Identification And Fuctional Study Of Differential MicroRNAs In Hepatocellular Carcinoma

【Background】Hepatocellular carcinoma (HCC) is one of the most common humanmalignancies worldwide, while the underlying mechanisms of HCCcarcinogenesis and progression are still unclear. MicroRNAs (miRNAs) are aclass of single-strand evolutionarily conserved small non-coding RNAs with19–22nucleotides in length. The specificity of miRNA targeting is based onWatson–Crick complementarities with the3’ untranslated region (UTR) of theirtarget mRNAs, which can down-regulate the expression of various target genesinvolved in the malignant process of cancer. For one thing, when the miRNAbinding shows perfect complementarities, the RISC induces mRNA degradation.For another, once an imperfect miRNA–mRNA target pairing occurs, translationinto a protein is blocked. Overall, miRNAs serve as expression regulators infundamental cell progression, including growth, proliferation, apoptosis,differentiation and metastasis. An increasing number of evidence shows that the aberrant expression of certain miRNAs in cancers plays significant roles in thetumorigenesis and progression.Because of miRNAs’ property of ‘one for all’, they are able to targetmultiple genes involved in cancer pathways so that cancer phenotypes can bemodified by targeting gene expression. However, targets of miRNAs intumorigenesis, progression and therapeutic intervention of HCC remains unclear.The corresponding mechanisms require more concentration and commitment oftargeting miRNAs in therapeutic strategies for HCC in future studies.【Aims】To screen and identify the aberrantly expressed miRNAs in HCC comparedwith matched non-cancerous tissues, and to investigate the effects involved inHCC carcinogenesis and progression, for better understanding the mechanisms ofHCC from a fresh perspective and providing a new theoretical basis for earlydiagnosis and intervention of HCC.【Methods】1. Based on our former experimental data and references, we selected5miRNAs: miR-199a-1, miR-125a、miR-125b、miR-373、miR34a for furtherstudy. The miRNAs levels in18pairs of HCC and corresponding non-tumortissues were detected by quantitative real time-PCR (qRT–PCR), andanalyzed the difference expression of each in HCC and non-tumor tissues.2. A complementary of22more paires of HCC and corresponding non-tumortissues was carried out with the same treatment as described above. Theexpression levels of miR-199a-1in these22HCC tissues and correspondingnon-tumor mucosa were detected by qRT–PCR. Correlations between themiR-199a-1expression level and clinicopathologic characteristics of HCCwere also analyzed. 3. The expression of miR-199a-1was also detected in HCC cell lines HepG2and SMMC-7721, and normal liver cell line Chang liver. MiR-199a-1wasfound to be lowly expressed in HepG2cells, and over-expression ofmiR-199a-1in HepG2cells using a miR-199a-1expression vector(pGenesil-1-miR-1) was used for further study. The effect of miR-199a-1expression on cell growth and proliferation in vitro and tumorigenicity invivo were determined by MTT assay, flow cytometry, flat cloningexperiments and nude mice.4. To determine the underlying mechanisms that miR-199a-1contributes to themigration and metastasis of HCC, computative predicting tools includingmiRanda, Pictar, and TargetScan were used for the prediction of potentialregulatory targets of miR-199a-1. FZD7was confirmed to be one of themost inmortant target genes by luciferase, qRT–PCR and western blot assays.The downstream genes of FZD7, including β-catenin, Jun, CyclinD1andMyc were also inverstigated by western blot analysas.【Results】1. MiR-199a-1was distinctly over-expressed among all the five selectedmiRNAs, indicating that miR-199a-1might serve as a key regulator in HCCcarcinogenesis.2. The results of qRT-PCR verified that the miR-199a-1expression level wassignificantly lower in HCC comapred with mathced non-neoplastic tissues.Further analysis showed the levels of miR-199a-1were significantlycorrelated with the patients’ clinical parameters including TNM stage,metastasis and poor prognosis of patients with HCC.3. Up-regulated miR-199a-1cell line was successfully established in HepG2cells. MTT assays revealed that miR-199a-1could obviously repress HCCcell proliferation. The flat cloning experimental data indicated that miR-199a-1transfected HCC cells had a significantly decrease in theircolony forming ability. Flow cytometry data demonstrated that theproliferation index (PI) of both negative control group and the miR-199a-1hypoexpressd HepG2parental one were60.7and63.9respectively, whichwere dramatically higher than miR-199a-1hyper-expressed HepG2cells,indicating that over-expression of miR-199a-1could obtain a G1arrest.Furthermore, in vivo experiments revealed that the tumor growth withinHCC BALB/c nude mice had sharply slowed down after40days of tumorcell injection. Above all, these data demonstrated that miR-199a-1couldsuccessfully inhibit HCC growth and proliferation in vivo and vitro.4. Bioinformatics predicted that FZD7may be a target for miR-199a-1. Tofurther test the hypothesis, we analyzed the influence of miR-199a-1onFZD7expression in HCC cell line HepG2. The results showed thatover-expression of miR-199a-1could significantly down-regulate expressionof FZD7, and its downstream genes including β-catenin, Jun, CyclinD1andMyc, indicating that miR-199a-1repressed the development of HCC partlythrough inhibiting FZD7passageway.【Conclusions】1. MiR-199a-1expression is significantly down-regulated in HCC, anddecreased expression of miR-199a-1was significantly correlated with themalignant progression of HCC and poor prognosis.2. Up-regulation of MiR-199a-1expression in HCC cells can significantlysuppress HCC growth and proliferation in vitro and in vivo through G1arrest.3. MiR-199a-1suppreses HCC progression partly through inhibiting the FZD7pathway.