALV-J epidemic isolates with HPRS-103, the ALV-J prototype virus, have revealed several distinct features related to its pathogenicity. For example, a 205-nucleotide (nt) deletion and E element in the ALV-J genome occurs naturally and contributes to its pathogenicity All animal experiments were approved by the ethical review board of Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Sciences (CAAS) and performed in accordance with approved animal care guidelines and protocols. The animal Ethics Committee approval number is Heilongjiang-SYXK-2006–032.DF-1 cells (ATCC accession number: CRL-12203) were grown in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum and 100 µg/ml of penicillin and streptomycin. Tumors from chickens raised in Shandong province, China XbaI and ClaI restriction enzymes, which were amplified separately. The primer sets used to amplify fragment I were 5′-CCCGGCCGTGTAGTGTTATGCAATACTCTTATGTAACGATGAAAC-3′ Proviral DNA was extracted from SD1009-infected DF-1 cells for use as a template for polymerase chain reaction (PCR) amplification. The SD1009 genome was cleaved into three fragments using the 5′-GGCCATTTTCATGTCTAGATT-3′ . The primer sets used to amplify fragment II were 5′-GGCGAGGGAATGGAATCT-3′  and 5′-GCGCCAGGAGTAAGAAATCGATG-3′ . The primer sets used to amplify fragment III were 5′-CGAGCAGCCATCGATTTCTTACTC-3′  andand 5′-CCCTCGAGTGAAGCCATCCGCTTCATGCAGGTGCTCGTAGTTGTCAGG-3′ . The amplicons were gel purified and subcloned into pMD-18T vectors  and ligated in order into pBlueScript II KS(+) plasmids (TaKaRa Biotechnology Co. Ltd.) via restriction enzyme digestion. The recombinant plasmid was named pBlu-SD1009. To delete the 19-nt insertion in the leader sequence, fragment I was replaced with a fusion product of two PCR fragments containing overlapping sequences spanning the deleted region. The primer sets were F1, 5′-CGAGCAGCCATCGATTTCTTACTC-3′ and R1, 5′-CTGGTGCGGACCACTCAGT-3′ and F2, 5′-ACTGAGTGGTCCGCACCAGGCGTGATTCTGGTCGCCCT-3′ and R2, 5′-CCCTCGAGTGAAGCCATCCGCTTCATGCAGGTGCTCGTAGTTGTCAGG-3′. Finally, the new fragments I, II, and III were ligated into the pBlueScript II KS(+) plasmid, in that order, to obtain the pBlu-SD1009△19 recombinant plasmid. The above construction for the two full-length cDNA clones is shown in Highly purified pBlu-SD1009 and pBlu-SD1009△19 DNA was obtained using Qiagen Plasmid Midi Kits  according to the manufacturer’s instructions. The purified plasmid DNA (4 µg) from pBlu-SD1009 and pBlu-SD1009△19 was introduced into DF-1 cells using Lipofectamine 2000 . The culture supernatant containing the viral stocks was harvested 48 h later and then blind-passaged into secondary DF-1 cells. The rescued viruses were named rSD1009 and rSD1009△19. To determine the specificity of the rescued viruses, the secondary DF-1 cell supernatants were analyzed using an enzyme-linked immunosorbent assay  with the ALV group-specific antigen p27. An indirect immunofluorescence assay (IFA) was performed with virally infected DF-1 cells. Briefly, DF-1 cells were grown to 60% confluence in a tissue-culture plate and then infected with supernatants from the passage of rSD1009 and rSD1009△19. After incubation for 48 h at 37°C, the cells were washed once with phosphate-buffered saline/Tween 20 (PBST) and then fixed with ethanol for 20 min at room temperature. The fixed cells were incubated with monoclonal antibodies specific for ALV-J gp85 g and 4°C for 5 min. The supernatant was collected and 10-fold serially diluted to 10−8 and then inoculated into 96-well plates along with DF-1 cells. Eight parallel well were created in each gradient. After incubation for 5 days, the cells were washed once with PBST and then fixed with ethanol for 20 min at room temperature. The fixed cells were incubated with monoclonal antibodies specific for ALV-J gp85 in a humidified chamber for 60 min at 37°C and reacted with fluorescein-labeled goat anti-mouse immunoglobulin G (IgG) antibody  in a humidified chamber for 60 min at 37°C. Next, the cells were washed with PBST. The number of fluorescent cells per well were counted using a fluorescence microscope, and the TCID50 was calculated using the Reed -Muench method rSD1009, rSD1009△19 and HPRS-103 was inoculated into DF-1 cell culture bottles, freeze-thawed three times and then centrifuged at 7000×6 cells/plate) was infected with approximately 0.1 ml of 102.5/ml 50% tissue culture infectious doses (TCID50) of rSD1009, rSD1009△19, and HPRS-103. Post-infection culture supernatants were collected daily for 6 days and RT activity was quantitated using a colorimetric reverse transcriptase assay . To determine the replication kinetics of rSD1009, rSD1009△19, and HPRS-103 in more detail, DF-1 cells in 60-mm plates were infected with approximately 0.1 ml per plate of 102.5/ml TCID50 of rSD1009, rSD1009△19, and HPRS-103, respectively. Infected cell cultures were harvested at various time points and the titers of infectious progeny were determined as TCID50 per milliliter using the Reed–Muench method as described above. Results are presented as mean values and standard deviations from three independent experiments.RT activity and the replication kinetics in the culture supernatants of rSD1009, rSD1009△19, and HPRS-103 were measured. Each 60-mm-diameter plate of DF-1 cells  and the constructs were named rSD1009-leader, rSD1009△19-leader, and HPRS103-leader after confirmation by sequencing.The luciferase-expression vector pGL3-Promoter Vector  was used as the parent vector for the leader sequences of rSD1009, rSD1009△19, and HPRS-103 reporter-analysis experiments. The leader sequences of rSD1009 (279 nts), rSD1009△19 (259 nts), and HPRS-103 (259 nts) were amplified by PCR. To facilitate cloning, 2.5/ml TCID50 of rSD1009 and rSD1009△19 viral stock per chicken. The titer used to inoculate the chickens in this study was based on a previous report that showed that similar titers could induce tumors One-day-old chicks of specific-pathogen-free (SPF) layers were purchased from the Experimental Animal Center of the Harbin Veterinary Research Institute, CAAS, China, and housed in negative-pressure-filtered air isolators. The animal experiments were approved by the Animal Ethics Committee of the institute. The SPF layers were infected on the day of hatching by intra-abdominal inoculation with approximately 0.25 ml of 10The statistical significance was determined by Student’s t test using GraphPad Prism (version 5.0) software .The ALV-J isolate SD1009, which was isolated from a layer chicken in the Shandong province in China 5′-GCGCGGTTCCGGTTGCTCT-3′), located between nt 573 and 574 according to the HPRS-103 sequence, was present in the leader sequence. The leader sequence from 10 ALV-J isolates  were analyzed  and rSD1009△19 (without the 19-nt insertion), were rescued using reverse genetics. An IFA was performed to identify the rescued viruses. The DF-1 cells infected with the rescued viruses were stained by monoclonal antibodies specific for ALV-J gp85. Primary antibody binding was detected using fluorescein isothiocyanate-labeled anti-mouse IgG, which exhibited green fluorescence . ElectroTo explore whether the 19-nt insertion in the leader sequence influenced the enhancer activity of the leader sequence, we constructed a luciferase vector with a leader sequence with and without the 19-nt insertion. We also constructed a luciferase vector with the same leader sequence as HPRS-103. As shown in in vitro replication of the rescued virus. DF-1 cell cultures were infected with equivalent amounts of rSD1009, rSD1009Δ19, and HPRS-103. The culture supernatants were harvested periodically thereafter for reverse transcriptase (RT) activity assays. For the first 2 days, rSD1009 displayed growth curves similar to rSD1009Δ19 before the RT activity could be detected. Subsequently, rSD1009-infected cells produced more viral particles than those infected with rSD1009△19. By day 6, the number of viral particles of both strains peaked. The results revealed that rSD1009 had a moderate growth advantage over rSD1009Δ19  and rSD1009△19 (7.6%). Details regarding the types of identified tumors are shown in in vitro and in vivo by construction and rescue of a pair of viruses (rSD1009 and rSD1009△19). The results indicated that the 19-nt insertion in the leader sequence of ALV subgroup J contributed to its replication in vitro, but was not related to its pathogenicity in vivo.Since 2008, ALV-J infections in chickens have become widespread in China It is important to monitor sequence changes in the leader sequence of ALV-J because the 5' leader sequences of avian retroviruses contain functional elements involved in key steps of the retroviral life cycle in vitro replication of the rescued viruses. Our results indicated that rSD1009 replicated more rapidly than rSD1009△19 in DF-1 cells (in vitro, which may be the reason that the insertion changed the length of the leader sequence, thereby re-adjusting its structure and ultimately influencing the cis-acting characteristics of the leader sequence in the ALV-J strain.We employed DF-1 cells, which are commonly used for propagating ALV-J -1 cells . In addi-1 cells . The ALVin vitro replication, it did not appear to affect viral replication in vivo, as demonstrated by the neutralizing-antibody response of infected chickens at 7 weeks of age (in vitro and in vivo has also been observed in other viruses, such as a single amino acid mutation in very virulent infectious bursal disease virus that could attenuate replication in vivo but increased replication in vitroin vitro and in vivo requires further investigation.Although the 19-nt insertion enhanced s of age . This diin vivo. A previous hypothesis suggested a link between in vivo replication and pathogenicity and proposed that viral replication and pathogenicity were positively correlated in vivo replication of rSD1009 and rSD1009△19 led to similar pathogenicity to the SPF layers, supporting the proposed hypothesis. Although a correlation between pathogenicity and effective viral replication has also been observed in other reports, including a study that linked the replication of Newcastle disease virus with its pathogenicity There was no significant difference in the prevalence of tumors in chickens following infection with either rSD1009Δ19 or rSD1009. In addition, the oncogenic spectrum of the two viruses was very similar . The oncin vitro, but was not related to its pathogenicity in vivo. This study not only demonstrated a unique molecular characteristic of ALV-J, but also generated data that may benefit the understanding of the relationships between variations in genomic sequences and the oncogenicity of ALV-J.ALV-J-induced disease among chicken flocks in China has been surprising and generated a large amount of attention worldwide


The HLA-G molecule plays an important role in immunomodulation. In a previous study carried out on a southern French population our team showed that HLA-G haplotypes, defined by SNPs in the coding region and specific SNPs located in 5′URR and 3′UTR regulatory regions, are associated with differential soluble HLA-G expression (sHLA-G). Furthermore, the structure of these HLA-G haplotypes appears to be conserved in geographically distant populations.The aim of our study is to confirm these expectations in a sub-Saharan African population and to explore additional factors, such as HLA-A alleles, that might influence sHLA-G expression.DNA and plasma samples were collected from 229 Malians; HLA-G and HLA-A genotyping were respectively performed by the Snap Shot® method and by Luminex™ technology. sHLA-G dosage was performed using an ELISA kit. HLA-G and HLA-A allelic and haplotypic frequencies were estimated using an EM algorithm from the Gene[Rate] program. Associations between genetic and non genetic parameters with sHLA-G were performed using a non-parametric test with GRAPH PAD Prism 5.Our results reveal a good conservation of the HLA-G UTR haplotype structure in populations with different origins and demographic histories. These UTR haplotypes appear to be involved in different sHLA-G expression patterns. Specifically, the UTR-2 haplotype was associated with low sHLA-G levels, displaying a dominant negative effect. Furthermore, an allelic effect of both HLA-G and HLA-A, as well as non genetic parameters, such as age and gender possibly linked to osteogenesis and sexual hormones, also seem to be involved in the modulation of sHLA-G.These data suggest that further investigation in larger cohorts and in populations from various ethnical backgrounds is necessary not only to detect new functional polymorphism in HLA-G regulatory regions, but also to reveal the extent of biological phenomena that influence sHLA-G secretion and this might therefore have an impact on transplantation practice. The role of the non-classical class Ib Human Leukocyte Antigen-G (HLA-G) in immune-tolerance has been well documented Contrary to the classical HLA class I loci, HLA-G is characterized by a low polymorphism in the coding regions. To this day, 50 HLA-G alleles have been identified, which encode 16 trans-membrane proteins  and two truncated proteins (HLAG*01:05N and G*01:13N) insertion/deletion polymorphism has been associated with differential sHLA-G expression (i.e. the ins/ins genotype displays a lower level of sHLA-G than the ins/del and del/del genotypes) Several studies have suggested an association between soluble (s)HLA-G expression and specific HLA-G alleles or SNPs in the non-coding regions. Notably, HLA-G*01:04 and G*01:05N have been respectively associated with high and low HLA-G secretion HLA-A is the closest functional gene to HLA-G. The genetic distance between these two genes is approximately 150 Kb Castelli et al. defined 8 UTR HLA-G haplotype groups using sequenced SNPs in the 5′URR, 3′UTR and coding regions in a Brazilian population ins/del exon 8, 3142 (C/G), 3187 (G/A) and 3196 (C/G)) and coding HLA-G alleles defined by eight SNPs. In this preliminary study we confirmed the conservation of the HLA-G UTR haplotype structure and its allelic association by identifying the eight previously defined UTR haplotypes ins/ins genotype, consensually associated with lower sHLA-G expression: the ins allele was present in both UTR-5 and UTR-2, which we showed to be respectively associated with high and low sHLA-G secretion. However, another team has contradicted these preliminary results, notably showing that UTR-2 and UTR-5 are respectively correlated with intermediate and low sHLA-G levels .Based on this study, our team investigated HLA-G UTR haplotype conservation and its association with the expression of sHLA-G in serum from Volunteer Bone Marrow Donors (VBMD) from South-eastern France On this basis, we propose to investigate the following hypotheses: (1) the restricted number of UTR HLA-G haplotypes and their structure may reflect selective forces associated with differential expression of sHLA-G and its biological significance. This supposition should be confirmed in a sub-Saharan African population since these populations generally display higher genetic diversity and lower levels of linkage disequilibrium compared to populations from other continents. (2) Accordingly, the association between specific UTR haplotypes and sHLA-G levels should be reproducible at plasma level in Malian samples. (3) Finally, the HLA-A gene, due to its proximity to the HLA-G gene, may influence the expression of sHLA-G. Thus, haplotype conservation might be extended to the HLA-A gene.Malaria Research and Training Center, Department of Epidemiology of Parasitic Diseases, Faculty of Medicine, Pharmacy and Dentistry, Bamako, Mali. Participants provide their written informed consent. Kin provide their written informed consent on the behalf of the children participants involved in this study. The study protocol and the consent procedure were approved by the Ministère de l'Enseignement Supérieur et de la Recherche in France and by the Comité d'éthique institutionnel de la faculté de médecine, de Pharmacie et d'odontostomatologie in Mali.Sample collection was conducted by the sex ratio of 1.2). The mean age was 47.7 years old (range: 26–88 years) for adults and 9.7 years old (range: 3–25 years) for children.Blood and plasma samples were collected from 229 unrelated Malians after informed consent, in the villages of: Bandiagara (n = 61), Binedama (n = 12) Madougou (n = 53), Manteourou (n = 51), N'Gono (n = 21) and Petaka (n = 31). The ethnic groups were: Dogon , Peulh , Tamashek  and Bambara . This cohort was composed of men and women  according to the manufacturer's instructions. Genomic analyses and serology were performed in Marseilles respectively on genomic DNA and plasma from the same cohort. 229 individuals were successfully analyzed for HLA-G coding alleles, 5′URR and 3′UTR polymorphisms and 195 individuals were analyzed for HLA-A coding alleles. sHLA-G level was determined in plasma samples from 219 individuals.ins/del exon 8 rs66554220; 3142 C/G rs1063320; 3187 G/A rs9380142 and 3196 C/G rs1610696) A homemade primer extension method was used to simultaneously analyse 16 SNP scattered along the HLA-G gene: 8 SNPs  defining HLA-G alleles (HLA-G*01:01 to G*01:09), 4 SNPs in the 5′ URR region  and 4 SNPs in the 3′UTR region (Luminex™ technology (HLA-A-One Lambda LABType® SSO) was used to determine HLA-A alleles at an intermediate resolution using the manufacturer's kit. The HLA-A allelic assignment is based on the HLA sequences listed in the official IMGT/HLA database 3.12.0 May 2013 in duplicate on plasma samples using the ELISA test  according to the manufacturer's protocol. For each ELISA test, 6 samples were used as controls, 2 with low levels, 2 with median levels and 2 with high levels, to evaluate the repeatability and reproducibility. Inter-plate sHLA-G value variability was normalized using the mean value of a control plate.Measurement of soluble isoforms HLA-G1 and -G5 was performed http://geneva.unige.ch/generate) HLA-G genotypes were automatically converted from output files (.txt) exported from GeneMapper 4.0 into coding alleles and UTR using an in-house computer program, readable by the ‘Phenotype’ application of the Gene[Rate] computer tool package  for all the 16 HLA-G SNPs were tested using a nested likelihood model Frequencies for HLA-G alleles, SNPs in the 5′ and 3′ regions, HLA-A alleles, UTR∼HLA-G and HLA-A∼HLA-G∼UTR haplotypes were estimated using an EM algorithm from the Gene[Rate] program Two-locus linkage disequilibrium (LD) was tested for the 16 HLA-G SNPs by a conventional goodness-of-fit test with the Arlequin v3.5.1.2 package www.fluxus-engineering.com/network). The HLA-A alleles were grouped into five lineages previously defined Gametic association between specific pairs of alleles is provided as a list of standardized residuals for each observed haplotype. The null hypothesis of independence of the loci implies a gaussian distribution of deviations and, by convention, absolute values over 2 are considered to be significant. In order to establish the relationships among the HLA-A and HLA-G haplotypes, a Median Joining (MJ) network Associations between sHLA-G and genetic polymorphism  or non genetic parameters (sex) were tested with non-parametric tests performed with GRAPH PAD Prism 5. Mann-Whitney t-test was used to test two modalities. Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test was used when there were more than two modalities. Statistical correlation between age and sHLA-G levels was tested using Spearman's rank test.ins/del, 3142 C/G, 3187 G/A and 3196 C/G) in the Malian samples are shown in Frequencies of the HLA-G alleles, the 4 SNPs in the 5′URR  and the 4 SNPs in the 3′UTR >G*01:04 (26.2%)>G*01:03 (15.2%)>G*01:05N (10.5%)>G*01:06 (2.2%). The five HLA-G alleles previously reported in the French population (VBMD) were all observed in Malians. Malian samples displayed a significantly higher frequency (p<0.001) for HLA-G*01:04, G*01:03, G*01:05N as compared to VBMD, while HLA-G*01:01 frequency was significantly higher in VBMD than in Malians (p<0.001); no statistical difference was found for G*01:06 .Frequencies of SNPs in the 5′URR and 3′UTR were significantly different between VBMD and Malians (p<0.05), except for −725 C.All SNPs of the HLA-G gene were in Hardy Weinberg Equilibrium (HWE), except the SNP at codon 130 that displayed a significantly higher heterozygosity than expected. A T in codon 130 specifically defines the HLA-G*01:05N allele.Two-locus Linkage Disequilibrium (LD) for SNPs in 5′URR, 3′UTR and coding alleles is shown in Global LD was observed between HLA-A and HLA-G alleles and between HLA-A alleles and HLA-G UTR, respectively (both p = 0 and quantile = 1000).UTR composition, their allelic association and their frequencies in the Malian samples are shown in ins/del exon 8, 3142 C/G, 3187 G/A and 3196 C/G), are restricted to a small number in the Malian samples (6 UTRs estimated out of 28 possible)  and four SNPs in the 3′UTR (ossible) . In compUTR-2 had the highest frequency in the Malian samples. UTR frequencies were as follows: UTR-2 (29.9%)>UTR-3 (23.9%)>UTR-5 (14.3%)>UTR-1 (13.4%)>UTR-6 (12.1%)>UTR-4 (5%). Malians displayed significantly higher frequencies (p<0.001) for UTR-2, UTR-3 and UTR-5 compared to VBMD and lower frequencies for UTR-1 and UTR-4 (p<0.001).Concerning the association between UTR and HLA-G alleles, the same association previously reported in the VBMD population was described in the Malian samples . UTR-3∼GHaplotype analysis was extended to HLA-A alleles in association with UTR and HLA-G alleles. These HLA-A∼UTR∼HLA-G haplotypes are in a limited number (43 HLA-A∼UTR∼HLA-G estimated out of 204 possible). HLA-A∼UTR∼HLA-G haplotypes estimated in the Malian samples and their frequencies are showed in Association between HLA-A and HLA-G was further investigated using the Median Joining (MJ) method based on protein sequences . UTR werThe overall distribution of sHLA-G fits a Gaussian distribution. The sHLA-G mean value in the 219 plasma samples was 143.18±31.05 UI/ml.No significant difference was found between gender and sHLA-G level even though men displayed lower values than women .However, a significant negative correlation  was observed between age and sHLA-G levels. When individuals were classified according to gender, a significant negative correlation was only found for women  . Both boSignificant associations were found between 5′URR −716 G/T (p = 0.03), −201 G/A (p = 0.03) and 3′UTR +3196 C/G (p = 0.03) and sHLA-G. The Dunn post-hoc test showed significantly higher sHLA-G levels for −716 T/T; −201 G/G and +3196 C/C genotypes. No significant associations were observed for HLA-G alleles and the other 5′URR and 3′UTR SNPs.ins/ins genotype did not show significantly lower sHLA-G than ins/del or del/del genotypes, thus confirming our previous results ins/ins 136±30.67 UI/ml, all except ins/ins 145±31.0 UI/ml, p = 0.144).The exon 8 sHLA-G mean values and standard deviation for each UTR genotype are shown in UTR-2 individuals displayed a significantly lower level of sHLA compared to non-UTR-2 individuals , but thiNo significant correlation was found between sHLA-G and the other UTRs.To assess the influence of HLA-A alleles on sHLA-G expression, we tested the correlation within and among the A lineages defined by Gu X et al. No significant differences in sHLA-G expression were found between HLA-A alleles from other HLA-A lineages.In this study we present results based on HLA-G and HLA-A genotypes and sHLA-G serological analyses performed on 229 Malian samples. UTR haplotypes described in the Malian samples show reduced diversity  and share a similar structure to those described in the French VBMD and in the Brazilian population Stronger LD between SNPs within 5′URR and 3′UTR was found in the Malian samples than in the French samples. Previous multi-loci studies have suggested that sub-Saharan Africans display a lower LD compared to populations from other continents Haplotype structure conservation was further confirmed when HLA-A alleles were considered since the number of HLA-A∼UTR∼HLA-G haplotypes was also greatly reduced in the Malian samples. The estimated haplotypes were limited to 43 compared to the 204 possible haplotypes according to the observed alleles. One should keep in mind that we studied 229 samples and that some rare haplotypes might not have been detected.The second objective of this study was to confirm the correlation between sHLA-G expression level and UTR haplotypes. Highly variable sHLA-G values have been reported with the Elisa kit using the MEM-G/9 antibody according to the biological fluid analyzed or the calibration standard used; our results are in accordance with published data based on plasma samples and expressing their results in U/ml according to the calibration standard displayed by the supplier We confirmed an association between the UTR-2 haplotype and lower sHLA-G levels in the Malian samples. UTR-2 individuals showed, both in homozygous and heterozygous state, a significant association with lower values. This result tends to show a Dominant Negative Effect (DNE) of UTR-2. Moreover, even though no significant association was observed between HLA-G alleles and sHLA-G levels, an allelic effect was still observed for UTR-2. Indeed, HLA-G*01:05N/G*01:05N showed higher (but not significantly) sHLA-G levels compared to other UTR-2 allelic combinations. This putative allelic effect of HLA-G*01:05N/G*01:05N on sHLA-G does not exclude a haplotype effect of HLA-A on UTR-2; indeed HLA-G*01:05N∼UTR-2 was only coupled with HLA-A*30:01. However, HLA-G*01:05N codes for a truncated protein since HLA-G*01:05N presents a stop codon in position +189, therefore the mRNA of G*01:05N translates only HLA-G5 and -G6 soluble isoforms No significant correlation has been found between the other UTRs and sHLA-G levels. However, UTR-1 and UTR-5 homozygous individuals displayed higher sHLA-G levels than the overall sample mean. Interestingly HLA-A*02 subtypes bearing UTR-5 displayed significantly higher sHLA-G levels compared to other HLA-A alleles bearing UTR-5. This differential sHLA-G expression may be due to an HLA-A*02 effect or to supplementary polymorphisms in regulatory regions of UTR-5. Indeed, 3 SNPs in the 5′URR, −646, −540 and −509 described by Castelli et al. (2011) can separate UTR-5 in 2 sub haplotypes Our analyses on HLA-A showed that HLA-A alleles display LD with both HLA-G alleles and UTR. HLA-A*23:01:01∼HLA-G*01:04∼UTR3 displayed the highest frequency, confirming the reported LD between HLA-A*23 (and HLA-A*24) and HLA-G*01:04 in different populations HLA-G*01:04 has been previously associated with a high sHLA-G production This study showed a negative correlation between age and sHLA-G levels in Malian women which remained significant when UTR-2 individuals were excluded. None of the previously published studies have shown correlation between age and sHLA-G levels, even related with gender. Moreover, we also found that boys and girls between 3–25 years old showed statistically higher sHLA-G values compared to men and women over 26 years old. It has recently been reported that mesenchymal progenitors and osteoblastic cells specifically express HLA-G5 during osteogenesis, with a key role in bone homeostasis Taken together, these data support the theory of a conservation of UTR haplotype structure in populations with different origins and demographic history, such as Malians, French and Brazilians. These UTR haplotypes appear to be implicated in different sHLA-G expression patterns. Particularly, the association between the UTR-2 haplotype and low sHLA-G levels seems to be further confirmed and preserved in different populations, displaying a dominant negative effect of UTR-2. However, the allelic effect of HLA-G and HLA-A genes, independent from UTRs, seems to be implicated in sHLA-G modulation. Moreover, results on age and gender indicate that both of these parameters should be further investigated in studies involving sHLA-G expression. Finally these data suggest that sHLA-G production is not only regulated by UTR but also potentially by specific microenvironments. For exemple, UTR-1 and UTR-3 have been associated with different levels of malaria infection under a recessive model These results may constitute essential elements on the one hand to optimize the selection of donors for organ transplantation and on the other hand the diagnosis and treatment of infectious and parasitic diseases. Further investigations in larger cohorts and in different populations are necessary not only to detect new functional polymorphisms in HLA-G regulatory regions, but also to reveal the extent of biological phenomena that influence sHLA-G secretion.Figure S1www.fluxus-engineering.com/network) based on protein sequences . UTR were added afterwards based on estimates for HLA-A∼HLA-G∼UTR haplotypes. Branch length represents the phylogenetic distance between HLA-A∼HLA-G haplotypes based on amino acid substitutions. The yellow circles represent haplotype frequencies. HLA-A lineages based on Gu X. and Nei M. 1999 Median Joining (MJ) network on HLA-A∼HLA-G haplotypes constructed using the Network program((TIF)Click here for additional data file.Figure S2Comparison between homozygous HLA-A*02  and HLA-A*68  (p = 0.002).(TIF)Click here for additional data file.Table S1HLA-A∼UTR∼HLA-G haplotype frequencies (Fq) estimated with the Gene[Rate] program in the Malian samples.(DOCX)Click here for additional data file.Table S2Observed (Obs) and expected (Exp) frequencies, differences (Diff) and standardized residuals (StdRes) for HLA-A and HLA-G and for HLA-A and UTR.(DOCX)Click here for additional data file.Table S3Soluble HLA-G mean and standard deviation (SD) are shown for each HLA-G UTR genotype.(DOCX)Click here for additional data file.


Arabidopsis thaliana, increased expression of a major borate transporter gene AtBOR1 or boric acid channel gene AtNIP5;1 improves plant growth under B-deficient conditions. In this study, we examined whether high expression of a borate transporter gene increases B accumulation in shoots and improves the growth of tomato plant, a model of fruit-bearing crops, under B-deficient conditions. We established three independent transgenic tomato plants lines expressing AtBOR1 using Agrobacterium-mediated transformation of tomato (Solanum lycopersicum L. cv. Micro-Tom). Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that two lines (Line 1 and Line 2) more strongly expressed AtBOR1 than Line 3. Wild-type plants and the transgenic plants were grown hydroponically under B-sufficient and B-deficient conditions. Wild-type and Line 3 (weakly expressing transgenic line) showed a defect in shoot growth under B-deficient conditions, especially in the development of new leaves. However, seedlings of Line 1 and Line 2, the transgenic lines showing strong AtBOR1 expression, did not show the B-deficiency phenotype in newly developing leaves. In agreement with this phenotype, shoot biomass under low-B conditions was higher in the strongly expressing AtBOR1 line. B concentrations in leaves or fruits were also higher in Line 2 and Line 1. The present study demonstrates that strong expression of AtBOR1 improved growth in tomato under B-deficient conditions.Nutrient deficiency in soil poses a widespread agricultural problem. Boron (B) is an essential micronutrient in plants, and its deficiency causes defects in both vegetative and reproductive growth in various crops in the field. In  Boron (B) is an essential micronutrient for plants . B media Arabidopsis thaliana roots, AtNIP5;1, a boric acid channel, plays a role in B uptake  and established AtBOR1-overexpressing tomato plants. Tomato is a model plant of fruit-bearing crops. The occurrence of B-deficiency in tomato cultivation fields has been reported in several countries from Europe, Asia, South America, and Africa  and hygromycin  were used for selecting the transformants carrying pTF467 and pHH104, respectively. Agrobacterium-mediated transformation of Micro-Tom was performed as described previously  and was referred to as the Actin-like gene. The primers specific to Actin-like were 5′-TGTTGCTATTCAGGCTGTGC-3′ and 5′-AATCACGACCAGCAAGATCC-3′.Genomic DNA was extracted from the leaf (about 5 mg) as described previously . Obtaine1) were germinated on vermiculite and grown for 14 days . T-DNA insertion was then examined by PCR as described above. PCR-positive transgenic plants and non-transgenic plants were transferred to MGRL hydroponic solution  were sown on vermiculite and grown for 19 days, after which the T-DNA insertion was examined by PCR as described above. PCR-positive transgenic plants and non-transgenic plants were transferred to MGRL hydroponic solution supplemented with 0.1 or 100 μM boric acid. The solution was renewed twice a week for the 0.1 μM boric acid treatment and once a week for the 100 μM boric acid treatment.Plants were incubated at 25°C under a 16 h light/8 h dark cycle. Seeds of non-transgenic and transgenic plants  for quantifying the B concentration . Plants t-test (p < 0.05).To verify the statistical significance of differences among the lines, the data were analyzed using the Student’s 0). The T-DNA integration into the genome was examined on these plants by PCR analysis using the specific primers for T-DNA . All tested regenerated T0 plants showed a band at the expected size. No band corresponding to the T-DNA fragment was obtained from non-transgenic plants using PCR. The Actin-like gene was used as a positive control and all plants (including non-transgenic plants) showed a band at the expected size. These results indicated that the regenerated plants contained the T-DNA insertion in their genome. These three lines were named L1, L2, and L3.After several batches of transformation, 11 independent candidate lines were obtained. Among them, three lines showed good fertility and more than 100 seeds were obtained from each line. These three lines were used for the following experiments. Genomic DNA was extracted from a leaf of regenerated plants . AtBOR1 expression was clearly observed after 20 cycles of PCR in L1 and L2, whereas no signal was detected in non-transgenic plants and L3. A relatively weak signal of AtBOR1 was obtained from L3 after 30 cycles of PCR compared to L1 and L2, but not from non-transgenic plants. These results suggest that introduced AtBOR1 is expressed relatively strongly in L1 and L2 compared to L3.Reverse transcription-polymerase chain reaction analysis was conducted to investigate whether the introduced AtBOR1 expression on growth of tomato plants under B-deficiency, non-transgenic plants and three transgenic T1 lines were grown hydroponically in the presence of 0.1 or 100 μM boric acid. Experiments were performed with at least four replications and representative individuals are shown in Figure 2. Under B-sufficient conditions (100 μM B), shoot growth and development were normal in all tested lines , although L3 showed relatively small shoots  compared to other samples . Phenotypic differences among lines were clear under the low-B conditions (0.1 μM B). In non-transgenic plants under the low-B conditions, shoot growth was retarded compared to the control . Curly leaves were observed and development of newly growing leaves was often inhibited by the B-deficient treatment . These phenotypes were also observed in L3 subjected to the low-B treatment , but not observed in L1 and L2 plants . In L1 and L2, development of new leaves was normal, even under the low-B condition . These results indicated that heterologous expression of AtBOR1 increases tolerance of tomato plants to B-deficiency stress.To investigate the effect of heterologous Figures 2H,I), was slightly higher than those of the non-transgenic plants and L3, although not significantly . This tendency was not observed in the 100 μM boric acid treatment.Shoot dry weight and B concentration were measured after 20 days of hydroponic culture under the B-sufficient and B-deficient conditions. L1 was not tested in this experiment due to the limited seed numbers. Shoot dry weight of L2, which showed improved B-deficiency-tolerance . Under the low-B treatment, shoot B concentration was 1.4-fold higher in L2 than in non-transgenic plants and L3. When plants were supplied with 100 μM boric acid, B accumulation in shoots did not differ significantly between the non-transgenic plants and L2. L3 tended to accumulate less B than non-transgenic plants.The B concentration in shoots was compared among the lines . For the low-B treatment, the fruits of L1 accumulated significantly higher B than non-transgenic plants . Fruit B concentration under the B-deficient condition appeared higher in L2 than in non-transgenic plants, although the difference was not significant. This tendency of an increased B concentration in L1 and L2 was not observed under B-sufficient conditions . The B concentrations in fruits of L3 was relatively lower than in non-transgenic plants under both B treatments. These results suggest that B accumulation in shoots and fruits is enhanced in the stronger AtBOR1 expression lines.We also examined fruit yields of non-transgenic and transgenic plants. In all lines, low-B treatment impaired fruit yield; however, no consistent result was obtained due to the large variation among plants (data not shown). We next measured B concentration in fruits harvested from the plants grown under B-sufficient and B-deficient conditions . These phenotypes are typical B-deficiency symptoms reported in early tomato seedlings, suggesting that our experimental condition was suitable for evaluating the effects of AtBOR1 overexpression in young tomato plants grown under limited B availability. We performed several batches of transformation experiments and obtained three independent transgenic tomato lines carrying P35S-AtBOR1  with substantial seed yields. Expression analysis of introduced AtBOR1 in these lines demonstrated that L1 and L2 were strongly expressing lines and L3 was a rather weakly expressing line . These strongly expressing lines did not show severe B-deficiency symptoms, which were observed in non-transgenic plants as well as in weakly expressing L3 . Furthermore, shoot and fruit B accumulation under the low-B conditions significantly increased in L1 and L2, respectively, compared to non-transgenic plants, but not in L3 . These results suggest that enhanced expression of a B transporter improves the root-to-shoot translocation of B in tomato under low-B availability, which contributes to maintaining proper shoot development. Since the effect of AtBOR1 expression on yield remains unclear due to the considerable variation observed under our hydroponic conditions, this should be further examined with an improved cultivation system and moderate B-deficiency conditions.In the present study, wild-type tomato plants showed retarded whole shoot growth, curled leaves, and abnormal and poor development of new leaves in the presence of 0.1 μM boric acid . Growth under the 100 μM boric acid condition was comparable among the non-transgenic and transgenic plants , suggesting that overexpression of AtBOR1 has a negligible adverse effect on tomato young seedlings under B-sufficient conditions. In A. thaliana roots, AtBOR1 protein levels are regulated by post-translational mechanisms, and protein degradation is promoted under B-sufficient conditions through ubiquitination of AtBOR1 (A. thaliana accumulates wild-type levels of B under excess B treatments (AtBOR1 expression in tomato, but similar mechanisms may regulate AtBOR1 levels in tomato under B-sufficient conditions.Optimal B concentrations in culture media are narrow, and overdose of B leads to increased B accumulation in plant tissues and plant growth inhibition . Excess f AtBOR1 . This reeatments . In thisIn the present study, we established transgenic tomato plants tolerant to B-deficient environments by upregulating a B-transporter gene. Since B-deficiency is a serious issue in various crop productions, our findings suggest that application of AtBOR1 or its ortholog to crop breeding may improve growth under B-deficient cultivation conditions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HLA-G is a non-classical HLA class-Ib molecule expressed mainly by the extravillous cytotrophoblasts (EVT) of the placenta. The expression of HLA-G on these fetal cells protects the EVT cells from immune rejection and is therefore important for a healthy pregnancy. The mechanisms controlling HLA-G expression are largely unknown. Here we demonstrate that miR-148a and miR-152 down-regulate HLA-G expression by binding its 3′UTR and that this down-regulation of HLA-G affects LILRB1 recognition and consequently, abolishes the LILRB1-mediated inhibition of NK cell killing. We further demonstrate that the C/G polymorphism at position +3142 of HLA-G 3′UTR has no effect on the miRNA targeting of HLA-G. We show that in the placenta both miR-148a and miR-152 miRNAs are expressed at relatively low levels, compared to other healthy tissues, and that the mRNA levels of HLA-G are particularly high and we therefore suggest that this might enable the tissue specific expression of HLA-G. HLA-G is a nonclassical HLA class Ib molecule that possesses unique features, such as low polymorphism, restricted expression and seven spliced isoforms NK cell activity is balanced by signals from activating and inhibitory receptors and in that regard, HLA-G inhibits NK activity mainly via the interaction with the inhibitory receptor LILRB1 Although the HLA-G protein shows low polymorphism in the human population, especially when compared with other HLA class-I molecules, it possesses high polymorphism in its promoter region and in its 3′untranslated region (UTR). A 14 bp insertion/deletion polymorphism in the 3′UTR of HLA-G has been associated with RNA stability, however it is not clear whether or not this polymorphism can indeed affect HLA-G expression MiRNAs are ∼22 nt long non-coding RNA molecules that usually negatively regulate gene expression post-transcriptionally, mainly by binding to the 3′UTR of mRNAs Here we demonstrate that both miR-148a and miR-152 down-regulate HLA-G expression and that this down-regulation has functional consequences, as it prevents HLA-G-mediated inhibition of NK cell killing, through the LILRB1 inhibitory receptor. We further show that, contrary to what has been suggested, the C/G polymorphism has no influence on HLA-G regulation by these miRNAs. Finally, we show that the expression levels of the HLA-G-targeting miRNAs in the placenta are low as compared to other tissues and that the mRNA levels of HLA-G in the placenta are high. Therefore, we suggest that this might allow the expression of HLA-G in the placenta and not in other healthy tissues.5′ CGGGATCCGCCGCCACCATGGTGGTCATGGCGCCC 3′. Rv HLA-G +3′UTR primer: 5′GGCATTCAAAGTTCTCATGTCTTCCATTTA 3′.We used the RKO cell line (obtained from ATCC) and the MHC class I-negative human B lymphoblastoid cell line 721.221 5′ tagctcagtggaccacaaatt 3′). The downstream fragment was amplified using an internal 5′ primer bearing the mutation (5′aatttgtggtccactgagcta 3′) and the 3′ HLA-G+3′UTR edge primer, described above. Next, both purified fragments were mixed together with the 5′ edge primer and the 3′ edge primer to generate the mutated full-length cDNA.The plasmid expressing HLA-G with a cytosine base in position +3142 in the 3′UTR was generated by PCR. First, the upstream fragment was amplified using the 5′ HLA-G edge primer, described above and an internal 3′ primer bearing the mutation , according to the manufacturer's instructions. The identity of NK cells was confirmed by FACS. Purity was >98%. LILRB1-positive NK clones were grown in culture in the presence of 10 ng/ml IL-15 (PeproTech) and then used for NK cytotoxicity assays.The following oligonucleotides were used to generate artificial short hairpin RNAs that function as orthologs of pre-miRNA hairpins :5′gatccccTCAGTGCACTACAGAACTTTGTttcaagagaACAAAGTTCTGTAGTGCACTGAtttttggaaa 3′Fw miR148a: 5′agcttttccaaaaTCAGTGCACTACAGAACTTTGTtctcttgaaACAAAGTTCTGTAGTGCACTGA ggg 3′.Rv miR-148a: Fw miR-152:5′gatccccTCAGTGCATGACAGAACTTGGttcaagagaCCAAGTTCTGTCATGCACTGAtttttggaaa 3′.5′agcttttccaaaaaTCAGTGCATGACAGAACTTGGtctcttgaaCCAAGTTCTGTCATGCACTGAggg 3′.Rv miR-152: 5′ gatcccc TCCTGTGGTGTTTGGTGTGGTTttcaagaga AACCACACCAAACACCACAGGAtttttggaaa 3′Fw miR-BART12: 5′ agcttttccaaaaa TCCTGTGGTGTTTGGTGTGGTT tctcttgaa AACCACACCAAACACCACAGGA ggg 3′Rev miR-BART12: The specific oligonucleotides of the different miRNAs were annealed and inserted into the pTendoplasmic reticulum vector and then were excised from the vector together with the H1 RNA polymerase III promoter into the lentiviral vector SIN18-pRLL-hEFIp-EGFP-WRPE, as described The institutional review board of Hadassah–Hebrew University Medical Centers approved the use of decidual and placental waste material from healthy elective first-trimester pregnancy terminations, according to the principles of the Helsinki convention. Immunohistochemistry for HLA-G was performed on first trimester human placental formalin fixed paraffin embedded sections For the firefly luciferase vector we used the pGL3 control vector (Promega). The 3′UTR of HLA-G (containing guanine or cytosine at position +3142) was amplified by PCR from the pcDNA3 plasmid expressing the HLA-G protein with its 3′UTR (described above) and inserted into XbaI site immediately downstream from the stop codon. The primers used for this amplification were:5′gctctagaattgaaaaggagggagctact 3′ and Rv 3′UTR XbaI: 5′gctctagaaagttctcatgtcttccattt 3′. For the generation of the mutated 3′UTR of HLA-G the seed binding region in the 3′UTR was mutated by PCR. First, the upstream fragment was amplified using the 5′ edge primer, used to amplify the 3′UTR and an internal 3′ primer bearing the mutation (mut middle: 5′agttatagcagtcgtgaccacaaa 3′). The downstream fragment was amplified using an internal 5′ primer bearing the mutation (mut middle 5′tttgtggtcacgactgctataact 3′) and the 3′ edge primer used to amplify the 3′UTR.Fw 3′UTR XbaI: RKO cells were transduced with the lentiviral vectors for miR-148a, miR-152 and the control miRNA. Cells in 24-well plates were transfected with LT1 transfection reagent (MiRus) with 200 ng firefly luciferase reporter vector and 50 ng renilla luciferase pRL-CMV  in a final volume of 0.5 ml. Firefly and renilla luciferase activities were measured consecutively with the Dual-Luciferase Assay system (Promega) 48 h after transfection. This system allows us to estimate the down-regulation of the 3′UTR of HLA-G (which is cloned downstream to the firefly luciferase gene) by the miRNAs present in the transduced RKO cells. As an internal control, the firefly luciferase activity is normalized to renilla luciferase activity (both constructs are co-transfected to the cells and their activity is measured from a single sample). To finally determine the effect of the miRNAs on the 3′UTR of HLA-G, the normalized firefly luciferase activity, which was measured in the transduced cells expressing either miR-148a or miR-152, is further normalized to the firefly luciferase activity which was measured in the transduced cells expressing the control miRNA.The following antibodies were used: DyLight 649-conjugated goat anti-mouse (Jackson ImmunoResearch), mouse anti human HLA-G , DyLight 649-conjugated goat anti-human IgG was used as a secondary Ab for the staining with the fusion protein. For staining of the NK clones, the mAb anti hILT2 (R&D systems) was used. The fusion protein used in the present study was LILRB1-Ig . All specimens were polyadenylated with poly(A) polymerase (Ambion). RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) and 0.5 g poly(T) adaptor. DNA was amplified with specific primers and Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen). Reaction primers were a 3′ adaptor primer and primers based on the microRNA sequences. The following primers were used in quantitative real-time PCR analysis of mRNA levels: Fw HLA-G: Recently, several studies suggested that HLA-G expression is regulated by the cellular miRNAs miR-148a, miR-148b and miR-152 which have predicted binding sites in its 3′UTR To verify that a binding site for miR-148a, miR-148b and miR-152 exists in the 3′UTR of HLA-G we used the RNAhybrid algorithm On the basis of the calculation of minimum free energy (mfe) it can be indeed predicted that both miR-148a and miR-152 can potentially target the 3′UTR of HLA-G . The anaTo demonstrate that miR-148a and miR-152 directly bind the 3′UTR of HLA-G, we performed a dual luciferase reporter assay. For this, we generated three firefly luciferase constructs: one contained the 3′ UTR of HLA-G with a guanine at position +3142 (G variant), one contained the 3′ UTR of HLA-G with a cytosine at position +3142 (C variant) and another in which the 3′UTR of HLA-G was mutated at the ‘seed sequence’ of the predicted miR-148a and miR-152 binding sites (the mutation included both binding sites). We transiently transfected these constructs into the RKO human colon cancer cells that had been transduced with miR-148a, miR-152 or a control miRNA. As can be seen in To demonstrate that miR-148a and miR-152 can target HLA-G we generated transfectants of 721.221 cells that express HLA-G with its 3′UTR . In addition, we cloned miR-148a, miR-152 and a control miRNA into lentiviral vectors that also contain a green fluorescent protein (GFP) cassette, which allowed us to monitor transduction efficiency. These constructs were then transduced into the 721.221/HLA-G transfectants. The lentiviral transduction was extremely efficient . We confWe next used our 721.221/HLA-G transfectants expressing HLA-G with or without its 3′ UTR and examined the expression levels of HLA-G in the presence or in the absence of miR-148a and miR-152 . As can HLA-G is mainly recognized by the inhibitory receptor LILRB1 We also transduced JEG-3 cells, which endogenously expresses HLA-G, with miR-148a and with miR-152 and, as expected, observed down-regulation of HLA-G (data not shown). However, we chose not to continue our experiments with this cell line, since it is not killed by NK cells as it does not express most of the killer ligands for the NK killer receptors To reveal the mechanism of the miRNA-mediated repression, we performed quantitative real-time PCR analysis of the 721.221/HLA-G cells transduced with either miR-148a, miR-152 or a control miRNA to evaluate the abundance of the HLA-G transcript. We observed that in the presence of both miR-148a and miR-152, the mRNA levels of HLA-G were substentially decreased . This suThe interaction between LILRB1 and HLA-G leads to inhibition of NK cell-mediated cytotoxicity EVT cells in the placenta express high levels of HLA-G . Our finWe next evaluated the expression of the HLA-G mRNA and miRNAs in various human tissues by using quantitative real-time PCR. Because the mRNA levels are probably different among various tissues, an internal normalizer control must be used. For that purpose mir-16 was used as a reference miRNA, since the expression of this miRNA is quite ubiquitous in almost all somatic tissues. As can be seen in HLA-G is a non-classical MHC-Ib molecule with a restricted distribution that is almost completely confined to the EVT cells of the fetus It has been suggested that the expression of HLA-G is controlled by miRNAs, which target the 3′UTR of HLA-G mRNA, thereby down-regulating its expression We show here that both miR-148a and miR-152 target HLA-G at its 3′UTR and hence down-regulate HLA-G expression. This down-regulation of HLA-G expression has functional consequences, as it diminished the binding of the inhibitory receptor LILRB1 to the transduced cells and consequently abolished the HLA-G mediated inhibition of NK cytotoxicity. Furthermore, we have found that the C/G polymorphism has no influence on HLA-G expression. Interestingly, while we show that the +3142 C/G polymorphism makes no difference to the miR inhibition of HLA-G, a different polymorphism at the 3′UTR of HLA-C did show an effect on miR-148a inhibition of HLA-C We further show the physiological implications of the miR-148a and miR-152 mediated down-regulation of HLA-G expression. As mentioned above, HLA-G expression is almost completely restricted to the EVT cells Several findings suggest that the regulation of HLA-G by miR-148a and miR-152 extends beyond normal physiological conditions. It has been suggested that HLA-G is important for a successful pregnancy not only due to its protective effect on the fetal cells (by inhibiting immune cell function) but also due to its effect on cytokine and angiogenic factor secretion by NK cells In summary, our study shows that both miR-148a and miR-152 specifically regulate HLA-G expression by targeting its 3′UTR and that this regulation may control NK-mediated killing, through the down-regulation of HLA-G.


The anticancer efficacy of topical and oral P-S was further evaluated in mice bearing A431 intradermal xenografts. Compared to the controls, topical P-S hydrogel inhibited the A431 xenografts by 70.5% (p<0.01), while oral P-S inhibited it by 43.4% (p<0.05), being significantly less effective than topical P-S (p= 0.017). Topical P-S hydrogel generated significant levels (>500 nmol/g tumor tissue) of intact P-S in the tumors, accounting for 92.5% of the total metabolites in the A431 xenografts. This local delivery of high levels of intact P-S to the A431 xenografts is an important contributor to the potent activity of topical P-S and no local or systemic side effects were noted in the treatment group. Thus, topical P-S is a promising treatment modality against non-melanoma skin cancer and merits further evaluation.Phospho-sulindac , a novel sulindac derivative, has shown superior anticancer efficacy and safety compared to sulindac. In this study, we investigated the efficacy of topical P-S hydrogel in the treatment of non-melanoma skin cancer in preclinical models. P-S is a potent inhibitor of A431 epidermoid carcinoma  Non-melanoma skin cancer is the most common type of cancer in Caucasian populations ,2. In thNon-steroidal anti-inflammatory drugs (NSAIDs) have demonstrated significant efficacy in the chemoprevention of colon  and skinTopical treatment regimens are considered to be effective alternatives for non-melanoma skin cancer. Skin delivery is non-invasive; achieves high local levels of drug; minimizes systemic exposure; and is more acceptable to patients. In our previous investigations, the effective delivery of P-S to tumors was limited by its inactivation by carboxylesterases ,12. We rIn this report, we demonstrate that P-S is a potent inhibitor of non-melanoma skin cancer cells, and topical delivery of P-S resulted in strong inhibition of skin cancer xenografts in mice. These findings suggest that topical P-S is a promising strategy for the treatment of non-melanoma skin cancer.P-S (OXT-328) was a gift from Medicon Pharmaceuticals Inc. . Cell culture reagents were purchased from Cellgro . Other reagents, unless otherwise stated, were obtained from Sigma-Aldrich .The human epidermoid carcinoma cell line (A431) was obtained from American Type Culture Collection (ATCC), and maintained in DMEM media containing 10% fetal bovine serum and penicillin/streptomycin. All experiments were performed with cells between passages 1 to 10.A mixture of Pluronic P123 and P-S, dissolved in tetrahydrofuran , was dialyzed for 24 h at room temperature through a membrane  in phosphate-buffered saline, which was replaced three times. The dialysis bag was then wiped with absorbent paper and placed under solid PEG  to absorb and concentrate the solution inside the bag until gel formation.The final drug loading onto the gel was 1.4% (w/w), while the polymer constituted 27–30% w/v of the gel. Control gel was prepared by a cold method where 28% w/v of pluronic P123 was dispersed slowly in PBS at 2–5°C.Cell viability was measured with the MTT assay (Roche Diagnostics) and cell proliferation with the 5-bromo-2′-deoxyuridine  assay, according to the manufacturer’s instructions. Apoptosis and necrosis were assessed by staining cells with Annexin V and propidium iodide (PI) and analyzing them by flow cytometry .6 each) suspended in 100 μl complete DMEM medium. When the average tumor size reached 120±40 mm3, the animals were divided into four groups (n=6–7), and were given the following treatments, respectively: i) none; ii) topical plain hydrogel (3 times per day); iii) P-S ; and iv) topical P-S hydrogel . The tumors were measured twice a week with a digital microcaliper, and tumor volumes were calculated using the following formula: tumor volume = [length × width × (length + width/2) × 0.56]. After treatment for 2 weeks, the animals were sacrificed and their tumors were removed. The levels of P-S and its metabolites in the tumors were determined by HPLC. This animal study was approved by the Institutional Animal Care and Use Committee of Stony Brook University.Female NOD/SCID mice (6–7 weeks-old) were purchased from Harlan Sprague-Dawley . At 7–8 weeks of age, the mice were inoculated intradermally on both flanks with A431 cells ] and 100% acetonitrile.The HPLC system consisted of a Waters Alliance 2695 Separations Module equipped with a Waters 2998 photo-diode array detector (328 nm)  and a Thermo BDS Hypersil C18 column  . The mobile phase consisted of a gradient between buffer A [HCell death and proliferation of paraffin-embedded A431 xenograft tissue sections were determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Ki-67 immunohistochemical staining, respectively, as previously described .Data are expressed as the mean ± SEM. Statistical analyses were performed by ANOVA. P-values <0.05 were considered statistically significant.in vivo efficacy of P-S against skin cancer, we treated SCID mice bearing A431 xenografts (n=6–7) topically with vehicle hydrogel, or P-S hydrogel, or orally with P-S, whereas the last group was left untreated. At equivalent dosage, topical P-S more effectively inhibited the growth of A431 tumors (p=0.017) compared to oral P-S  of sulindac or sulindac sulfide only weakly inhibited cell growth (<40%). Cell cycle analysis revealed that PS treatment induced cell cycle arrest at G2/M phase  of sulindac or sulindac sulfide had no measurable impact on cell proliferation or apoptosis.Moreover, treatment of A431 cells with P-S at 1.5×ICWe next determined cell proliferation and apoptosis in tumor tissue sections. Compared to control, topical P-S decreased cell proliferation (determined by PNCA staining) by 25% and induced apoptosis (TUNEL) by 30% (p<0.05) . These fTo evaluate the delivery of P-S via the topical route, we measured blood and tumor drug levels one hour after drug administration . TopicalNo intact P-S could be detected in the blood of the topical P-S treated animals. Sulindac is the major metabolite found in the blood, albeit at lower levels (7-fold lower) compared to those after oral administration . These dOur study demonstrates that P-S is a strong inhibitor of nonmelanoma skin cancer in pre-clinical models. Topical P-S strongly suppresses the growth of A431 skin cancer xenografts in mice, an effect mediated by i) the potent cytokinetic effect of P-S on A431 skin cancer cells, and ii) direct delivery to the skin tumors of intact P-S, the biologically most active molecule.in vitro (36-fold more potent than sulindac). A strong cytokinetic effect underpins the inhibitory potency of P-S, which is a result of inhibition of cell proliferation, induction of apoptosis and cell cycle arrest (G2/M). The induction of apoptosis appears to be the predominant mechanism, as P-S profoundly triggers apoptosis in A431 cells (4 to 10-fold over control) in vitro; whereas an equimolar level of sulindac, the parent NSAID, has no significant effect.P-S is a potent inhibitor of the A431 epidermal skin cancer cell line In vivo, topical application of P-S strongly suppresses the growth of A431 xenografts by 70.5%, compared to the control. Oral administration of P-S, on the other hand, only resulted in a moderate inhibition of xenograft growth (43.4%). Interestingly, the anti-tumor efficacy of the topical route is significantly better than that of oral administration (differs by nearly two fold). Topical P-S also effectively induces apoptosis in A431 xenografts. These results indicate that P-S is an efficacious agent against non-melanoma skin cancer, and topical delivery of P-S appears to confer a significant therapeutic advantage compared to oral administration. Dilution effects, intestinal absorption and the metabolism of oral P-S can account for its lower efficacy.P-S can be incorporated in a pluronic polymer to form a hydrogel for topical application. in vivo. P-S is considerably (8- to 36-fold) more cytotoxic towards A431 cells than its metabolites, sulindac, sulindac sulfide and sulindac sulfone (An important contributing factor to the potent activity of topical P-S is the improved delivery of intact drug to tumors  sulfone , or the  sulfone . However sulfone ,12 with Carboxylesterase 1, a broadly-specific carboxylesterase highly expressed in the intestine, liver and plasma, is primarily responsible for the hydrolytic inactivation of P-S ,12. The Topical drug delivery is a valuable strategy of limiting systemic exposure, thereby lowering the risk of undesirable side effects. Indeed, topical therapy has been proposed to reduce the potential gastrointestinal and cardiovascular side effects of conventional NSAIDs in the treatment of actinic keratosis , pain 22 and artIn conclusion, topical application of P-S-incorporating pluronic hydrogel is effective in inhibiting the growth of nonmelanoma skin cancer, and has superior efficacy compared to oral administration. Our results indicate that direct skin delivery of P-S is a promising modality for the treatment of skin cancer which merits further investigation.


The Goto-Kakizaki (GK) rat is a well-studied non-obese spontaneous type 2 diabetes (T2D) animal model characterized by impaired glucose-stimulated insulin secretion (GSIS) in the pancreatic beta cells. MicroRNAs (miRNAs) are short regulatory RNAs involved in many fundamental biological processes. We aim to identify miRNAs that are differentially-expressed in the pancreatic islets of the GK rats and investigate both their short- and long term glucose-dependence during glucose-stimulatory conditions.Global profiling of 348 miRNAs in the islets of GK rats and Wistar controls  using locked nucleic acid (LNA)-based microarrays allowed for the clear separation of the two groups. Significant analysis of microarrays (SAM) identified 30 differentially-expressed miRNAs, 24 of which are predominantly upregulated in the GK rat islets. Monitoring of qPCR-validated miRNAs during GSIS experiments on isolated islets showed disparate expression trajectories between GK and controls indicating distinct short- and long-term glucose dependence. We specifically found expression of rno-miR-130a, rno-miR-132, rno-miR-212 and rno-miR-335 to be regulated by hyperglycaemia. The putative targets of upregulated miRNAs in the GK, filtered with glucose-regulated mRNAs, were found to be enriched for insulin-secretion genes known to be downregulated in T2D patients. Finally, the binding of rno-miR-335 to a fragment of the 3′UTR of one of known down-regulated exocytotic genes in GK islets, Stxbp1 was shown by luciferase assay.The perturbed miRNA network found in the GK rat islets is indicative of a system-wide impairment in the regulation of genes important for the normal functions of pancreatic islets, particularly in processes involving insulin secretion during glucose stimulatory conditions. Our findings suggest that the reduced insulin secretion observed in the GK rat may be partly due to upregulated miRNA expression leading to decreased production of key proteins of the insulin exocytotic machinery. MicroRNAs (miRNAs) are ∼21-23 nucleotides (nt) long, non-coding RNAs which negatively regulate expression of genes via mRNA degradation, mRNA deadenylation and/or translational repression The link between miRNA and diabetes began with the discovery of a highly-expressed pancreatic islet miRNA, miR-375. It was then shown to be involved in exocytosis via the targeting of the myotrophin gene in a still unclear mechanism N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex proteins has been shown in GK rat islets The Goto-Kakizaki (GK) rat, selectively bred from Wistar rats with high blood glucose levels, is a well-studied non-obese spontaneous type 2 diabetes animal model The contribution of miRNAs to the development of diabetes in the GK rat has been largely unexplored. Insulin resistance in target tissues is one aspect of the GK diabetic phenotype and miRNA profiling studies on skeletal muscle, liver and adipose tissues of the diseased animal have been performed N = 10) compared to the age-matched Wistar controls  (p = 7.5×10−5) (Consistent with previous studies on 8-week old Stockholm colony GK rats .5×10−5) . The pla.5×10−5) , though vs 8 mmol/L in Wistar control). After 120 minutes, the blood glucose in the GK was still twice its fasting level clearly indicating an impaired insulin response Another well-known phenotype of GK rat is insulin resistance in target tissues. In the aforementioned study of Ostenson and co-workers, results of intra-peritoneal glucose tolerance test showed rapid increase in blood glucose to a maximum 20 mmol/L   . = 0%). Employing SAM without large fold-change threshold requirement allowed for the identification of differentially-regulated miRNAs with small but consistent differences among the individual animals from each group.Hierarchical clustering of the global miRNA profiles comprising 348 rat miRNAs allowed clear separation of the GK rats from the controls . SignifiUsing miRNA-specific stem-loop qPCR assays Since one of the major diabetic phenotypes of GK rats is impaired GSIS, we investigated whether the differentially-expressed miRNAs would show glucose-dependent regulation both in the short-term (1 h) and long-term (24 h) exposure to varying glucose concentrations. The three glucose concentrations used were 2.8 mM (2.8G), 8.3 mM (8.3G) and 16.7 mM (16.7G), representing hypoglycaemic, near-physiological and hyperglycaemic environments.vs 2.8G in Wistar), i.e. GK miRNAs significantly-upregulated in the microarrays consistently exhibited higher levels of expression than the Wistar miRNAs, whereas rno-miR-708 which was found to be downregulated in GK was minimally expressed. Among the miRNAs tested only rno-miR-130a, rno-miR-132, rno-miR-212 and rno-miR-335 responded to glucose stimulation at either 8.3 mM or 16.7 mM in the healthy Wistar islets . In fact, a number of miRNAs are markedly decreased at 8.3G. In virtually all upregulated miRNAs in the GK, their levels at 16.7G are significantly greater than the levels found in Wistar. Even rno-miR-375 which was non-responsive at stimulatory conditions in Wistar significantly increased in GK at 16.7G  were observed upon glucose stimulation at 8.3G, especially for rno-miR-124, rno-miR-142-5p and rno-miR-409-3p   or do not change (rno-miR-409-3p), whereas for miRNAs whose levels decrease with increasing glucose concentration in Wistar islets, the GK islet miRNAs is also reduced (miR-375) .These results indicate compensatory mechanisms in the GK islets striving to reset the levels of miRNAs to that of the controls during prolonged exposure to hyperglycaemic condition. This is clearly seen for rno-miR-212, rno-miR-132, and rno-miR-130a, whose expression levels in the GK and Wistar islets eventually coincide at 16.7G . HoweverNext we were interested to find potential targets proteins of miRNAs up-regulated in the GK rat. The poor overlap between different miRNA target prediction algorithms is well known and the lack of rationale behind filtering of targets by using multiple target prediction algorithms has been demonstrated To reduce the amount of false positive hits, the predicted targets were then filtered by intersecting with a list of glucose-regulated mRNAs expressed in isolated rat islets incubated in low, medium and high glucose levels Stxbp1 (Syntaxin-binding protein 1) or Munc18-1 plays an important role in the fusion of insulin secretory granules in the plasma membrane of the beta cell by modulating the folded conformation of syntaxin 1A We have shown differential miRNA expression profiles in the isolated pancreatic islets of mildly hyperglycaemic GK rats compared to that of healthy Wistar controls. One of the strengths of this work lies in the use of LNA-based arrays. In such arrays, the capture oligonucleotide probes were adjusted within a narrow melting temperature range by introducing LNA monomers yielding optimal hybridisation conditions. Recently, the utility and reliability of these arrays were shown to be comparable, if not superior to next generation high-throughput sequencing approaches to expression profiling Four of the glucose-regulated miRNAs found in this study, mir-124, mir-212, mir-132, and mir-409-3p, were previously reported to be upregulated in the mouse insulin secreting cell line, MIN6, after 16 h stimulation in 25 mM glucose Interestingly, miR-212 and miR-132 have also been shown to be upregulated in isolated pancreatic islets in the obese phenotypes of both the diabetes-resistant (B6) and diabetes-susceptible (BTBR) mouse models per se since they responded to glucose stimulation even in the Wistar islets.We found that incubation of isolated rat islets in different glucose concentrations and lengths of time revealed differences in both magnitude and direction of miRNA expression between the model animals and the controls. We saw changes in miRNA expression manifested within a short temporal window of only one hour, such as for rno-miR-130a, rno-miR-132, rno-miR-212 and rno-miR-335. These miRNAs are most likely regulated by hyperglycaemia rather than the GK phenotype e.g. V- or inverse-V- shaped across four glucose concentrations. Furthermore, statistically-significant changes in gene expression were predominantly observed between G5 and G10 indicating active gene regulation mechanisms aimed at keeping optimal islet function during physiological glucose levels A further evidence of active regulation of miRNAs during short-term fluctuation of glucose levels is the prominent decrease of some miRNAs at 8.3G, which is a clear deviation from the typical up or down glucose concentration-dependent mode of regulation. Interestingly, this was also reported in the work of Bensellam and co-workers in which more than 400 mRNA probe-sets displayed atypical expression patterns, Rapid modulation of cellular miRNAs was previously observed in human hepatoma cell line and primary murine hepatocytes where IFNβ treatment lead to induction of specific miRNAs within just 30 minutes of incubation Islets incubated for 24 h at 2.8G and 16.7G represent exposure to chronic states of hypo- and hyperglycaemia, respectively. Such extended exposure of islets to non-physiological glucose environments is more aimed at studying the effects of nutrient availability and in the case of higher glucose concentrations, also imposes effects of gluco-toxicity on islet function. A longer incubation time also allows for the observation of constitutively regulated miRNAs. Notably, during this period we found evidence of dosage compensatory mechanisms attempting to bring the expression levels of specific miRNAs within the islets of the GK animals towards that of the controls. This trend was particularly distinct for both miR-212 and miR-132, which belong to the same gene cluster being only 200 nt apart and containing identical seed sequences. Additionally, the genes encoding both miRNAs harbour a cAMP-response element binding protein (CREB) site indicating common regulation by the same transcription factor The presence of deregulated miRNAs in the GK rat islet is strikingly clear, but how do miRNAs ultimately contribute to the islet's specialized function as a hormone-secreting factory? In a recent review, D.P. Bartel (2009) outlined different scenarios of miRNA-mediated regulatory effects, wherein miRNAs may act as binary off switches, rheostats or as facilitators of neutral interactions It is well-known that the lower capacity of GK islets to secrete insulin under glucose stimulatory conditions is not due to reduced stores of islet hormones since insulin content in GK islets and controls are comparable in vitro experiments performed on isolated pancreatic islets may only partially reflect the dynamic nature of miRNA regulation happening in vivo. Nonetheless, the potential role of miRNAs in regulating the insulin exocytotic machinery will pave further investigation about the contribution of deregulated miRNAs in T2D. Translating these findings into the context of human T2D and further characterization of other hypothesized miRNA-target interactions would certainly contribute to understanding the roles of these relatively novel regulatory molecules in the aetiology of the disease.To conclude, we hypothesize that the imbalance in the miRNA network in the GK rat islets may contribute to the impaired GSIS characteristic of the animals, though it cannot be excluded that such perturbation may serve as a resetting/compensatory mechanism to counteract the disease phenotype. It should also be noted that the All animal experimentations were approved by the local ethics committee of Lund University under permit number M 185 06 and M 73 08.http://www.taconic.com). Non-fasting glucose levels were measured from intra-cardiac blood using a glucose meter  immediately after killing. Plasma insulin was measured by RIA. Due to insufficient amount of islets from individual animals, separate batches of animals were used for in vitro insulin-secretion assay and global profiling of miRNA expression.Female GK/MolTac and control Wistar rats, 60 days old, were purchased from Taconic Europe. The GK inbred model was developed at Tohoku University, Japan in 1975 2, the islets were transferred in new plates with fresh medium at desired glucose concentrations and incubation time. In parallel, batches of 10–12 intact islets were transferred into new tubes in quadruplicates for 1 h or 24 h GSIS. Samples were retrieved from the tubes for insulin secretion assays using RIA. The islets from plates were collected and processed immediately for total RNA preparation and stem loop qPCR.Pancreatic islets were isolated by collagenase digestion and the islets were handpicked in cold Hank's buffer with 1 mg/mL bovine serum albumin (BSA) prior to secretion assays. Islets from three individual animals (no pooling) were seeded in separate wells in plates containing Krebs buffer with 2.8 mM glucose. After 1 h pre-incubation at 37°C with 5% COTotal RNA from freshly isolated islets from individual animals was extracted using the Qiagen miRNeasy isolation kit . The quality of the total RNA was evaluated both by spectrophotometry and electropherogram profiles using Nanodrop and Experion's automated electrophoresis system (Bio-Rad). All samples have Abs 260/280 ∼2.0, Abs 260/230 >1.4 and Experion's RNA Quality Indicator (RQI) >7.0, indicating high-quality total RNA preparations and consistent extraction procedure (data not shown). A reference pool was made from mixing equal amount of total RNA from the individual animals. 500 ng of total RNA from each sample and reference pool were labelled with miRCURY™ Hy3™ and Hy5™ fluorescent label . The Hy3™-labeled samples and a Hy5™-labeled reference pool RNA samples were mixed pair-wise and hybridized to the miRCURY™ LNA array v.11.0  according to manufacturer's protocol for Maui hybridisation chamber. The LNA array contains capture probes targeting 348 rat miRNAs listed in the miRBASE version 12.0 release N = 3), each in triplicate wells in 384-well plates using Applied Biosystems 7900HT RT-PCR system.Total RNA from all islet samples were prepared and quality checked as above. MiRNA expression was measured by stem-loop qPCR using primers and probes from TaqMan MicroRNA assays (Applied Biosystems). For each experimental condition, three separate real-time PCR runs were performed from islet preparations from different individuals  TM) were purchased . The sequences included position 1368 to 1547 (TargetScan numbering) of the 3′UTR of Stxbp1 (NM_003165). To facilitate directional insertion into the pmiRGLO dual luciferase miRNA target expression vector , PmeI and NotI restriction sites were also added in the 5′end and XbaI site in the 3′end . The sequences of the 195 nt strands are: i) sense strand: AAACTAGCGGCCGCCTTCTTTTGTCCCTTCTCTCTGGTCAAGCAATGCTCTTGCTTCAGGACCTTGTTTGTCGAACATGTGGGGTTTCCTTTATGTTATTTATATAAATAATTTCTCAAATGGATATTTAAAAAAAAAGCTAGTCTGTCTTGAAACTTGTTAACTTGAAATCTTGACATCTCAGTGTTTAAAGTT5′- and ii) antisense strand: CTAGAACTTTAAACACTGAGATTCAAGAGTTTCAAGTTAACAAGTTTCAAGACAGACTAGCTTTTTTTTTAAATATCCATTTGAGAAATTATTTATATAAATAACATAAAGGAAACCCCACATGTTCGACAAACAAGGTCCTGAAGAGAGCCAATTGCTTGACCAGAGAGAAGGGACAAAAGAAGGCGGCCGCTAGTTT5′-. These strands were allowed to form a duplex prior to ligation within the multiple cloning site downstream of the luc2 reporter gene in the linearized vector. This resulted to the dual-luciferase reporter containing wildtype rno-miR-335 binding seed sequences (bold sequences). Another pair of ultramers containing mutations (bold-underlined mutated into complementary bases) in the two rno-miR-335 seed sequence binding sites is likewise inserted into a separate pmiRGLO vector resulting to the mutant construct. Sequences and orientation of inserts in both constructs were verified by sequencing .To generate the inserts encompassing the two rno-miR-335 binding sites in the proximal region of Stxbp1-3′UTR, single-stranded oligonucleotide sequences  supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10% heat inactivated fetal bovine serum (Sigma #7524), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma # P0781) while ∼30000 INS1-832/13 cells were seeded in 96-well plate containing RPMI 1640 (Gibco Cat #21875) with 11.1 mM D-glucose supplemented with 10% heat inactivated fetal bovine serum (Sigma #7524), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma # P0781), 10 mM HEPES (Sigma #H0887), 2 mM L-glutamine (Sigma #G7513), 1 mM sodium pyruvate (Sigma #S8636) and 50 µM ß-mercaptoethanol, cultured at 37°C and 5% CO2 in a humidified atmosphere. The empty vector and plasmid constructs were then co-transfected with either pre-miR-335 or pre-miR-scrambled negative control #1 (Applied Biosystems Europe BV) to 60–70% confluent HeLa or INS1-832/13 cells using the Attractene transfection reagent . Transfection experiments were done independently for both cell lines two times in triplicate wells.Renilla signal. Luminescence was acquired using the Magellan software on Tecan Infinite M200 plate reader.The Firefly luciferase activity was measured 2 days post-transfection using the Dual-Glo Luciferase assay system . Both cell lines exhibited ∼90% confluence by the time of assay. Transfection efficiency was normalized using the Student's t test. Values of p<0.05 were considered as statistically significant. For LNA arrays, Significant Analysis of Microarrays (SAM) was performed as implemented in MeV v4.3 for two class unpaired without using the fold-change criterion d, from the original and permuted data for each gene. The observed vs expected d values may be adjusted using a Delta parameter allowing for identification of significant genes even with small but consistent differences. SAM also estimates the False Discovery Rate (FDR) giving the proportion of significant genes most likely identified by chance. In this study, we set the Delta parameter to a conservative level corresponding to median FDR = 0%, i.e. the median number of false positives calculated during the procedure is equal to zero.Insulin secretion, luciferase assays and qPCR data are presented as mean ± SEM and p values are from two-tailed Figure S1Relative abundance of selected miRNAs in the pancreatic rat islet.A. Quantile plots of miRNA signals from LNA (locked nucleic acid) arrays of total RNA of Wistar and GK pancreatic islets. The Hy3 fluorescence  of miRNAs from six arrays of independent biological replicates from each animal group were averaged. B. Significant Analysis of Microarray (SAM) was performed on Hy3/Hy5 ratios . Significant hits identified by SAM have fold-changes of at least 1.5.(PDF)Click here for additional data file.Figure S2Evaluation of stability of endogenous controls for qPCR and array signals of the selected miRNAs.A. Distribution of the average raw Ct values of each miRNA in the 14 qPCR conditions. B. Quantile plots of the average raw Ct values showing median expression levels and scatter of data points.(PDF)Click here for additional data file.Table S1List of 1342 rat genes which are intersection of glucose-responsive genes  and collective miRNA targets in this study.(XLS)Click here for additional data file.Table S2List of 125 rat genes which are enriched for Transport and Secretory Processes with their corresponding putative miRNA regulator.(XLS)Click here for additional data file.