Ectopic Expression Of MiR-125a Inhibits The Proliferation And Metastasis Of Hepatocellular Carcinoma By Targeting MMP11and VEGF

【Background】Hepatocellular carcinoma (HCC) is a primary cancer in the liver. It is the third leadingcause of cancer death in man worldwide. Different from other tumors, the incidence andmobility rate of HCC are still increasing in many countries. Because of the lack ofunderstanding of liver carcinogenesis and the failure to develop interventions that aretargeted at blocking or reversing the steps of malignant transformation, the prognosis ofHCC patients remain poor. Although, global human gene expression profile studies haverevealed the activation of several pathways that promote uncontrolled proliferation and aberrant survival signaling. Several key pathways have been identified. Recently, sorafenib,the mixed kinase inhibitor, has been approved for treatment in HCC. However, there are nonovel gene markers or molecular prognostic factors which have been used for clinicalpractice, emphasizing the need for new markers for diagnosis and prognosis of HCCpatients. MicroRNAs are small non-coding RNAs of17-25nucleotides in eukaryotic cells.It has been reported that miRNAs play an important role in carcinogenesis through negativeregulating the tumor related genes. Some experts even considered that miRNAs can becomea better new diagnostic and therapeutic target than genes. The results of human miRNAmicroarrays from two different labs have indicated that HCC tissues exhibit lowerexpression of miR-125a. However, the mechanisms of how miR-125a involving in HCCcarcinogenesis still remain unknown.【Aims】1. We examined the expression level of miR-125a in HCC tissues and the adjacentnon-tumor tissues. And the difference between miR-125a expression level and patients‘clinical parameters and prognosis were analyzed.2. The functional role of miR-125a involved in HCC carcinogenesis was studied.3. The mechanisms miR-125a involved in regulating HCC proliferation and metastasis.【Methods】1. Quality real-time PCR was employed to examine the expression level of miR-125a inHCC tissues and adjacent non-tumor tissues. We further analyzed the relationship betweenmiR-125a expression level and HCC patients‘clinical parameters.2. Lentivirus-mediated miR-125a was constructed. And HCC cell lines were infected withlenti-miR125a or lenti-control virus to obtain the stable expression cell lines. Toinvestigate the effects of miR-125a on the proliferation of HCC, MTT, soft agar and invivo tumorigenicity experiments were used.3. Transwell, migration and tail vein injection of nude mice were used to examine theeffects of miR-125a on the migration of HCC cells. 4. Inhibitor of miR-125a was used to examine the effects of downregulate endougnenesmiR-125a on HCC cells. MTT and Trasnwell experiments were employed to test the effectof it on HCC proliferation and migration.5. To explore the mechanism underlying miR-125a involvement in the progression andmetastasis of HCC, miRanda and Pic Tar algorithms were used to search for the targetgenes. Predicted target genes of miR-125a were determined by dual-luciferase reportingassay, qRT-PCR, and western blot analyses.6. IHC was used to examine the target genes‘expression in HCC patients. Therelationship of miR-125a expression level and its target genes in HCC was furtheranalyzed.【Results】1. We demonstrated that miR-125a expression was decreased in77.5%(62/80) of HCCtissues compared with matched adjacent liver tissues, with an average of4.72-foldreduction in expression, and that lower expression of miR-125a was significantlyassociated with the progression and poor prognosis of patients with HCC.2. Infected by lenti-miR-125a or lenti-miR-control, HCC cell lines can highly expressmiR-125a than control cells. MTT assays showed that HCC cells with miR-125aoverexpressed exhibited much slower growth than their corresponding controls afterculture for four days. The colony formation assay results showed that ectopic expressionof miR-125a markedly decreased the colony numbers of control cells.By subcutaneous injection in nude mice, tumors appeared much smaller at the sitesinjected with overexpressed miR-125a cells compared with each control.3. Overexpression of miR-125a significantly inhibited the migratory and invasive abilitiesof HCC cells in vitro. Consistent with the in vitro observations, the animal experimentresults showed that liver and lung metastases were apparent in mice injected withHCC-control cells, but few were observed in mice injected with miR-125a overexpressedcells. 4. Downregulated miR-125a expression can promote HCC cell proliferation andmetastasis by MTT and Transwell assay.5. Luciferase assays showed that the3‘UTRs of both MMP11and VEGF, but not others,causing a significant reduction in the luciferase activity. This result indicated that MMP11and VEGF could be miR-125a‘s target genes. To further testify this hypothesis, qRT-PCRresults showed that miR-125a could downregulate MMP11and VEGF mRNA expressionlevel. These findings were further verified by the results of western blot analyses.6. To further study whether miR-125a was correlated with MMP11and VEGF expressionin clinical HCC tissues, IHC was performed to detect MMP11and VEGF expression inHCC samples from the80patients. Spearman‘s rank test showed that a significantnegative correlation was found between miR-125a and MMP11and VEGF proteinexpression.【Conclusion】1. Decreased miR-125a was observed in HCC tissues, which was associated with patients‘aggressive pathologic features.2. Ectopic expression of miR-125a can significantly inhibit HCC cell proliferation andmetastasis in vitro and in vivo.3. Down-regulating miR-125a could promote HCC cell growth and migration.4. Up-regulating miR-125a significantly inhibited the malignant phenotypes of HCC byrepressing the expression of MMP11and VEGF.5. miR-125a expression was inversely correlated with both MMP11and VEGF expressionin HCC tissues and the expression level of miR-125a and its target genes associated withprognosis of HCC patients.