Effects Of MiR-122a On The Radiosensitivity Of Human Liver Cancer Cells

Background and objectiveHepatocellular carcinoma (HCC) is one of the most common malignant tumorsworldwide.The morbidity and mortality rate were about50%in China. Many studies showthat HCC is moderately sensitive to radiation.Its sensitivity is equivalent to lowdifferentiated squamous cell carcinoma. In recent years, following with the development ofthree-dimensional conformal radiotherapy, the low radiation tolerance of normal liver tissueis gradually overcame. The radiotherapy combined with gene therapy against tumor is anextremely important and new trend.MicroRNAs (miRNAs) are a class of about19~25nucleotides (nt) in length, non-protein-coding single-stranded small RNAs, which are cleaved from the70~80nt partiallyduplexed precursor by the RNase III Dicer.miRNAs are widely distributed in viruses, plantsand mammals and are known to negatively regulate gene expression via incomplete orcomplete matching with the3’UTR of their target genes at the post-transcriptional level.Evidence suggests that miRNAs may contribute to cancer pathogenesis,and they may serveas oncogenes or anti-oncogenes during carcinogenesis.Recent studies have showed that thedifferential expression of miRNAs is related to the radiosensitivity of tumors,however,themechanism of miRNAs on radiosensitivity is still unknown.Of course,if the molecularmechanism could be clarified, miRNAs combined with radiotherapy will become a verypotential and clinical value of treatment.miR-122a is located at18q21.31of human chromosome, and it is a liver-specificmiRNA which accouts for about70%of the liver total miRNAs.miR-122a involved inmany biological processes such as the differentiation, proliferation and apoptosis of hepaticcells.Many research have proved that miR-122a is downregulated in about70%HCCtissues and hepatoma cell lines.The result also indicated that miR-122a plays an importantroles during the occurrence and development of HCC. In our previous studies,we detected the different expression profiles of miRNAs in HepG2and LO2cell lines by miRNAmicroarray. Along with reports of the different expression patterns of miRNAs in livercancer cells, this allowed us to isolate miR-122a as a study target, as it is downexpressed inHepG2cell.Futhermore recent reports have showed that the expression of miR-122a isassociated with the chemotherapy sensitivity of hepatocellular carcinoma cells.However,the relationship between miR-122a and the radiotherapy of HCC is still unknown and needus further study.The key to study the function of miRNAs is to find out its target genes and investigatethe interaction between miRNAs and the corresponding targets. We predicted the miR-122atarget genes by miRGen3.0, and analyse target genes of miR-122a using enrichmentanalysis(GO-Analysis), signal transduction pathway enrichment analysis (Pathway-Analysis) and protein interaction network analysis.In our study, we detected whether miR-122a can influence the biological behaviors ofhepatocellular carcinoma cells and the radiosensitivity of hepatocellular carcinoma cellsusing the different experiments including Cell cycle analysis,MTT,Clony formation assayand Cell apoptosis analysis.The results showed that miR-122a overexpression lead to G2/Marrest of Hep3B(p53-/-) cell,rather than HepG2(wtp53) cell. miR-122a overexpression caninhabit the proliferative ability and promote apotosis of hepatocellular carcinoma cellsfollowing with different dose of radiation.In order to understand the function of miR-122a,we further analyzed cyclin G1,the potential target genes of miR-122a, which may beinvolved in the radiosensitivity of hepatocellular carcinoma cells.The results will not onlyhelp us understand the role of miR-122a during the radiosensitivity of hepatocellularcarcinoma cells, but also provide a new target for the treatment of HCC.Methods1. The target genes of miR-122a were predicted and analyzed by the differentbioinformatics ways such as GO-Analysis, Pathway-Analysis and Protein interactionnetwork analysis.2.Constructing miR-122a overexpression lentiviral vector.3.Cell cycle analysis,MTT,Clony formation assay and Cell apoptosis analysis wereperformed to investigate the miR-122a influences the biological behaviors of different livercancer cells. 4. Western blot was performed to detect the relationship between miR-122a andcyclinG1.5. Dual luciferase reporter assay was performed to detect whether APE1was the targetgene of miR-122a.Results1. There are1104target genes of miR-122a were predicted at present,and most ofthese are involved in cell cycle, signal transduction, cell proliferation, differentiation,apoptosis, and so on.2. Overexpression of miR-122a could inhibit the cell cycle process of Hep3B cell, butnot HepG2cell.3. Overexpression of miR-122a could inhibit hepatocellular carcinoma cellsproliferation and promote cell apoptosis following with ionizing radiation.4. The expression of cyclin G1is downreulated in Hep3B cells with overexpression ofmiR-122a,but the expression of cyclin G1is not changed in HepG2cell withoverexpression of miR-122a.5. The expression of APE1is not changed in Hep3B and HepG2cells with overexpressionof miR-122a.Conclusion1. According to the bioinformatics analysis,the target genes of miR-122a involved incell cycle, signal transduction, cell proliferation,differentiation,apoptosis and otherbiological processes.2. Overexpression of miR-122a may interact with cyclinG1to increase theradiosensitivity of hepatocellular carcinoma cells which is associated with p53expression.