Hsa-mir-331-3p Inhibits Vhl Expression by Directly Targeting Its Mrna 3'-utr in Hcc Cell Lines

Dysregulation of miRNA is widely involved in human cancers, including hepatocellular carcinoma (HCC). Array data for miRNAs indicated that miR-331-3p might be one of the disorderly expressed miRNAs in HCC cell lines, but the function of miR-331-3p in HCC remains unclear. In this study, quantitative real time polymerase chain reaction (qRT-PCR) results indicated that miR-331-3p was up-regulated in HepG2.2.15 cells, Ad-HBV-HepG2 cells and pCH9/3091transfected SMMC7721 cells compared with their control group, respectively. miRNA target prediction software was used, and VHL was found to be one of the target genes of miR-331-3p. qRT-PCR and western blot analysis indicated VHL expression was decreased when miR-331-3p was over-expressed and increased when miR-331-3p was inhibited in SMMC7721 cells. The luciferase reporter activity was inhibited in SMMC7721 cells when co-transfected with miR-331-3p expression vector and VHL 3'-UTR wild type vector and increased in HepG2.2.15 transfected with miR-331-3p inhibitor compared to its control group respectively. When co-transfected with miR-331-3p expression vector and VHL 3'-UTR mutated type vector in SMMC7721 cells the lucif-erase reporter activity was recovered. All of these results show that HBV up-regulated miR-331-3p expression in HCC cell lines and miR-331-3p could inhibit VHL expression by directly targeting its 3'-UTR. This provided useful information in exploring the mechanism of HCC induced by HBV infection.


INTRODUCTION
MicroRNAs (miRNAs) are small single-stranded noncoding RNA molecules composed of 21-22 nucleotides and account for about 1% of the entire genome.miR-NAs widely exist in eukaryotic cells and regulate gene expression by interacting preferentially with the 3'untranslated regions (3'-UTRs) of target mRNAs, which may cause either inhibition of translation or degradation of the targeted mRNA (Bartel, 2004;Calin & Croce, 2006).miRNAs and their target mRNAs form complex regulatory networks, involved in cells proliferation, apoptosis, cell differentiation, stress response and other complex regulatory networks (Gaal & Olah, 2012;Lin et al., 2013).A large number of researches indicates that deregulation of miRNAs is common in human tumors.miRNAs can inhibit target mRNAs which are involved in the occurrence , development and progression of cancer as either oncogenes or tumor suppressors (Zhang et al., 2007).miRNAs expression disorder maybe a common cause of human tumor (Croce, 2009).
HCC is one of the most common and typical malignancies, and most cases are attributable to persistent hepatitis B virus (HBV) infections.It has also been reported that virus infection can interfere with cellular miRNA expression (Lin & Flemington et al., 2011).Lots of reports indicate that HBV can induce carcinogenesis in HCC in a miRNAs involved pathway (Wang et al., 2011;Xu et al., 2013;Zou et al., 2014).Different miRNAs expression patterns between HepG2 cells and HepG2.2.15 (a HBV stable expression HCC cell line based on HepG2) cells have been shown by a miRNA microarray analysis in a previous study (Zhang et al., 2011).There, we found that miR-331-3p was up-regulated in HepG2.2.15 cells compared to its control HepG2 cells.It has also been reported that miR-331-3p plays an important role in tumors, like prostate cancer, gastric cancer and glioblastoma multiforme (Guo et al., 2010;Epis et al., 2011;Epis et al., 2012;Epis et al., 2014), but whether miR-331-3p is involved in HCC development and progression remains unknown.
The Von Hippel-Lindau (VHL) syndrome is a dominantly inherited familial cancer syndrome predisposing to a variety of malignant and benign tumors.The basis of familial inheritance of VHL syndrome is a germline mutation of the VHL gene (Maher & Kaelin, 1997).This gene encodes a component of a protein complex including elongin B, elongin C, and cullin-2, and it possesses ubiquitin ligase E3 activity.VHL protein is associated with the ubiquitination and degradation of hypoxia inducible factor (Cockman & Masson, 2000).
Here, we detected the expressions of miR-331-3p in different HCC cell lines and confirmed VHL was the target gene of miR-331-3p.These data will be helpful for investigating the potential association between miR-331-3p and HBV.
Luciferase reporter assay.For the luciferase reporter assay, SMMC7721 cells were seeded in a 24well plate at a density of 45% and co-transfected with 250 ng pTARGET-miR-331-3p or pTARGET vector, 150 ng of pGL3-Control-VHL-WT or pGL3-Control-VHL-MUT constructs and 25 ng pRL-TK plasmid expressing renilla luciferase (Promega, Madison, WI).HepG2.2.15 cells were seeded in 24 well plates at a density of 60% and co-transfected with 70 pmol miR-331-3p inhibitor or inhibitor NC, 150 ng of pGL3-Control-VHL-WT and 50 ng pRL-TK.Cells were collected 48 h after transfection and analyzed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI).Relative luciferase activity was normalized to renilla luciferase activity.Transfections were done in triplicate and repeated at least 3 times in independent experiments.
Statistical analysis.Data are expressed as mean standard deviation (S.D.).Statistical analysis was performed by using the independent t-test.P value of less than 0.05 was considered statistically significant.
These results indicated HBV could promote miR-331-3p expression in HCC cell lines.

DISCUSSION
In recent years, miRNAs have been reported frequently to undergo different expression patterns in many biological events, especially tumor genesis (Ambros, 2004;Griffiths-Jones et al., 2008).miRNA expression profiling studies have shown that HBV infection resulted in alterations of many miRNA expression (Gao et al., 2011;Yip et al., 2011).As HBV is the main cause of HCC, maybe HBV plays an important role in HCC occurrence and development in miRNA-related manners.According to our studies, miR-331-3p was up-regulated by HBV, but whether miR-331-3p was involved in the HBV-miRNA-HCC mode of action needs a further research.
It has been reported that VHL mutation is associated with several types of tumors like pheochromocytoma, clear-cell renal cancer, central nervous system and retinal angiomas, which suggests that VHL gene may play a role as a tumor suppressor in those cancers (Latif et al., 1993;Kim & Kaelin, 2004).According to miRNA bioinformatics analysis, VHL was predicted to be a target gene of miR-331-3p.We detected VHL expression after overexpression or inhibition of miR-331-3p in SMMC7721 cells by qRT-PCR and western blot.As we expected, VHL was inhibited by miR-331-3p.To further investigate its mechanism, SMMC7721 cells were co-transfected with the expression plasmid of miR-331-3p and VHL 3'-UTR wild type or 3'-UTR mutated type to detect their luciferase activities.Luciferase reporter gene assay results show that miR-331-3p can decrease the luciferase activity of pGL3-Control-VHL-WT and has little influence on pGL3-Control -VHL-MUT.Luciferase activities of HepG2.2.15 cells transfected with miR-331-3p inhibitor were higher than inhibitor NC.These indicated the inhibitory effect of miR-331-3p on VHL depended on its 3'-UTR, so we confirmed that VHL is one of the target genes of miR-331-3p.
MiR-331-3p were reported to be down-regulated in several types of tumors like glioblastoma multiforme, prostate cancer and gastric cancer and plays a role as a tumor suppressor (Guo et al., 2010;Epis et al., 2011;Epis et al., 2012;Epis et al., 2014).But interestingly, our studies showed that miR-331-3p was up-regulated in HepG2.2.15 cells in comparison to HepG2 cells, and inhibited the expression of tumor suppressor gene VHL.It means miR-331-3p might act as a promotion factor in HCC cells.This may be due to its different roles in dif-ferent cancers.More detailed mechanisms and functions of miR-331-3p need to be further elucidated.As VHL is a target gene of miR-331-3p which can be regulated by HBV in HCC cell lines and we also observed that VHL was down-regulated in HepG2.2.15 cells and recovered in HepG2.2.15 cells transfected with miR-331-3p inhibitor, but we did not observe the same variation tendency in HBV transient transfection HCC cell lines, so whether VHL is involved in HBV-related HCC still needs further study.