TP53, MDM2, MDM4, the polymorphisms of HPV-associated genes MTHFR, CASP8, CCR5, and HPV infection with survival of LC patients. SNPs were genotyped using PCR-RFLP. qRT-PCR was used to detect, identify, and quantify HPV. No statistically significant differences were detected between individual SNPs and patient survival with stage I-IV LC. Cluster analysis of SNPs in genes MDM4 A/A, CCR5 wt/Δ32, MTHFR C/T, MDM2 T/T showed possible association with the worse survival. Patients who were diagnosed with C/T polymorphic variant of gene MTHFR tend not to survive stage III-IV LC (P = .12). There is a tendency between MDM2 gene T/T variant and worse survival of patients diagnosed with late stage LC (P = .11). HPV infection is very rear among LC patients (3 of 92). Overall, there is a link, although statistically insignificant, between specific SNPs and LC patient survival frequency and time, meanwhile the combination of specific SNPs showed a statistically significant measure. In conclusion, we determined statistically significant (P = .04) link between the poor survival of LC patients after surgery and the combination of polymorphic variants C/T of the MTHFR and T/T of the MDM2 genes, whereas individually these SNPs do not show significant relationship with the survival of patients after surgery.Lung cancer (LC) is the second common and with the highest mortality oncological disease. Specific biomarkers for its diagnostics, treatment, and prognosis are still under the investigations. Aim of our study was to evaluate the relationship between the polymorphisms of TP53 pathway genes  Lung cancer (LC) is one of the most common malignant tumors  and the There are two main pathological types of LC: small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). NSCLC is the most common form of LC, where about 85% of cancers are classified as NSCLC, while SCLC occurs in approximately 13–15% of patients . The maiIt is well known that tobacco is considered to be the main risk factor for LC development. The length of time spent smoking and type of tobacco used may increase the relative risk of acquiring this disease , 8, 9. ATP53 (rs1042522), MDM2 (rs2279744), MDM4 (rs4245739), MTHFR (rs1801133), CASP8 (rs3834129), and CCR5 (rs333) alone or in combination, could affect survival in advanced non-small cell lung cancer (NSCLC) patients [However, not only tobacco smoking or various infections are responsible for LC development. Various genes and their mutations are closely associated with LC. Large studies were performed to find the exact biomarkers for precise diagnostic and treatment or follow-up of LC patients. On the other hand, multiple biologically relevant polymorphisms may have more accurate prediction power of cancer prognosis compared with single polymorphism because of the modest effect . The funFR rs18013, CASP8 TP53 pathway plays a crucial role in preventing carcinogenesis through its ability to impose cell cycle arrest and apoptosis following DNA damage and oncogene activation [TP53 gene and the most frequently studied is the G to C change at codon 72 in exon 4, resulting in the amino acid substitution of Arg to Pro . This SNP has been shown to alter TP53 function because it is located in the proline-rich region that is required for TP53 to induce apoptosis. Emerging evidence has shown that the TP53 Arg/Pro polymorphism is not only associated with LC development, but also affects individual sensitivity to platinum-drug chemotherapy and patients’ survival [in vitro and in vivo studies have indicated a greater susceptibility to degradation by HPV E6 for the p53 protein produced by the Arg allele at codon 72 than for the p53 protein produced by the Pro allele [Combined effect of HPV infection and gene polymorphism could have an important role in the development of NSCLC. Recently, it has been reported that high-risk HPV 16/18 3E6 protein is associated with p53 protein degradation in LC . The tumtivation , 16. Thetivation . Multiplsurvival . A tendeMDM2/MDM4) or viral proteins, leading to its degradation [(MDM2) is a key negative regulator of the TP53 pathway and usually is overexpressed in many cancers as oncoprotein. MDM2 oncogenic protein is the principal cellular antagonist of the TP53 gene product [MDM2 SNP at the 309th nucleotide in the first intron (rs2279744), with a T to G change, could increase the affinity for stimulatory protein 1 binding and result in increased MDM2 expression and subsequent attenuation of the TP53 pathway. This polymorphism has been associated with several cancers including LC [In most, if not all, cancers lacking mutation, wild-type p53 is inactivated by interaction with cellular  in the MDM4 3′ untranslated region has been reported to create a target site for hsa-miR-191, resulting in decreased MDM4 mRNA levels [MDM4, together with TP53, MDM2 and HPV E6 oncoprotein, may play a critical role in HPV-associated carcinogenesis [The MDM4 protein plays an important role in the negative regulation of the TP53 through its interaction with MDM2. A levels . Therefoogenesis , 22 and MTHFR) is a key enzyme in metabolism of folate and nucleotides to maintain DNA stability and prevent cancers through catalysing irreversible conversion of 5,10-methylenetetrahydrofolate  to 5-methyl tetrahydrofolate (5-methyl THF) [MTHFR gene is located on the short arm of chromosome 1 (1p36.3) and its cDNA has a total length of 2.2 kb [MTHFR is involved in DNA methylation and synthesis as an important enzyme in the folic acid metabolic process [MTHFR T/T genotype exhibited a significantly increased risk of LC compared to the MTHFR C/C and C/T genotype [Folate is essential for the synthesis of nucleotides, its deficiency induces double-strand breaks and increases cancer risk . Methylehyl THF) , 25, 26.f 2.2 kb . MTHFR i process . The mos process . The MTHgenotype .CASP8 gene) is crucial in generating cell death signals and eliminating potentially malignant cells. Genetic variation in CASP8 may affect susceptibility to cancer [CASP causes cell death by nuclear membrane breakdown, DNA fragmentation, chromatin condensation, and the formation of apoptotic genetic polymorphisms for genes controlling the cell cycle or apoptosis [CASP apoptotic pathway have been increasingly recognized. Several studies have demonstrated that some variants in apoptosis pathway genes are associated with the susceptibility to various human cancers, especially LC [CASP8-652 6N ins/del (rs3834129) polymorphism has been previously reported to influence the progression to several cancers. Finally, this suggests that the del allele, carrier and ins/del genotype of the -652 6N ins/del polymorphism in the CASP8 gene may be protective factors for cancer development [Caspase-8 , which is a 32-bp deletion in the coding region, encodes a truncated inactive receptor that is not expressed on the cell membrane. Moreover, molecular epidemiological studies showed that Δ32 polymorphisms are associated with cancer development [Chemokines are a family of small cytokines that regulate leukocytes in tissues, thus playing an important role in regulation of immunological processes . Chemokielopment .HPVs are small non-enveloped DNA viruses that usually infect squamous epithelial cells . It is wTP53 Arg72Pro (rs1042522), MDM2 T309G (rs2279744), MDM4 (rs4245739), MTHFR (rs1801133), CASP8 (rs3834129), and CCR5 (rs333) on the advanced LC patients` survival.Summing up, all these above described genes or SNPs alone or in combination could affect survival of patients with advanced LC. In this study, we for the first time investigated the impact of a set of HPV and such different SNPs as The mean age of the patients included in the study was 63 years (SD 8.7). Tumor cases from 76 (82.6%) male and 16 (17.4%) female patients were collected during the surgical operation. Histological type of cancer distribute as follows: 41 squamous-cell carcinoma (45.1%), 31 adenocarcinoma (34.1%), 6 pleomorphic-cell carcinoma (6.6%), 5 large-cell carcinoma (5.5%), 4 small-cell carcinoma (4.4%), 2 carcinoid (2.2%), 1 giant-cell carcinoma (1.1%), 1 chorio-carcinoma (1.1%), one case histology type has not been established. 92 lung cancer patients were divided into groups according to the stage of diagnosis: stage I – 36 (39.1%), stage II – 28 (30.4%), stage III – 23 (25.0%), and stage IV – 5 (5.4%). Regarding the smoking status, 16 patients were non-smokers, 75 patients smoked, while in 1 case the data was not given.TP53 Arg72Pro (rs1042522), MDM2 T309G (rs2279744), MDM4 (rs4245739), MTHFR (rs1801133), CASP8 (rs3834129), and CCR5 (rs333) polymorphisms in 92 Lithuanian LC patients. After analysis the TP53 gene Arg72Pro genotype distributions were as follows: the majority of sampled patients were identified as Arg/Pro polymorphic variant (95.7%) and only in a few samples genotype of Arg/Arg (4.4%) was found. Pro/Pro polymorphic variant was not detected in our investigated cases. The allele frequencies for MDM2 gene (-410T-G) SNP  were 50.0%, 43.5%, and 6.5%, respectively. MDM4 gene A>C SNP distributions  were recorded to be 59.8%, 32.6%, and 7.6%, respectively. MTHFR gene C677T SNP distribution was as follows: C/C 58.7%, C/T 36.9%, T/T 4.4%. CASP8 gene (-652 6N ins/del) ins/ins, ins/del, and del/del genotype distribution was 28.3%, 66.3%, 5.4%, respectively. CCR5 gene-delta32 deletion SNP distribution was: wt/wt 83.7%, wt/∆32 14.1%, ∆32/∆32 2.2%. The frequency distributions of all genotypes among patients are presented in Table In our study we investigated the g72Pro rs042522, MMDM2 SNP distribution and patient sex  (Table MDM2c.-5+309 G>T G/G polymorphic variant was detected in higher frequency (18.8%) among female patients (3 of 16 patients), while in samples of male patients accounted only for 4.0% (3 of 76 patients). T/T polymorphic variant was detected in 52.6% of male samples (40 of 70 patients) and in 37.5% of female samples (6 of 16 patients). MTHFRc.677 C>T C/C polymorphic variant was characterized by the greater frequency (75.0%) in women samples (12 patients out of 16), in comparison to 55.3% of male samples (42 patients out of 76). C/T polymorphic variant was detected more frequently (40.8%) in male patients (31 patients out of 76), while in female patients it amounted for 18.7% . However, all these differences were not statistically significant .Survival analysis according to TP53c.215 G>C (Arg72Pro), MDM2c.-5+309 G>T, MDM4c.1q32 A>C, MTHFRc.677 C>T, CASP8c.-652 6N ins/del, CCR5c.-∆32 genes SNPsSubgroup analysis revealed marked relation between TP53c.215 G>C (Arg72Pro), MDM2c.-5+309 G>T, MDM4c.1q32 A>C, MTHFRc.677 C>T, CASP8c.-652 6N ins/del, CCR5-∆32 and LC patients’ survival. Due to the lack of uncensored values of Arg/Arg and Pro/Pro in SNP case, survival rate curves according to TP53 gene Arg/Pro, Arg/Arg, and Pro/Pro SPNs were not presented. However, it should be noted that 3 (75.0%) LC patients out of 4 diagnosed with Arg/Arg polymophic variant survived until the last day of this study. Meanwhile 51 (57.9%) patients out of 88 survived until the last day of trial with Arg/Pro polymorphic variant detected.The main task of our study was to determine the relationship between polymorphic variants of genes MDM2c.-5+309 G>T gene in the test group it was found that patients with T/G polymorphic variant had a better survival rate over patients with G/G and T/T variants P = .38)  Figure . For patMDM4c.1q32 A>C gene SNP A/A and 48.5% with SNP A/C at 1600 days post-surgical operation, however, it showed no significant differences in survival groups regarding all tested polymorphic variants (P = .84)     Figure . C/C pol) Figure . T/T pol) Figure . Del/del) Figure . Δ32/Δ32P < .01), and survival rates on 1494 day of study were 72.2%, 37.4%, 39.2%, 20.0%, respectively. Overall, analysis showed that patient survival rates had a trustworthy correlation with the staging of LC. After statistical analysis of tested genes SNPs, no reliable differences were found between patients diagnosed with early (I-II) and late (III-IV) stages of LC      Figure . At the ) Figure . Surviva) Figure .MDM2 T/T and CCR5 wt/wt (43.4%), MDM4 A/C and MTHFR C/C (43.3%) polymorphic variants. The lowest mortality rates were observed in patients who were identified as CCR5 wt/Δ32, MDM2 T/G, MTHFR C/T, and CASP8 ins/del genetic polymorphic variants with numerical expressions 30.7%, 38.4%, 39.4%, and 40.0%, respectively. For patients who were identified as MDM4 A/A, CASP8 ins/ins, TP53 Arg/Pro polymorphic variants, mortality rates were 40.7%, 42.3%, and 42.5%, respectively.Finally, cluster analysis was performed on the basis of the patients’ mortality rates and survival times to determine which of the analyzed SNPs resulted in the best patient survival. Table Q1 and Q3  and with two times longer survival than patients from cluster (I); and the second group (IIB) includes patients with identified MDM4 A/C, CCR5 wt/wt, MTHFR C/C, CASP8 ins/del, MDM2 T/G gene polymorphism variants and their survival was between 498–572 days.It should be emphasized that clasterisation of each SNP mostly depended on survival time median, MTHFR gene and T/T polymorphic variant of MDM2 gene are the most closely correlated with the poor survival of patients. The rate of both genes MTHFR C/T  and MDM2 T/T  had a tendency to decrease in samples of patients diagnosed with stage III-IV cancer. This could be attributed to the worse survival of patients in the early stages of the disease. Therefore, it is worthwhile to check an importance of these genes in the first stages of the disease and in the period after the surgical treatment. Our results showed that the combination of polymorphic variants C/T of the MTHFR gene and T/T of the MDM2 gene are significantly linked to the poor survival of LC patients after surgery (P = .04). Meanwhile, individual SNPs of genes have no significant effect on the survival of patients after surgery: MTHFR C/T (P = .38), MDM2 T/T (P = .49). Moreover, no significant link (P = .96) was detected between the combination of MDM2 T/T, MTHFR C/T and combinations of other polymorphic variants of MDM2 and MTHFR genes in regard to survival of LC patients after chemotherapy or other treatments strategies applied. Patients with MDM2 T/T and MTHFR C/T and who underwent surgery had 37.5% survival rate for one year, while it was 90.0% for patients with the same combination of SNPs and who received chemotherapy. Significance of the tested genes SNPs for the survival of lung cancer patients analysed with Cox model has not showed the single SNP effect, but SNPs combination of genes MDM2 T/T, MTHFR C/T (P = .14) along with the stage of patients with lung cancer (P = .02) were associated with different periods of survival of lung cancer patients . In one case HPV of A9 phylogenetic line  was identified, in the other A5/A6 (51 and 56 types of HPV) was detected. In the third sample infection with two A9 and A7  phylogenetic lines were stated. However, the exact type of virus was not specified. But in all three samples the viral copy number in the cell was determined. In sample with A9 phylogenetic line the number of virus copies was 0.98 lg HPV copy/cell, in sample with A5/A6 phylogenetic lines the viral load comprised 2.2 lg HPV copy/cell. And in sample with two A9 and A7 phylogenetic lines number of virus copies amounted to 2.7 HPV copy/cell. It is interesting to note that two out of three patients who were identified as HPV infected were smokers. One smoker was infected with A9, another with both A9 and A7 phylogenetic line viruses. Men patients harbored A9 HPV, whereas women A9, A7, A5/A6 HPV phylogenetic lines. Moreover, in all three HPV positive samples TPTP53c.215 G>C (Arg72Pro), MDM2c.-5 + 309 G>T, MDM4c.1q32 A>C, MTHFRc.677 C>T-652, CASP8c.6N ins/del, CCR5-Δ32 and patients with LC survival rates were highly controversial. All our samples of LC patients were investigated to identify TP53c.215 G>C (Arg72Pro), MDM2c.-5+309 G>T, MDM4c.1q32 A>C, MTHFRc.677 C>T-652, CASP8c. 6N ins/del, and CCR5-Δ32 genes polymorphic variants.Literature research data on relationship between SNPs of genes TP53c.215 G>C (Arg72Pro) dominates and the frequency distribution is as follows: Arg/Pro 95.65%, Arg/Arg 4.35%. The high difference in the distribution of these alleles was shown in other populations: in the Iranian population, gene TP53 Arg/Arg, Arg/Pro, Pro/Pro frequencies were respectively 42.6%, 49.6%, and 7.8% [Sreeja et al. Kaplan-Meier survival analysis showed a significant difference in LC patients’ survival between TP53 variant genotypes and overall survival. Cox regression analysis showed p53 Arg72Pro heterozygous genotype was overall an independent prognostic factor, suggesting Pro72Pro genotype to be a potential risk factor favoring the development of lung carcinoma [Our study showed that heterozygous Arg/Pro polymorphic variant of gene and 7.8% . Sreeja arcinoma .MDM2 309 T/G polymorphism and risk for LC development are inconclusive [Javid et al. revealed that MDM2 gene G/G polymorphic variant might be associated with increased expression of MDM2, which operates p53 expression changes [Chua et al. published a study of Singaporean non-smokers LC patients. It was revealed that in female samples not the G/G, but T/T polymorphism in position 309 of gene MDM2 increases the risk 2.1 times, and combination with the TP53 Pro/Pro allele increases the risk 2.5 times. There was, however, no effect of either polymorphism on age at diagnosis of LC or on overall survival [Enokida et al. published a case-control study showing gene MDM2 T/T, T/G, G/G polymorphic variants frequencies in samples to be 21.1%, 49.7%, and 30.2%, respectively [MDM2c.-5+309 G>T gene T/T, T/G, G/G showed that T/T polymorphic variant dominates and frequencies are 50.00%, 43.48%, 6.52%, respectively. Dong et al. found that the MDM2 SNP309 (rs2279744) GT/TT genotypes are associated with a significantly worse survival [Enokida et al. found that the overall survival of adenocarcinoma patients with pathological stage I disease and the MDM2 T/T genotype was significantly shorter than that of those with the T/G or G/G genotypes (P = .02) [MDM2 T/G or T/T polymorphic variant (respectively 38.4% and 43.4%). The similar tendency is observed in the assessment of survival time (respectively 572 and 437 days). However, patients with advanced stages of LC and T/T variant of the MDM2 gene has the worst overall survival rate in the end of follow-up, but data were not statistically significant  [MDM4c.1q32A>C dominates in LC samples. The frequency of A/A, A/C, C/C distribution is respectively 59.8%, 32.6%, and 7.6%. We showed that for patients identified with gene MDM4 A/A SNP mortality rate is lower than with A/C . Patients with A/A polymorphic variant have 1.6 times shorter survival rate after surgery compared with patients who were identified with A/C polymorphic form (355 days for A/A and 572 days for A/C). However, the data also were not statistically significant.Analyses suggested that the rs4245739 polymorphism was significantly associated with overall cancer risk . In YangP = .04) . Our resMTHFR C677T polymorphism and LC risk [Cui et al. determined frequencies of MTHFRc.677 polymorphic C/C, C/T, T/T variants to be 34.5%, 48.5%, and 17.0%, respectively. MTHFR 677 C/T and T/T genotypes showed a weak protective effect on LC development, compared with homozygous C/C genotype, although these results were not statistically significant [Cheng et al. showed MTHFRc.677 C>T polymorphic variants C/C, C/T, T/T distribution to be respectively 27.7%, 35.1%, 37.2% and found T/T genotype link with LC [Al-Motassem et al. established distribution of MTHFRc.677 C>T polymorphic variants as follows: C/C 59.6%, C/T 33.0%, and T/T 7.4%. Authors announced that patients who have C/C polymorphic variant also have an increased risk for LC [MTHFRc.677 C>T SNP distribution in LC patients were showed in our study: C/C polymorphic variant was dominating in our samples, C/C, C/T, T/T polymorphisms frequencies were respectively 58.7%, 37.0%, and 4.4%. However, for our patients who were identified with MTHFR C/T gene polymorphic variant mortality rate is almost the same as for those with C/C polymorphic variant , but their survival time stands out (respectively 375 and 557 days). Moreover, patients with advanced cancer stages (III-IV) and harbouring C/T polymorphism of the MTHFR gene had the shortest survival rate in regard to other MTHFR SNP variants . This difference was not statistically significant as well.Definite conclusions cannot be drawn from studies analysing the relation between  LC risk , 28, 55.nificant . Cheng e with LC . Al-Motak for LC . SimilarCASP8 -652 6N gene have shown no clear association with LC. Sun et al. showed that CASP8 -652 6N del polymorphism is associated with decreased LC development risk in China’s population [Ji et al. found that patients with CASP8 -652 6N del polymorphism have greater susceptibility for LC development [CASP8c. 6N-652 ins/del polymorphic ins/ins, ins/del, del/del polymorphisms frequency analysis showed that ins/del dominates and frequencies are respectively 28.3%, 66.3%, and 5.4%. We showed that mortality rate is lower for patients identified with CASP8 ins/del polymorphic form (40.0%) than for patients with ins/ins polymorphic variant (42.3%), but they survived shorter (542 days) than patients who were identified as ins/ins polymorphic variants (723 days).In the analysed literature, SNPs studies of pulation . Ji et aelopment . In our CCR5-Δ32 polymorphic variants interface with LC development. It is known that patients identified with CCR5-Δ32/Δ32 genotype have a 4.58 times higher risk for HPV infection [CCR5-Δ32 in LC cancerogenesis. Our results indicated that wt/wt polymorphic variant of gene CCR5-Δ32 dominates in samples and wt/wt, wt/∆32, ∆32/∆32 frequency distribute respectively 83.7%, 14.1%, and 2.2%. We showed that for patients identified with gene CCR5 wt/Δ32 polymorphic variant mortality rate is lower (30.7%) than for wt/wt polymorphic form (43.4%), and their survival is lower (413 days) than for patients identified with wt/wt polymorphic form (498 days). HPV infection, as mentioned above, was found only in 3 analysed samples, so statistical analysis of combined effect on HPV and analysed genes SNPs was not performed.To our best knowledge, there are no literature data on nfection . Due to TP53 Pro/Pro and MDM2 G/G genotypes had a supermultiplicative interaction with respect to lung adenocarcinoma risk [p53, MDM2 or MDM4 polymorphic variants, some of which have been further advanced to the stage of clinical trials [Other authors published several studies on combined effect of different SNPs and effect on LC development and disease progression. One of the study showed that combined oma risk . P53 patl trials . SNPs inMTHFR and other genes (TS and ERCC1) polymorphisms on effectiveness in first-line platinum and pemetrexed therapy in NSCLC patients was evaluated in Poland [MTHFR 677C>T polymorphism, however, progression-free and overall survival were not affected by MTHFR gene polymorphism. After combined analysis of TS VNTR (variable number of tandem repeats)and MTHFR 677C>T polymorphisms, a shorter progression-free survival in patients harboring C/C in MTHFR gene was shown [TS gene and C/C genotype of MTHFR gene have significantly shorter overall survival  and significantly higher risk of death  [Smit et. al [MTHFR gene have longer progression-free survival time compared to patients with C/C or C/T polymorphic variants. Li et. al [MTHFR heterozygotes yield better clinical benefit (P =.03) after platinum based chemotherapy, however, overall survival and progression-free survival of LC patients do not differ regarding MTHFR polymorphic variants. In our study we performed cluster analysis which showed that the combination of MTHFR C/T and MDM2 T/T are significantly linked to the poor survival of LC patients after surgery (P = .04). However, the effect of SNPs of our investigated genes on the effectiveness of chemotherapy was not evaluated in our study. Nevertheless, genetic factors  may have a high predictive and prognostic value for LC survival after surgery or chemotherapy applied.Combined effect of n Poland , 71. Autas shown . Later at et. al  showed, i et. al  showed tSyrjänen et al. in 1979 introduced the idea that HPV infection may be one of the factors involved in process of lung carcinogenesis [Sarchianaki et al. survey data showed that HPV types identified in LC patients could be such as 16, 11, 33, 31, 18, 6, 59. The most commonly found type is 16 (42.1%) and 11 (15.8%) [TP53c.215 G>C Arg/Pro, CASP8c.-652 6N ins/del, CCR5-Δ32 wt/wt polymorphic variants occurred. Jain et al. study of India’s population showed that out of 40 LC patients 2 were infected with HPV and were identified with TP53c.215 G>C (Arg72Pro) gene Arg/Arg polymorphic variant  [Amaral et al. connection analysis of cervical lesions, HPV, oral contraceptive, and MDM2c.-5+309 G>T polymorphism found that there is a link between high-grade cervical lesions, contraceptive use, and MDM2 gene T/G polymorphic variant [MDM2c.-5+309 G>T, MDM4c.1q32 A>C, MTHFRc.677 C>T-652, CASP8c. 6N ins/del, CCR5-Δ32 polymorphic variants, lung cancer, and HPV. Unfortunately, due to the small number of HPV infected patients in our study it was not possible to evaluate combined effect of HPV infection and different genes polymorphisms on the survival of LC patients. Therefore, here we conducted a study of TP53c.215 G>C (Arg72Pro), MDM2c.-5+309 G>T, MDM4c.1q32 A>C, MTHFRc.677 C>T-652, CASP8c.-652 6N ins/del, and CCR5-Δ32 polymorphic variants frequency analysis in LC samples (n = 92), regardless of the HPV infection. It is worthwhile to continue research in this direction with larger patient groups in order to clarify HPV implication in LC.Regarding viral infection and lung cancer, the first evidence that there is a link between HPV infection and bronchial lesions was determined in 1979 by Rubel and Reynolds . Syrjäneogenesis . Over thogenesis . Recentl (15.8%) . In our samples) . Lately, variant . To our P = .04) link was determined between the poor survival of LC patients after surgery with combination of polymorphic variants C/T of the MTHFR and T/T of the MDM2 genes, whereas individually these SNPs do not show significant relationship with our LC patients survival. However, it is important to notice that genetic factors  may have a high predictive and prognostic value for LC survival after different treatments strategies applied.Finally, there is a link between specific SNPs and LC patient survival frequency and time, meanwhile the combination of specific SNPs showed a statistically significant measure. In conclusion, statistically significant  with diagnosis of primary LC from Department of Thoracic Surgery at the National Cancer Institute  were asked to participate in the study. Study protocol was accepted by the Vilnius Regional Committee for the Biomedical Research (11-06-2013 No. 158200-13-638-204). All patients signed Informed consent before the surgery. Patients were included into the study according to the following criteria: all of them had primary lung cancer and none of them had received radiotherapy or chemotherapy prior to surgery. After surgery all tumors were examined by the pathologist and the diagnosis was confirmed by expert pathologists. Remaining material after the diagnosis was collected to the Biobank at the National Cancer Institute and stored till the scientific examination. All samples previously had been analyzed for the presence of HPV. Then the SPN analysis of various previously described genes was performed.DNA was purified by organic extraction method according to the approved standard operating procedures at the Biobank of the National Cancer Institute. Firstly, after pathology diagnosis, all tumor tissue samples were cut into small pieces (30–50 mg) and were snap froze in the liquid nitrogen. Control cut was also performed for the evaluation of amount of cancer cells in the sample. Before DNA extraction tumor sample was crushed and melted in liquid nitrogen. Melted material was placed in a test-tube, lysis buffer  and proteinase K solution (10 mg/ml) were added. Tube was incubated in 55ºC for 12 hours. DNA purification was carried out with phenol-chloroform solutions and precipitation with 70% and 96% ethanol solutions. After that DNA was dried. Later the dried DNA was dissolved in 500 µl of sterile distilled water at 4°C. DNA purification quality was measured using spectrophotometer . The quantitative DNA analysis was performed by measuring optical density at 280 nm wavelength. According to the ratio of A260/A280, isolated DNA of all samples used in this study was of adequate quality being in a range from 1.7 to 1.9.TP53 Arg72Pro (rs1042522), CASP8-652 6N (rs3834129), and CCR5 ∆32 (rs333) genes. For gene TP53 Arg72Pro (rs1042522) allele 4 different specific primers were used: Arg allele forward: 5′→ TCCCCCTTGCCGTCCCAA→3′ and reverse: 5′→CTGGTGCAGGGGCCACGC→3′, Pro allele forward: 5′→GCCAGAGGCTGCTCCCCCC→3′ and reverse: 5′→ CGTGCAAGTCACAGACTT→3′, which had revealed the following results of 141 bp and 177 bp products [CASP8-652 6N (rs3834129) gene 2 primers were used: forward: 5′→AGTGAAAACTTCT CCCATGGCCTC→3′ and reverse: 5′→ GATTGATACTG GCACAGTATACTTACC→3′, and two specific primers: : 5′→GTAATTCTTGCT CTGCCAAGCTG→3′;  5′→CCAAGGTCA CGCAGCTAGTAAG→3′, which had revealed the following results of 139 bp, 291 bp, and 396 bp fragments [CCR5- Δ32 (rs333) gene 2 specific primers were used: forward: 5′→ACCTGCAGCTCTCATTTTCC→3′ and reverse: 5′→GCAGATGACCATGACAAGCA→3′, which had revealed the results of 111 bp and 79 bp fragments [From each tumor sample extracted DNA was used for PCR analysis of products . Likewisragments . After PMDM2 T309G (rs2279744), MDM4 (rs4245739), and MTHFR (rs1801133) genotypes were determined by polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP). A 89 bp of fragment covering MDM2 gene was amplified by a set of primers: forward: 5′→TTCGGAGGTCTCCGCGGGAGTTCAG→3′ and reverse: 5′→TGCGATCATCCGGACCTCCCGCGTC→3′ [MDM4  (rs4245739) was digested with restriction enzyme MspI. Polymorphic variants were identified by existence of 134 bp, 111 bp, and 23 bp fragments [MTHFR (rs1801133)  [GCGTC→3′ . The PCRragments . FinallyGGAG→3′)  was digeDM4 rs424739, and E1 - E2 gene-based primers. In A9 phylogenetic line HPV types 16, 31, 33, 35, 52, 58; in A7 phylogenetic line HPV types 18, 39, 45, 59; and in phylogenetic line A5/A6 HPV types 51 and 56 were attributed. Amplification was performed using the Rotor-Gene Q amplificator and Rotor-Gene Q software (version 2.1.9.9). Endogenic control with β–globin was performed for each PCR cycle. Number of copies of the virus in the cell was calculated using the formula: log (HPV DNA copies/human DNA copies) × 200,000 = log .Phylogenetic group of HPV, viral copy number, and the total number of copies of the virus in cells were determined by RT-PCR. AmpliSens HPV screen-titre-FRT PCR kit (AmpliSens) was used to determine HPV A9, A7, and A5/A6 phylogenetic groups. The kit was composed of PCR-mix-1-FRT HPV A9, A7, and A5/A6 phylogenetic groups according to HPV P value < 0.2 were selected for Cox regression analysis. For SNPs analysis clusterisation in a multi-dimensional space , median (M), and quartile 3 (Q3) of patient survival time) was performed computing Euclidean distances between objects and linking several objects by Ward’s method. A P value of < .05 was considered statistically significant. Statistical analysis was performed using SigmaPlot 12.3 and STATISTICA 10.0 software.Statistical comparisons among groups were performed by Chi-square test. Survival was estimated by Kaplan-Meier method. The statistical difference between the survival curves was determined using the log-rank test. The variables with log-rank test


By further analysis, we found that the muscle key regulator MyoD was significantly enriched on this subset of genes and played a repressive role during myogenic differentiation. Among these genes, we identified the pluripotency gene Patz1, which is repressed during myogenic differentiation through direct binding of MyoD to promoter elements. These results point to the importance of integrating histone modifications and MyoD chromatin binding for coordinated gene activation and repression during myogenic differentiation.Post-translational modifications of histones play a key role in the regulation of gene expression during development and differentiation. Numerous studies have shown the dynamics of combinatorial regulation by transcription factors and histone modifications, in the sense that different combinations lead to distinct expression outcomes. Here, we investigated gene regulation by stable enrichment patterns of histone marks H3K4me2 and H3K4me3 in combination with the chromatin binding of the muscle tissue-specific transcription factor MyoD during myogenic differentiation of C2C12 cells. Using  Understanding the molecular and epigenetic regulation of gene expression during myogenic differentiation is important to gain insights into the mechanism underlying the pathogenesis of muscular dystrophies ,2. Epigeed genes . For exaed genes . In humaed genes ,7. Moreoed genes . In geneed genes ,10. Duried genes . The reped genes –13. Howeed genes . Furthered genes ,15. Inteed genes ,17.MyoD belongs to the myogenic basic helix-loop-helix (bHLH) family of transcription factors that activate muscle-gene expression during skeletal myogenesis through DNA binding to the consensus E-box motif (CANNTG) . Upon thHere, we investigated a stable enrichment pattern of the histone marks H3K4me2 and H3K4me3 in combinations with muscle tissue-specific transcription factor MyoD during myogenic differentiation. We performed chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) in two stages of myogenic differentiation in C2C12 cells. To study the impact on gene expression, we further analyzed genome-wide expression data (RNA-seq) from the ENCODE project . Finally2 and 37°C in Dulbecco's modified Eagle's medium  supplemented with 1% penicillin/streptomycin (Gibco) and 10% fetal bovine serum (Biochrom). Mononucleated C2C12 myocyte cells were harvested before reaching 70% confluence. To induce differentiation, cells were cultured with Dulbecco's modified Eagle's medium and 2% horse serum (Biochrom) and maintained for 48 hours, when more than 90% of the cells had fused into myotubes.C2C12 myoblasts were obtained from Professor Jakob Schmidt  and cultured at 5% COIMR-90 and BJ-CRL2522 primary human fibroblasts were purchased from ATCC. IMR-90 fibroblasts were cultured in growth medium supplemented with 10% fetal bovine serum. Muscle differentiation was induced by incubating cells in DMEM supplemented with 2% horse serum, 0.01 mg/ml bovine insulin and 0.0055 mg/ml human transferrin. BJ fibroblasts were cultured in growth medium supplemented with 10% fetal bovine serum, non-essential amino acids, pyruvate and penicillin–streptomycin. The differentiation was induced by incubating cells in DMEM supplemented with 2% horse serum.2 and 37°C in Dulbecco's modified Eagle's medium  supplemented with 1% penicillin/streptomycin (Gibco) and 10% fetal bovine serum (Biochrom).HEK293 cells (ATCC) were cultured at 5% COIMR-90 fibroblasts were transfected with a MyoD expression construct by electroporation. Cells were selected with 2 mg/ml of puromycin for six nights. MyoD expression was induced by 200 ng/ml of doxycycline in growth medium for 24 hours. No doxycycline was added in control cells. Differentiation was induced by culturing cells in differentiation medium for three days with/without doxycycline for 72 hours.To transfect BJ fibroblasts, MyoD ectopic expression was achieved by Adenoviral infection with Adeno-MyoD at MOI 800 for one hour. Adeno-Track was used as control virus. Then, cells were placed in growth medium for 24 hours and differentiation was induced by changing cells to differentiation medium for 56 hours. MyoD expression levels were measured by real-time PCR 24 hours after infection.™ Chromatin Immunoprecipitation System  was used with some modifications. Sonication was performed using the Biorupter UCD300 (Diagenode) to obtain chromatin fragments of approximately 100–300 bp. The following antibodies were used for ChIP: anti-H3K4me2 (Abcam ab7766), anti-H3K4me3 (Abcam ab8580), and anti-MyoD . Sequencing libraries were prepared using the NEXTflex™ ChIP-Seq Kit  according to an in-house modified protocol. The libraries were 51 bp single-end sequenced on an Illumina HiSeq 2000 platform. Base calling was performed with the Illumina Casava pipeline version 1.8.0. Initial sequencing quality assessment was based on data passing the Illumina Chastity filter. The raw and processed ChIP-seq data was submitted to GEO (GSE63716).ChIP was performed as described before . BrieflyHprt. For ChIP samples, enrichments were measured relative to input.Total RNA from C2C12, IMR-90 and BJ cells was extracted with Trizol (Invitrogen), according to manufacturer’s instructions. 0.5–1 μg of RNA was retro-transcribed using the Taqman reverse transcription kit (Applied Biosystems). Real-time quantitative PCR was performed using a 7500 Fast Real-Time System machine to analyze relative gene expression levels using SYBR Green Master mix (Applied Biosystems) and following manufacturer’s indications. Gene expression was calculated using the delta-CT method with normalization to the housekeeping gene Patz1 promoter  was cloned into the pGL3 basic vector (Promega). For luciferase assays, approximately 104 HEK293 cells were transiently transfected with 50ng of reporter vector, 5 ng of Firefly luciferase vector for internal normalization of transfection efficiency and 10–200 ng of MyoD expression vectors using Transfast (Promega). Activity was measured by Dual-Luciferase assay (Promega) after 48 hours in a Centro LB960 Luminometer (Berthold). All measurements were performed in triplicates.A minimum 400 bp Sequencing of DNA libraries resulted in ~29–66 million reads per sample . QualityP value cutoff of 10−4 was used.For signal detection (H3K4me2/3) and peak calling (MyoD), we used MACS (v1.4.2) , which pTo assign MyoD peaks to genes and histone signals to TSS, RefSeq genes (mm9) from the UCSC genome browser were used, containing 23,358 genes corresponding to 30,089 transcripts. MyoD peaks were assigned to the genes if they are located within 10 kb upstream of the TSS or in the transcribed region. For the analysis of histone signals, we focused on unique transcripts with at least 4 kb in length. The filtering resulted in 19,904 TSS corresponding to 24,051 transcripts . For eacK-means clustering was performed in R (v3.0.2) using the “kmeans” function with centers = 6 and iter.max = 1000000. We tried centers = 4,5,6,7 clusters and manually visualize if the clusters can be further separated with increasing centers. We defined six clusters for histone marks based on the visual inspection.Before clustering, the input signal was subtracted from the H3K4me2/3 signal for each TSS region (6 kb) and regions with signal ≤ 0 were discarded . K-meansP value ≤ 0.01 using the Benjamini-Hochberg method for controlling the false discovery rate was set as significant for GO terms in biological processes. To validate the results, we used 324 genes related to “muscle organ development” and 406 genes related to “muscle tissue development” based on the Mouse Genome Informatics (MGI) database [P values < 10−7).GO analysis was conducted using the DAVID functional annotation tool . An adjudatabase . Using tRNA-seq data was used from the ENCODE project . The 75 General bioinformatics and statistical analyses were conducted using R (including Bioconductor packages) and Perl.ChIP-seq data are available from the Gene Expression Omnibus (GEO) repository at NCBI (accession number GSE63716). RNA-seq data in C2C12 cells from ENCODE  are avaiC2C12 mouse myoblasts  provide To analyze the distribution of histone modifications around transcriptional start sites (TSS), we filtered for a defined set of gene transcripts longer than 4 kb, resulting in 24,051 transcripts with 19,904 unique TSS . We furtk-means clustering with six clusters using the filtered set of TSS . The lowest expression was found for genes detected in cluster 6  using the MGI database [Compared to H3K4me2, we observed a higher mean enrichment of H3K4me3 directly downstream of the TSS in both undifferentiated and differentiated C2C12 cells . Moreovedatabase .P value < 2.2x10-16, Given that muscle-related genes  are enriched in cluster 1 of H3K4me2 and H3K4me3, we analyzed the overlap between these clusters in undifferentiated C2C12 cells. We found a significant overlap of genes . In this cluster, we found approximately 30% of genes bound by MyoD in Undiff and 67% in Diff C2C12 cells , Frmd6 (FERM domain containing 6), Patz1 (POZ (BTB) and AT hook containing zinc finger 1), Ptbp1 (polypyrimidine tract binding protein 1) and Tmpo (thymopoietin).The percentage of genes bound by MyoD for each common H3K4me2/3 cluster is given in 12 cells . Focusin12 cells . Given t12 cells . The pre12 cells , of whic12 cells , includiPatz1, which was previously shown to have an important role in maintenance of the embryonic stem cell (ESC) phenotype and its knockdown leads to differentiation of murine ESCs into endoderm and mesoderm lineages at different time points [To further investigate the repression role of MyoD on these 14 down regulated genes during myogenic differentiation, IMR-90 human fibroblasts were converted to skeletal muscle cells by induction of MYOD. The expression of MYOD was induced ~400-fold in growth medium (GM) and ~800-fold in the differentiation medium (DM) . Interese points .Patz1 expression pattern, as a representative gene among those exhibiting H3K4me2/3 profile and bound by MyoD only in Diff C2C12 cells  show a differential MyoD binding, while 36% 21 genes) show a constitutive MyoD binding. The differential expression of the latter could possibly be explained by other cofactors necessary for transcriptional regulation. For example, several studies have shown that MyoD can activate gene transcription in cooperation with other factors such as E-proteins and the chromatin remodeling factor Baf60c –21. genes shPatz1 during myogenic differentiation. This observation was further confirmed in MyoD driven differentiation of fibroblasts to muscle cells. These findings might provide an important regulatory mechanism to promote myogenic differentiation.In summary, we identified a subset of highly expressed genes related to muscle development, which show a stable H3K4me2 enrichment over the gene body and H3K4me3 enrichment towards the gene body during myogenic differentiation. Our study reveals a significantly higher binding of MyoD to this particular subset of genes that correlate with repression of transcription. Interestingly, further analysis and experiments revealed that MyoD binds and down-regulates S1 TableSequence reads (single-end 51 bp) were mapped to the mouse reference genome (mm9).(XLSX)Click here for additional data file.S2 TableMuscle-related GO terms are marked in red.(XLSX)Click here for additional data file.S3 TableMuscle-related GO terms are marked in red.(XLSX)Click here for additional data file.S4 TableMuscle-related GO terms are marked in red.(XLSX)Click here for additional data file.S5 TableMuscle-related GO terms are marked in red.(XLSX)Click here for additional data file.S6 TableMuscle-related GO terms are marked in red.(XLSX)Click here for additional data file.S7 TableMuscle-related GO terms are marked in red.(XLSX)Click here for additional data file.S8 TableMuscle-related GO terms are marked in red.(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 Fig(A) Average profile of H3K4me2 and H3K4me3 in Undiff C2C12 and (B) Diff C2C12 cells around the transcription start site (TSS).(TIFF)Click here for additional data file.S3 Fig(A) H3K4me2 profiles identified by k-means clustering. The clustering is based on the transcription start site (TSS) and the corresponding number of genes is given for each cluster. Genes with multiple TSS can be present in more than one cluster. (B) The box plot (25% to 75% quartile) shows the levels of gene expression  of the different H3K4me2 clusters in Diff C2C12 cells. The expression of cluster 1 and cluster 2 genes was compared using the Mann-Whitney U test. (C) Overlap of genes between the clusters of H3K4me2 in Undiff and Diff C2C12 cells.(TIFF)Click here for additional data file.S4 Fig(A) H3K4me3 profiles identified by k-means clustering. The clustering is based on the transcription start site (TSS) and the corresponding number of genes is given for each cluster. Genes with multiple TSS can be present in more than one cluster. (B) The box plot (25% to 75% quartile) shows the levels of gene expression  of the different H3K4me3 clusters in Diff C2C12 cells. The expression of cluster 1 and cluster 2 genes was compared using the Mann-Whitney U test. (C) Overlap of genes between the clusters of H3K4me3 in Undiff and Diff C2C12 cells.(TIFF)Click here for additional data file.S5 Fig(A) Overlap of H3K4me2 and H3K4me3 cluster 1 genes in Diff C2C12 cells. The P value is based on a hypergeometric test. (B) GO enrichment analysis of common cluster 1 genes using the DAVID database. The top ten biological process terms with an adjusted (Benjamini-Hochberg) P value ≤ 0.01 are indicated. GO terms related to muscle development are highlighted in red.(TIFF)Click here for additional data file.S6 Fig(A) The enrichment of H3K4me2 and H3K4me3 on selected muscle-relevant cluster 1 genes. The TSS is marked by an arrow. The y-axis indicates the ChIP-seq signal. (B) ChIP-qPCR validation of H3K4me2 and H3K4me3 occupancy on the selected genes.(TIF)Click here for additional data file.S7 Fig(A) MYOD expression was measured by qPCR before and after induction in growth medium (GM) and differentiation medium (DM) IMR-90 fibroblasts. The expression of MYOD in control GM was set to 1. (B) Expression levels (mRNA) of PATZ1 in BJ fibroblasts. The expression of PATZ1 in control GM was set to 1. P value was calculated using Student's t-test based on at least three independent experiments.(TIFF)Click here for additional data file.


T1-weighted variable-flip-angle 3D TSE has emerged as a promising intracranial vessel wall imaging technique. To increase spatial coverage and cerebrospinal fluid (CSF) attenuation, a whole-brain 0.5-mm-reoslution protocol based on an inversion-prepared 3D TSE sequence has recently been proposed at 3T. However, its 11-12-min scan time renders it clinically impractical. This work aimed to develop an expedited protocol and validate it on patients.Elliptical data sampling and prolonged echo train length (ETL) can be exploited to expedite the 3D TSE acquisition. However, this would reduce SNR and compromise vessel wall delineation. On the other hand, SNR is intimately related to the refocusing flip angles that are calculated for a prescribed signal evolution of a tissue with specific T1 and T2 values (denoted here as simulation T1 and T2). We hypothesized that an appropriate choice in the combination of ETL and simulation T2 may help achieve an efficient protocol.On a 3T system, the effects of simulation T2 and ETL on wall SNR, wall-CSF CNR, and white-gray matter CNR (indicative of T1 contrast weighting) were first explored on 9 healthy subjects. Simulation T2 varied  while ETL was held fixed at 36 (5 subjects) or 60 (3 subjects), and ETL varied  while simulation T2 was held fixed at 170 ms (1 subject). The range of potential protocols (i.e. combinations of ETL and simulation T2) was then narrowed. Specifically, ETL = 52 combined with a simulation T2 of 140, 170, and 200 ms were respectively tested on 7 healthy subjects. In addition, a combination of ETL = 36/T2 = 100 ms was used as a reference. An optimal imaging protocol was determined from the four scans and finally applied to a pilot study comprising 18 patients with various known arterial wall disease.Increasing simulation T2 boosted SNR and CNR Figure . As expeWhole-brain intracranial vessel wall evaluation at 3T is feasible within a clinically acceptable scan time - 8 min. A large-scale trial on using the technique for diagnosis of stroke etiology is underway to establish its clinical usefulness.


A target molecule responsible for cell cycle arrest by metformin was discovered using a gene chip array in lung cancer cells and the effect of metformin on E2F8 was assessed. The siRNA-mediated knockdown of E2F8 significantly suppressed G1-S progression while ectopic expression of E2F8 relieved metformin-induced G1 arrest. The mRNA levels of p21 were found to be inversely related to those of E2F8 in lung cancer cells while siRNA-mediated knockdown of p21 partly rescued siE2F8-induced arrest of the cell cycle. Metformin had no effect on degradation of E2F8 mRNA. Activation and inhibition of AMPK by AICAR and Dorsomorphin, respectively, did not affect E2F8 suppression by metformin. The clinical significance of E2F8 was analyzed in The Cancer Genome Atlas (TCGA) data. One hundred six (13%) of 848 TCGA lung cancers overexpressed E2F8 mRNA. The overexpression of E2F8 was associated with poor overall survival . The present study suggests that metformin may induce cell cycle arrest at the G1 phase by suppressing E2F8 expression in lung cancer cells. In addition, E2F8 may be associated with poor overall survival in lung cancer patients irrespective of histology. Lung cancer is the leading cause of cancer-related death in the world. Despite recent advances in molecular target therapy, the cure rate of advanced lung cancer remains low. Recently, oral antidiabetic drug metformin has emerged as an attractive agent for lung cancer therapy. Metformin is associated with improved overall survival in patients with diabetes with stage IV non- small cell lung cancer (NSCLC) . CombinaThe E2F family is a core transcriptional axis crucial for cell cycle transitions by regulating gene expression, including expression of cyclins and CDKs. Traditionally, E2Fs are categorized into three groups based on their transcriptional activity: activators (E2F1-E2F3), canonical repressors (E2F4-E2F6), and atypical repressors (E2F7-E2F8). Upon mitogenic stimulation, activated E2F1-E2F3 will accumulate in the late G1 phase and initiate a transcriptional program that drives cells into S phase. The G1/S-specific transcriptome is then attenuated by the action of E2F7 and E2F8 –16. HoweIn this study, we analyzed global gene expression in metformin-treated lung cancer cells and found that E2F8 was significantly downregulated by metformin. The expression levels of E2F8 were inversely correlated to those of p21, and metformin-induced overexpression of p21 was independent of p53. In addition, E2F8 overexpression was found to be significantly associated with poor overall survival in lung cancer.To investigate the effect of metformin on cell proliferation and cell cycle as well as the expression of several cell cycle-related genes in lung cancer cells, H1299 Figure  and A549To find novel targets involved in metformin-induced cell cycle arrest in lung cancer cells, we analyzed mRNA levels using GeneChipÒ Human Gene ST Arrays in A549 cells treated with metformin. Genes that were 1.5 fold up- or down- regulated compared to the control were classified using DAVID  –7 24]. . 24]. ApTo further demonstrate the inhibitory effect of metformin on cell proliferation through E2F8 and p21, we overexpressed E2F8 in H1299 cells and treated cells with metformin. We first analyzed E2F8 expression during cell cycle progression. Like other E2F family members, E2F8 was principally expressed in the nucleus Figure  where itde novo mRNA synthesis by actinomycin D. However, no difference in mRNA level of E2F8 was observed between control and metformin-treated cells  times poorer than that in patients without E2F8 overexpression after adjusting for pathologic stage, age, histology, and recurrence  lung cancer data. The clinicopathological characteristics of 848 participants are described in Metformin exerts anticancer effects mainly by suppressing cell proliferation and inducing cell death in lung cancer cells. To understand the molecular mechanisms underlying metformin-induced inhibition of cell proliferation, we analyzed expression changes in mRNA levels using the Affymetrix GeneChip® Human Gene 1.0 ST Array. Metformin down-regulated the expression of cell cycle-related genes such as E2F8, CCNF, CCND3, CCNB3, and CDC6 but up-regulated the expression of apoptosis-related genes such as CHAC1, DDIT4, TRIB3, TP53INP1, and TP63. Among them, we focused on E2F8 because of its dramatic down-regulation by metformin. Metformin down-regulated four E2F family members, E2F1, E2F2, E2F7, and E2F8 in lung cancer cells, with E2F8 being the most prominently down-regulated one . In thisIt is currently unclear what is responsible for the extra bands shown in Figure E2F8 is known as a repressor of E2F activators such as E2F1 . During p21 by activated Ras requires transcription factor E2F1  assay on the third day after transfection as described previously . CellTitin vitro transcription was performed to produce biotin-labeled cRNA using GeneChip One-Cycle Target Labeling and Control Reagents  according to the manufacturer's instructions. After fragmentation, the cRNA was hybridized with the Affymetrix GeneChip® Human Gene 1.0 ST Array (Affymetrix). GeneChips were then scanned in a GeneChip Scanner 3000 (Affymetrix). Normalization, filtering, and gene expression analysis of the data were performed with the Affymetrix GCOS software.A549 cells were cultured in RPMI 1640 medium supplemented with 5 mM metformin for 48 h. Total RNA was isolated from these cells using the PureLink RNA Mini Kit . Double-stranded cDNA was synthesized from total RNA and To knockdown target genes, cells were transiently transfected with 40 nM of gene-specific siRNA  or non-targeting siRNA (BioNeer) as a negative control. The siRNA sequences used for knockdown experiment are shown in The pCMV6-Entry vector expressing GFP-tagged human E2F8  was used to analyze gene expression and Lipofectamine 2000  was used to transfect siRNA or plasmid into cells according to the manufacturer's protocol. At 48 h post-transfection, gene expression was measured using quantitative real-time PCR or western blot analysis.mRNA level was analyzed by quantitative real-time PCR. Total RNA was isolated using the PureLink RNA Mini Kit  and reverse transcription was carried out using the SuperScript VILO cDNA Synthesis Kit . Quantitative real-time PCR was performed with SYBR green dye  using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) under the following conditions: initial denaturation for 5 min at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. PCR primers were designed using Primer Express 3  and their specificity was checked by BLAST analysis. PCR primer sequences used for qRT-PCR are listed in Total proteins were extracted from cultured cells using a lysis buffer containing protease inhibitor cocktail . Cell lysates were heated at 95°C for 5 min, loaded on 10% sodium dodecyl sulfate-polyacrylamide gels, and transferred to a PVDF membrane . After blocking with a 3% solution of fetal bovine serum, membranes were probed with antibodies . DNA in cells was stained with DAPI.The cell cycle was analyzed using the FITC BrdU Flow Kit  or propidium iodide (PI) solution (50 μg/mL) according to the manufacturer's protocol. Briefly, cells were cultured in 6-well plates and transfected with siRNA or plasmid vectors. On the third day post-transfection, cells were incubated with 10 μM BrdU for 2 hours before harvest. Cells were resuspended, washed twice in phosphate-buffered saline (PBS), and fixed. DNA was stained with 7-AAD or PI and then analyzed using a FACS Calibur system  and CELLQuest software .p-value threshold of > 0.01.The transcription factors (TFs) responsible for downregulation of genes (<1.5) by metformin were analyzed using TRANSFAC and ExPlain . TRANSFAC and ExPlain use F-Match to search for putative TFBSs and TFs by a weight matrix-based algorithm through comparing a Yes-set  to a No-set (control or background set) . PromoteThe effect of E2F8 overexpression on survival was estimated by Kaplan–Meier survival curves and the significance of differences in survival between two groups was evaluated by the log-rank test. Cox proportional hazards analysis was performed to estimate hazard ratios of independent factors for survival after controlling for potential confounding factors. All statistical analyses were two-sided with a 5% type I error rate.


Actinidia chinensis) is a popular fresh fruit with a high market value due to its unique color, caused by anthocyanin accumulation. The R2R3-MYB transcription factors (TFs) have important roles in plant development and anthocyanin metabolism. In this first comprehensive study of R2R3-MYBs in kiwifruit, a total of 93 R2R3-MYB genes, including five novel previously unannotated AcMYBs, were identified. Their phylogenic relationship, exon-intron structures, and conserved motifs were analyzed. Based on transcriptome data, 60 AcMYBs were expressed (FPKM > 1) across seven developmental stages of kiwifruit, revealing five expression patterns. One of the 5 newly identified R2R3 TFs, AcMYB75, showed an anthocyanin accumulation-linked expression pattern during fruit development. AcMYB75 localized to the nucleus and has an active transactivation domain, verifying it as a transcription factor. AcMYB75 protein specifically bound the promoter of the anthocyanin biosynthesis gene ANS in yeast one-hybrid system and in vivo. In 35 S:AcMYB75 Arabidopsis plants, anthocyanin significantly accumulated in leaves, and the expression of anthocyanin biosynthetic genes was greatly up-regulated. Together, these results suggest that AcMYB75 is involved in anthocyanin biosynthesis in kiwifruit. These findings will increase our understanding of AcMYBs involved in anthocyanin biosynthesis, and also benefit further functional characterization of R2R3-MYB genes in kiwifruit.Red kiwifruit ( Flower and fruit pigmentation is largely derived from anthocyanins. Anthocyanins are potent antioxidants with health promoting properties, the benefits of which are increasingly of interest to consumers and make colorful fruits more favorable in the market2. Plant breeders have picked up nature’s work to augment fruit for the human consumer. Research has led to the understanding that fruit development and anthocyanin biosynthesis are largely controlled by specific transcription factors (TFs)4, especially R2R3-MYB TFs7.The rise of colorful flowers and fruits is a crux of plant biology and evolution. Fruit that is more attractive to insects and animals aids in the dissemination of genes and improves the survival rate of progeny10. MYB TFs are usually classified as 1R-MYB, R2R3-MYB, 3R-MYB or 4R-MYB, according to the number of conserved DNA-binding domains consisting of imperfect repeats (R) at the N terminus. Each repeat forms the hydrophobic core in a helix-turn-helix (HTH) structure, and the recognition helices in two repeats bind cooperatively to a specific DNA sequence motif. R2R3-MYBs usually possess an N-terminal DNA-binding domain -W-(X19)-W-…-F/I-(X18)-W-(X18)-W-) and a C-terminal transactivation or repression domain8.The structural characteristics of R2R3-MYB TFs have been extensively described for many species16. Recent reports on the regulation of anthocyanin accumulation in fruits by R2R3-MYBs include the study of MdMYB10 in apple20, VvMYBA1 and VvMYBA2 in grape22, PavMYB10.1 in cherry23, PcMYB10 and PyMYB10 in pear25, FaMYB10 in strawberry26 and AdMYB110a in kiwifruit flower pigmentation27. These studies reveal potential approaches for breeding and/or biotechnological development of diverse colors in horticultural fruits.R2R3-MYB TFs are widely involved in plant development, secondary metabolism, hormone signal transduction, cell apoptosis, disease and abiotic stress responses. Specific R2R3-MYB TFs have regulatory roles during fruit development and ripening and in determining fruit ascorbic acid level, carotenoid, flavonoid, lignin, and terpenoid levels30. However, no detailed bioinformatics analysis of the MYB family in kiwifruit (Actinidia Lindl.) has been reported. Over the past 30 years, kiwifruit has become a globally important horticultural cash crop. Furthermore, there are niche markets selling uniquely colored kiwifruit at premium prices. Both breeders and molecular biologists are interested in the mechanisms behind such diverse colors, such as yellow and red kiwifruit. The draft kiwifruit genome sequence (http://bioinfo.bti.cornell.edu/cgi-bin/kiwi/home.cgi)31 and our high-quality transcriptome data from the red-fleshed Hongyang kiwifruit32 provide an excellent opportunity for genome-wide analysis of kiwifruit genes.Searches of high-throughput genome sequences in different species has identified large numbers of MYB TFs based on conservation of the highly conserved DNA-binding MYB domainAcMYBs during kiwifruit development. Five previously unannotated R2R3 AcMYB TFs have been identified. Of these, we were most interested in AcMYB75, which showed high sequence similarity to anthocyanin-related R2R3-MYBs from other species and an expression pattern related to anthocyanin accumulation. AcMYB75 was found to localize to the nucleus and transactivate the anthocyanin synthase (ANS) gene promoter in yeast one-hybrid assay, and alter the anthocyanin accumulation when over-expressed in Arabidopsis. These results suggest that AcMYB75 is involved in anthocyanin biosynthesis in kiwifruit.In this study, a comprehensive genome-wide analysis was conducted to identify all R2R3-AcMYB TFs in kiwifruit. Analyses included phylogenetic relationship, gene structures, motif features and expression of R2R3 http://bioinfo.bti.cornell.edu/cgi-bin/kiwi/home.cgi) and our transcriptome data32 based on the classification in Arabidopsis thaliana33. Subsequently, domain detection confirmed that all of the identified R2R3-MYB TFs harbored the conserved MYB domain, which is the basic characteristic of the MYB family. Of these, five R2R3-MYB genes  were identified through a Tophat-Cufflinks pipeline using our previous transcriptome data and they were unannotated in the public genome database. Subsequently, manual checking with Integrative Genomics Viewer (IGV) further confirmed that the pair-end reads of each of these five genes were mapped on a genome region without gene annotation, respectively, and they were identified in different samples of transcriptome data  were well separated into different groups, except that the members of the original subfamily G9 and G20 were divided into two groups . The results showed that subfamilies G12, G18, G21, G22, G23 and G24 contained the most AcMYBs (≥4), with the maximum (7) in subfamily G21. There were no AcMYBs in subfamily G2, G3, G7, G26, G32 and G34, indicating an unexpected event during evolution. In G33, G35, G36, G37-1 and G38 families, there were only MYB genes from kiwifruit and tomato, indicating that these MYBs might participate in certain pathways particularly conserved in these two species.All 93 R2R3 AcMYBs were assigned into 36 subfamilies , together with other R2R3-MYB proteins from Arabidopsis: AcMYB20 closely clustered with its Arabidopsis homolog AtMYB20 and its tomato homolog SlMYB20 in subfamily G31; AcMYB70, AcMYB73, and AcMYB77 closely clustered with their homologs in both Arabidopsis  and tomato  in subfamily G22; and AcMYB75 in G6 closely clustered with two functionally characterized genes, AtMYB75 (PAP1) and AtMYB90 (PAP2), which are involved in flavonoid and anthocyanin biosynthesis in Arabidopsis34. This similarity suggested that AcMYB75 might have similar functions in flavonoid and anthocyanin biosynthesis in kiwifruit.The five newly annotated MYBs in kiwifruit seemed to have close homologs in tomato and/or AcMYB genes were identified based on their evolutionary relationships to annotated genes -W-(X19)–W -…-F/I-(X18)-W-(X18)-W-’. Motif 1, 6 and 9 were also identified as MYB domains, while other motifs  were of function unknown. Notably, all the AcMYBs had a MYB domain, and proteins of each AcMYBs subfamily shared some common motifs  exhibited high transcriptional abundance  during each of the 7 developmental stages, indicating key roles for these genes in fruit development. Genes in groups B and C showed decreasing expression levels from S1 to S7. Specially, AcMYB57 and AcMYB15 were not present at later stages of kiwifruit development. AcMYB5, AcMYB52, AcMYB89, AcMYB27, and AcMYB53 had low transcript levels at S5. S5 is the stage with the highest anthocyanin accumulation in kiwifruit32, suggesting that these 5 AcMYBs may not be directly committed to anthocyanin biosynthesis. There were 17 AcMYBs in group D that overall were present at low levels throughout fruit development. For example, transcripts for AcMYB26, AcMYB59, AcMYB12, and AcMYB64 were almost absent at S1, while the others had lower transcript levels at other stages. The 15 genes  in group E shared a pattern of the highest transcriptional levels  only at S1, with expression dropping across the other six stages. The transcript levels of these genes were higher at S1 than those in group D. The transcript levels of the newly identified genes AcMYB75 and AcMYB73 were high across all of fruit development, hinting that these genes might be involved in fruit development and formation of qualities such as flavor, color and taste. On the other hand, AcMYB20, AcMYB70, and AcMYB77 in group E might specifically relate to blooming and preliminary development of fruit. For this comparison of the expression profiles of the AcMYB genes at different developmental stages, it can be concluded that AcMYB members play differential roles in fruit development.To seek insights into the roles of nes Fig. . Ten of Arabidopsis thaliana and AcMYB75 also had the higher expression levels during anthocyanin accumulation in kiwifruit, AcMYB75 was studied further for its regulation on anthocyanin biosynthesis in kiwifruit. The full-length cDNA of AcMYB75 was cloned and sequenced. The result verified that AcMYB75 had 666 nucleotides and contained an open reading frame (ORF) encoding a deduced protein of 221 amino acids. The deduced AcMYB75 protein contained R2 (motif 3 and 4) and R3 (motif 2) MYB domains  and a motif typical of anthocyanin-related regulator (box)27  in motif 1 and motif 2 (R3) were related to the bHLH interaction35. There are 49 amino acids different between AcMYB75 and AcMYB110a, four of which are in the conserved R2, while those in the R3 MYB domains are completely the same. In the 8 amino acid motif of anthocyanin-related regulators, only three of the residues  were the same between AcMYB75 and AcMYB110a. Our phylogenetic tree showed the bootstrap value of AcMYB75 and other MYBs regulating anthocyanin accumulation, including PAP1 and PAP2 from Arabidopsis36, AcMYB110a from kiwifruit27, PyMYB10 from Pyrus pyrifolia var. culta25 and VvMYBA from Vitis vinifera37, of which 99% supported the clustering of AcMYB75 and AcMYB110a  and AtMYB90 (PAP2) . In addition, we found only yeast clones expressing either AcMYB75 and pMBS-AbAi (MYB binding sequences (MBS) cloned into the bait vector pAbAi) or the positive controls could grow on SD/-Leu selective medium containing AbA  and LUC activity is expected to increase when TFs bind the promoter. The same construct contains a luciferase gene from Renilla (REN) driven by a 35 S promoter that served as a control for normalization. Activity is identified as a ratio of LUC to REN-luc activity. It showed that the AcMYB75 cDNAs was able to induce an approximately 2-fold increase in luciferase (LUC) enzyme activity when co-infiltrated with construct containing the AcANS promoter fused to the firefly LUC gene  Arabidopsis plants had purple leaves, especially leaves in the rosette compared to those from wild type (WT) and vector only (VE) plants   Fig. , while t−1) Fig. .Figure 7CHS, CHI, F3H, and F3′H) and late  flavonoid biosynthetic genes. As a whole, the results showed that the expression levels of these genes were up-regulated in AcMYB75 over-expressing transgenic Arabidopsis plant. Particularly, the expression levels of AtANS and AtF3GT that determine the last steps of anthocyanin biosynthesis were about 7 and 5 fold higher in OE plants than in WT plants  originates from China and has become an important commercial horticultural crop due to its pleasant flavor and nutritional components benefiting human health38. For example, the red-fleshed Hongyang kiwifruit, derived from A. chinenesis var. chinensis39, is becoming a favorite of consumers, breeders and growers due to its unique phenotype, excellent agricultural characteristics, and high premium price at market. However, research progress on kiwifruit was relatively slow compared to other important horticultural crops, such as grape, apple or tomato. A comprehensive analysis of the R2R3 AcMYB gene family has yet to be reported, and the functions of most R2R3 AcMYBs are still unknown. In this study, we comprehensively analyzed the R2R3 AcMYB family, by looking at their phylogenetic relationships, gene structures, protein features, and expression profiles during fruit development.Kiwifruit (AcMYB genes identified in kiwifruit (93) was lower than in Pyrus bretschneideri (105)7, Glycine max (244)30, Malus pumila Mill. (222)40, Arabidopsis thaliana (126)8, Zea mays (157)41, and Solanum lycopersicum (127)42 but higher than in Cucumis sativus (55)43 and Orzya. sativa (88)28. The phylogenetic relationship of MYBs among kiwifruit, Arabidopsis and tomato indicated that the three species displayed both conserved and specialty evolution of MYB members, particularly in kiwifruit and tomato. As examples, group C12 contained genes only from kiwifruit and tomato; group C1 had no genes from kiwifruit; and groups C3, C8 and C17 had more MYB genes than other groups. These results suggested that species-specific MYB genes were either acquired in kiwifruit or lost in Arabidopsis lineages after divergence from their most recent common ancestor.The number of R2R3 Notably, most genes within the same group generally share similar exon-intron patterns in our genes structure analysis. Differences in exon-intron patterns, lengths or presence of proteins motifs may suggest hints at the function of these genes and how they evolved.44. Functional analysis of MYB transcription factors has been performed in A. thaliana and other species46. AtMYB77 and AtMYB44 are associated with fruit development, stress response, and also auxin and abscisic acid signal transduction in Arabidopsis48, suggesting that AcMYB70, AcMYB73, and AcMYB77, which closely clustered with AtMYB77 and AtMYB44 in the same subfamily , we investigated the interaction between AcMYB75 and bHLH type transcription factors from Arabidopsis through the yeast two-hybrid system. The result showed that AcMYB75 had interaction with N-terminal domains of TT8 snd GL3 GTTA) and MBSII (aaaAGTTAGTTA) were the binding sites of Ph3 in Petunia hybrid and involved in flavonoid biosynthetic genes regulation56. We also speculated that the TT residue in the MBS might be related to the binding of AcMYB75 with AcANS. However, the whole or key MYB binding sequences in flavonoid biosynthetic genes in kiwifruit should be verified furthermore.Furthermore, the MYB binding sites in kiwifruit should be verified further in the future. As reported, TAACTG was the MYB recognition sequence of A. chinensis) were collected from the Wuhan Botanical Garden, Hubei Province in 2015. Samples from seven stages of fruit development from 7 (S1), 70 (S2), 90 (S3), 100 (S4), 110 (S5), 120 (S6), and 140 (S7) days after anthesis were harvested according to the development and the anthocyanin accumulation of kiwifruit as our previous research32. Briefly, the highest anthocyanin is kiwifruit ovary at S1, but disappears at S2. During the expansion of kiwifruit, anthocyanin accumulation starts again at S3. The deepest red color appears from S4 to S5, but anthocyanin will lost a little from S6 to S7 when fruit matures.Red-fleshed Hongyang kiwifruit  using Cuffdiff (v2.1.1)59.Transcriptome data from seven stages of fruit was obtained from our previous experimentshttp://bioinfo.bti.cornell.edu/cgi-bin/kiwi/home.cgi)31 by local HMM-based searches with E-values <0.0160. Other R2R3-MYB protein sequences from Arabidopsis and tomato (Solanum lycopersicum) were included from UniPort (http://www.uniprot.org/) and Phytozome (http://www.phytozome.net/slycopersicum.php)42. Subsequent, BLAST searches were performed to check the predicted kiwifruit R2R3 MYBs with all the Arabidopsis and tomato R2R3 MYBs as queries. In addition, pair-end reads from our transcriptome data set were used to identify novel MYB candidates as previously described58. Briefly, clean reads were mapped to kiwifruit genome using Tophat v2.0.10 allowing novel splice junctions identification (read mismatch = 1)61. Novel transcripts were identified by cufflink and cuffcompare in the Cufflinks v2.2.162. For each transcript sequence, the coding regions and related protein sequences were predicted by TransDecoder v3.0.0 (http://transdecoder.github.io/). Finally, all candidate protein sequences were further examined by the CDD (http://www.ncbi.nlm.nih.gov/cdd/)  and PFAM (http://pfam.sanger.ac.uk/)  databases.To identify putative R2R3-MYB genes in kiwifruit across the kiwifruit genome, the MYB_DNA-binding (PF00249) family from PFam (http://pfam.sanger.ac.uk/) was used to query the kiwifruit protein sequences obtained from Kiwifruit Genome Database  was employed to detect the molecular weight and isoelectric points of the R2R3 AcMYB proteins65. Exon-intron structures were predicted with Gene Structure Display Server 66. The conserved motifs of kiwifruit MYB proteins were identified using the MEME program (http://meme.ncbr.net/meme/cgi-bin/meme.cgi), with optimum motif width ranging from 11 to 50 and the maximum number of motifs set to ten67. All identified motifs were annotated with interProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/). Hierarchical cluster analysis was performed through MeV software from the TM4 suite (http://www.tm4.org/mev.html)68.The ExPASy proteomics server . First-strand cDNAs were synthesized using the FastQuant RT Kit (with gDNase) , according to the manufacturer’s instructions, before use as templates for real-time PCR amplification.® Premix Ex TaqTM , and 0.2 μM of the forward and reverse primers for each gene. Reactions were run on an Applied Biosystems StepOne Real-Time PCR system . The conditions for each PCR reaction were as following: 95 °C for 30 s, followed by 40 cycles of 5 s at 95 °C, 20 s at 58–62 °C depending on primer TM, and 20 s at 72 °C. At the end of each experiment, a melt-curve analysis was performed using the default parameters . The relative expression levels of the target genes were calculated by the 2−ΔΔCt method. The β-actin 2 (At3g18780) and glyceraldehyde-3-phosphate dehydrogenase A (GAPDH) (At3g22650) genes were used as internal references to normalize the transcriptional levels of target genes. All genes involved in the flavonoid pathway in Arabidopsis36 were chosen. Genes and primers are listed in Supplementary Table The PCR mixture contained 1 μl of the cDNA template, 10 μl of 2 × SYBRAcMYB75 was amplified using the following primers: F: 5′-TGC TCT AGA ATG GAA AGT GTT ACT TTA GG-3′, R: 5′-CG GGA TCC ATC ATC ACC TAA GAG ATC CC-3′ and cloned into the plant expression vector of pCAMBIAI1300 under the control of CaMV35S promoter. The histone H3 acetyltransferase gene  was used as a positive reference. Agrobacterium tumefaciens, LAB4404 with the plasmids of CaMV35S::AcMYB75-GFP, CaMV35S::AtH3-GFP or CaMV35S (vector only) were infiltrated into tobacco (Nicotiana benthamiana) epidermal cells, respectively. Infiltrations were performed according to the methods of Li et al.68. After 3–5 d of incubation at room temperature, the tobacco cells were observed under a confocal microscope .The full length cDNA of Saccharomyces cerevisiae Y187:ura3–52, trp 1–901, Ura3::GALUAS-Gall TATA-LacZ) . The full-length of AcMYB75 cDNA was fused with the GAL4 DNA-binding domain to generate pGBKT7-AcMYB75 through EcoRI/BamHI. The gene PERIANTHIA (PAN), reported as a bZIP transcription factor, was used as the positive control69. The pGBKT7-AcMYB75, pGBKT7-AtPAN and pGBKT7 constructs were transformed by PEG/LiAc method into yeast strain Y187 according to the yeast transformation system 2 user manual . The reporter genes LacZ is controlled by the G1 promoter (the GAL4 binding domain responding cis-element). The Y187 transformants were selected on SD/-Trp at 30 °C for 3 d. X-α-gal was used for the confirmation of transcriptional activation.The GAL4 one-hybrid system was used for identifying the transactivation of AcMYB75 in Y187 yeast (AUR-1C) which is the reporter on the bait vector pAbAi. Activation of the AbA resistance gene (AbAr) occurs, if the binding domain of transcription factors bind to the bait sequence. Three tandem repeats of the putative MYB binding sequence (MBS) from anthocyanin synthetase (ANS) (Achn361621) promoter (273 bp before ATG). promoter were synthesized  (5′-TGGATCTCTATCTCACGACGGTTAGACCACCTCGTGCTTGTCTCACGACGGTTAGACCACCTCGTGCTTGTCTCACGACGGTTAGACCACCTCGTGCTTGTGCCATCCAG-3′) and cloned into the SacI/XhoI on the pAbAi vector under the yeast iso-1-cytochrome C minimal promoter as the bait (pMBS-AbAi). The full length of AcMYB75 was fused with the GAL4 transcription activation domain to generate pGADT7-AcMYB75 through EcoRI/BamHI under the constitutively active ADH1 promoter (PADH1) as the prey.The Matchmaker Gold Yeast One-Hybrid Library Screening System  employing strong selective power of Aureobasidin A (AbA) resistance to produce screens with very low backgrounds was used for the identifying of the DNA-binding domain in AcMYB75. Resistance to AbA is conferred by the AbAr gene . The recombinant Y1HGold yeast cells with bait and prey plasmids were selected on SD/-Ura medium with 200 ng ml−1 AbA, at 30 °C for 3 d.The sequence with three ‘TT’ pairs mutated to three ‘CC’ pairs at positions 22–23, 52–53, and 82–83, respectively, designated as pMBSmt-AbAi, was used as negative control. The plasmids pMBS-AbAi, pMBSmt-AbAi or p53-AbAi were integrated into yeast strain Y1HGold by homologous recombination to construct the bait yeast according to the user manual. The concentration of 200 ng mlANS promoter was insterted into the plasmid of pGreenII70 to construct the plasmid of pGreenII-AcANS. The Agrobacterium tumefaciens strain LBA4404 cultures transformed with pGreenII-AcANS were mixed with the Agrobacterium tumefaciens cultures transformed with constructed plasmids of pCAMBIAI1300-AcMYB75 according to v/v (1:5) for co-infiltration. Nicotiana plants were grown under glasshouse conditions under natural light with 16 h daylight for transient transformation. The Agrobacterium mixtures were injected into tobacco leaves according to Li’s method68.The Three days after infiltration, 1 cm leaf discs  were collected and the activity of luciferase and REN-Luc were measured according to Dual-Luciferase Reporter Assay System manufacture’s manual . The binding ability of AcMYB75 to the promoter of AcANS were represented by LUC/REN (ratio of LUC to REN-luc).Arabidiopsis, AtTT8 (At4g09820) and AtGL3 (At5g41315)72. The two DNA binding domains of AtTT8 and AtGL3 were cloned into pGBKT7 vector to generate pGBKT7-AtTT8-1, pGBKT7-AtTT8-2, pGBKT7-AtGL3-1, and pGBKT7-AtGL3-2, respectively, as the binding domain plasmids. The yeast strain AH109 with four report genes of LacZ, HIS3, ADE2, MEL1 under the control of GAL1, GAL2 and MEL1 promoters was used for the screening73. The constructed activation domain plasmids (pGADT7-AcMYB75) and four binding domain plasmids  were co-transformed into AH109, respectively, according to the yeast transformation system 2 user manual . The transformants were selected on SD/-Leu/-Trp at 30 °C for 3 d. and then the positive transformants were again selected on SD/-Ade/-His/-Leu/-Trp at 30 °C for 3-5 d. The positive transformants from SD/-Ade/-His/-Leu/-Trp were ultimately selected through X-α-gal with SD/-Ade/-His/-Leu/-Trp again at 30 °C for 3–5 d.The yeast two-hybrid screening was used to verify the interaction between AcMYB75 and endogenous bHLH transcriptors from Arabidopsis thaliana ecotype Columbia (Col-0) plants were grown in growth rooms at 22 °C in long (16-hr of light) or short (9-hr of light) days. The constructed plasmids of CaMV35S::AcMYB75 for subcullular localization analysis were electroporated into Agrobacterium tumefaciens LBA4404 and then transformed into A. thaliana plants by the floral dipping method74. Four independent single copy insert lines  were taken to the third generation for use in experiments. Seeds of individual self-fertilized T2 lines were collected and single-copy insertion lines were selected based on a Mendelian segregation ratio. All healthy leaf material and seeds of T2 lines were collected from individual plants and immediately frozen in liquid nitrogen and stored at −80 °C.2O/acetic acid (80:20:1 v/v/v) in an Ultra-Turrax homogenizer for 30 minutes and extraction overnight at 4 °C. Anthocyanin extraction and detection were as described75. Anthocyanin concentration was calculated from the peak area at 530 nm as cyanidin 3-O-glucoside.Samples (1 g for leaves) were ground in liquid nitrogen before anthocyanin extraction using 5:1 (v/w) ethanol/HSupplementary TablesSupplementary Figures