MiR-192 Inhibits Nucleotide Excision Repair By Targeting ERCC3 And ERCC4 In 2.2.15 Cells

Objective The maintenance of genomic integrity through efficient DNA repair is essential for propagation of cellular life.Un-repaired and mis-repaired lesions lead to chromosomal aberrations (CAs). Nucleotide excision repair (NER) is one of the most versatile DNA repair system for elimination of bulky DNA adducts caused by ultraviolet irradiation, environmental carcinogens and some endogenous free radicals. It involves more than 30 polypeptides within two distinct pathways that are different in the modes of DNA damage recognition:the global genome DNA repair (GGR-NER) and transcription-coupled repair (TCR-NER). Some viruses inhibits NER to increase the frequency of mutations. Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. In Asia and Africa, chronic HBV infection is the most important cause of this disease. The integration of HBV genomic fragments into the human genome promoted by aberrant DNA repair was thought to be one of the major mechanisms of HBV to trigger tumorigenesis. HBx was reported to inhibited NER through both p53-dependent and p53-independent pathways. MicroRNAs (miRNAs), a class of small non-coding RNAs, modulate gene expression by binding to the 3’-untranslated region (3’UTR) of target mRNAs to promote mRNA degradation and/or repress protein translation. They play pivotal roles in a gamut of cellular processes including cell development, differentiation, apoptosis, signal transduction, metabolism and tumorigenesis. Several miRNAs involved in DNA repair have been identified. Here we hypothesized that the existence of HBV products may interfere with cellular NER through microRNA-mediated gene regulation. To test this hypothesis, a genome-wide miRNA microarray was performed to identify differentially expressed miRNAs between 2.2.15 cells and HepG2 cells. Some miRNAs were chosen for further investigation on the function and putative targets identification in NER pathway.Methods We examined the NER capacity of HBV-transfected 2.2.15 cells compared with HepG2 cells. A genome-wide miRNA microarray was then performed to identify differentially expressed miRNAs between these two cells. Bioformatics analysis using three prediction programs (miRanda, TargetScan and PicTar) were further performed to search for miRNAs which potentially played a role in DNA repair. MiR-192 was found to target some key proteins involved in NER pathway and was chosen for further investigation. To verify the results obtained from microarray profiling, Taqman miRNA RT-PCR assay was performed to examine miR-192 expression. The real targets of miR-192 were verified by using luciferase reporter assay, SYBR Green RealTime PCR and Western Blot. Finally, we assessed the NER capacity of HepG2 cells transfected with miR-192 or miR-NC using host cell reactivation assay.Results Compared with HepG2 cells, much lower luciferase activities were detected in 2.2.15 cells. The relative repair capacity for UV-induced DNA damage in 2.2.15 cells was decreased, which indicated that the NER capacity in 2.2.15 cells was greatly reduced. Differentially expressed miRNAs between 2.2.15 and HepG2 cells were identified using miRNA microarrays. Twelve of 723 human miRNAs examined were found to be significantly dysregulated (>100-fold difference). Six of them (miR-194,-429,200b,-215,-146a, and miR-192) were upregulated, yet the other six (miR-30a*,-99a,-100,-130a,-30a and-125b) were downregulated. According to Bioformatics analysis, miR-192, the most differentially upregulated miRNA in 2.2.15 cells, was found to target some key proteins involved in NER pathway:XPA, ERCC3 and ERCC4. To determine if XPA, ERCC3 or ERCC4 are real targets of miR-192, a luciferase reporter assay was performed. The relative luciferase activity of the vector that contained wild-type 3’UTR of ERCC3 or ERCC4 was significantly suppressed when miR-192 was cotransfected. In contrast, the relative luciferase activities of the mutant vectors were unaffected by cotransfection of miR-192. However, the luciferase activity with XPA 3’UTR or its mutant form was not altered after miR-192 transfection. We further investigated the role of miR-192 in regulating endogenous expression of ERCC3 and ERCC4. Real-time RT-PCR demonstrated that ERCC3 and ERCC4 mRNA levels were reduced in miR-192 transfected cells compared to negative controls. In accordance with the changes of mRNA level, the protein levels of ERCC3 and ERCC4 were also substantially decreased when miR-192 was overexpressed. Taken together, miR-192 could suppress the expression of ERCC3 and ERCC4 by directly binding the target sites in their 3’UTRs. Compared with the negative control, the relative repair capacity for UV-induced DNA damage was reduced to when miR-192 was cotransfected. These data indicated that miR-192 inhibited cellular NER in HepG2 cells.Conclusions Altered miRNAs were identified by comparison of the expression profiling of miRNAs in 2.2.15 cells versus HepG2 cells. These miRNAs could play a role in cellular nucleotide excision repair by regulating their target proteins. Deficient nucleotide excision repair contributes to genetic integrity so as to cause the development of HBV-related HCC. Our findings thus offer a new perspective in understanding the pleiotropic functions of HBV and its contribution to HCC development.