MiR-145 Inhibits The Growth And Metastasis By Targeting CDK6 In Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC) is the fifth most common malignancy and the third most frequentcause leading cancer-related death worldwide. Although lots of treatmentsincludedsurgery, chemotherapy, ablation and sometimes radiotherapy are used to HCC patients, the recurrent and metastatic HCC still havea much poorprognosis because of the typical symptoms in advanced stage. The genetic and epigenetic changes resulting in the abnormal proliferation and differentiation is associated with the development of HCC, but the underlying molecular mechanisms about the migration and invasion acknowledged as important lethal attributes for advanced HCC are largely unknown.Micro RNAs(mi RNAs) as a class of 22 nt non-coding RNAs may regulate a large amount of protein-coding genes in animals and plants. It has been confirmed that mi RNAs have an important role in many cancer-related cellular processes involved in cell growth, proliferation, differentiation, migration, invasion and apotosis. Recently, growing evidences showed that mi RNAs are tightly associated with HCC cancer progression, diagnosis, prognosis and response to therapy. An increasing number of mi RNAs were proved to be involved in growth and metastasis of HCC, such as mi R-491, mi R-26 a, mi R-200 a, mi R-222, mi R-126, mi R-106 a, mi R-340 and mi R-218. In previous studies, they found mi R-145 was decreased in HCC, and low expression of mi R-145 has been associated with metastastic potential of HCC and ensuing poor prognosis. Although growing studies focus on the biogenesis and mechanisms of mi R-145 in HCC, the exact mechanisms of mi R-145 dysregulation in HCC are still largely unclear.In the present study, we validated that mi R-145 in HCC tissues were significantly low expression compared with corresponding noncancerous liver tissues and mi R-145 down regulation significantly correlated with positive metastasis in HCC patients.Meanwhile, the univariate analysis showed the overall survival was poorer in HCC patients with low expression of mi R-145. In vitro,mi R-145 overexpression significantly decreased cell proliferation,invasion and migration in HCC cells. Furthermore, our data showed that mi R-145 directly down-regulated CDK6 expression by binding to CDK6-3’-UTR and the expression of CDK6 was negatively correlated with the expression of mi R-145 in HCC tissues. Moreover, mi R-145 inhibited tumor growth of HCC. Our findings indicate that mi R-145 might be a potential biomarker to predict the prognosis of HCC and be targeted for novel therapeutic.ObjectiveThis study aims to investigated the expression of mi R-145 in human HCC and explore the function and mechanism of mi R-145 in tumor invasion and metastasis of HCC. We also analyzed the relationship between mi R-145 target gene CDK6 and HCC clinicopathologic significance.Methods1. We choose four human HCC cell lines(Hep G2, HLE, H2 Pand SMMC) and a normal liver cell line(L02) to detect the expression levels of mi R-145 expression. Furthermore, the expression levels of mi R-145 in the 50 HCC tissues and adjacent matched tissues were also detected by q PCR.2. SMMC and HLE cells were transfected with mi R-145 mimic or mi R-NC by use of Lipofectamine 2000. mi R-NC was a Negative control. The expression levels of mi R-145 were detected by q PCR. The effect of mi R-145 mimic on SMMC and HLE cells proliferation was evaluated by the CCK8 assay. The migration and invasion assays were performed using the Transwell assay.3. Three bioinformatics softwares( mi Rbase, Target Scan) were utilized to predict CDK6 might be the downstream target gene and Luciferase Reporter Assay was used to comfirm the relationship between mi R-145 and CDK6.mi R-145 mimic was transfected into SMMC and HLE cells, the expression level of CDK6 was detected by Real-time PCR and western blot.4. The expression levels of CDK6 in the 50 HCC tissues and adjacent matched tissues were detected by q PCR.The relative expression of CDK6 and mi R-145 in HCC tissues was analyzed.Results1. The expression levels of mi R-145 in four HCC cell lines(Hep G2, HLE, H2 P and SMMC) were significantly lower than human normal liver cells(L-02). Real-time PCR analysis showed the levels of mi R-145 expression in the HCC sections(1.9943±0.8400 were lower than in the adjacent tumors tissue(2.8222 ±0.8052, P<0.001).2. After transfection, we found that mi R-145 expression in SMMC and HLE cells transfected with mi R-145 mmic was significantly increased by q RT-PCR. The results of CCK8 showed that mi R-145 could significantly suppressed the proliferation of SMMC and HLE cells compared with the negative control.3. Over-expression of mi R-145 reduced the invasive capacity of SMMC and HLE cells to 48% and 48% that of the NC cells respectively, Over-expression of mi R-145 and HLE cells also suppressed the migratory ability of SMMC and HLE cells to 67% and 54% that of the NC cells respectively(P<0.01).4. CDK6 is a novel target gene of mi R-145 by the two bioinformatics softwares( mi Rbase, Target Scan) and Luciferase Reporter Assay. The m RNA and protein expression of CDK6 were significantly decreased by mi R-145 in SMMC and HLE cells.5. The average level of CDK6 m RNA in HCC tissues(2.7910±1.6093) was significantly higher than the corresponding normal tissues(1.7082±0.9804, P<0.01). Furthermore, CDK6 m RNA level was negatively correlated with mi R-145 level(r=-0.257,P<0.01).Conclusions1. mi R-145 was significantly decreased in HCC cell lines, Hep G2, HLE, H2 P and SMMC, compared with the human normal liver cells L-02. The expression levels of mi R-145 in the HCC tissues was significantly decreased compared with the paired nontumor tissues. In vitro, mi R-145 over expression significantly decreased cell proliferation,invasion and migrationin SMMC and HLE cells. mi R-145 could be a potential biomarker to predict the prognosis of HCC and be targeted for novel therapeutic in the future.2. CDK6 is a novel target gene of mi R-145 By using the bioinformatics softwares and luciferase reporter assay. mi R-145 can significantly decrease the m RNA and protein expression of CDK6 in SMMC and HLE cells.3. The average level of CDK6 m RNA in HCC tissues was significantly higher than the corresponding normal tissues. Furthermore, CDK6 m RNA level was negatively correlated with mi R-145 level.