| Background and objective:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and a major cause of mortality wordwide:there is a clear ascendant trend in the incidence of HCC and it’s the fifth most common cancer (748000 new cases each year) and the second cause of cancer death (nearly 700000 cases each year). Among all the cases, China alone accounts for more than 50% of the world’s cases. Despite great progress in surgical technique, diagnostic method and new chemotherapy regimens, HCC remain the second cancer killer in our country, threatening people’s health and life.More and more studies suggest that the incidence and development of HCC is a multi-step, gradual process. It plays an important role for the expression regulation disorders of oncogenes and tumor suppressor genes among them. Many studies have shown the process is related to these disorders, for examples, the inactivation of tumor suppressor genes such as TP53, AXIN1/2, APC and PTEN, et al., or the upregulation or mutation of oncogenes such as β-catenin,TGF-β and c-myc et al.Even though, few of these genes are helpful for early diagnosis, and the molecular mechanisms underlying development and progression of HCC remain poorly understood.Thus, studying the molecular mechanism of developing liver cancer, looking for liver cancer diagnosis and treatment of new molecular markers is still an important part of the study of liver cancer. So, further understanding its molecular mechanisms and identifying both early diagnostic markers and novel therapeutic targets are of great clinical value.MicroRNAs (miRNAs) are a class of highly conserved small(18-25bp) noncoding RNA molecules that function as critical regulators of gene expression post-transcriptionally.So far, growing number of miRNAs were found to be of great importance in many physiological processes.Besides, these miRNAs could regulate cell proliferation, differentiation, angiogenesis and apoptosis by targeting the genes involved in the development and progression of human cancers.And these could represent the alteration in biological characteristics.Futhermore, the aberrantly expressed miRNAs could act as molecular biomarker for diagnosis, treatment and prognostic prediction. Therefore, exploring the role of differentially expressed miRNAs in HCC would provide new insight into the molecular mechanisms of HCC.In our study, we at first used RT-PCR to explore the expression level of miR-128a in the HCC tissues. Then we focused on the role and its mechanism of miR-128a in hepatocellular carcinoma to analyze whether miR-128a could serve as a new biomarker for diagnosis, treatment and prognostic prediction of HCC.Methods1.The expression of miR-128a in HCC tissues.We applied qRT-PCR to detect the expression level of miR-128a in 19 cases of HCC tissues and adjacent tissues. Paired-sample t tests were used to compare the difference between the expression level of miR-128a in tumor tissues and adjacent tissues.2. The biological effect and the mechanism during proliferation of miR-128a in HCC cells.The miR-128a inhibitor or mimic was transfected into HCC cells to decrease or increase the expression of miR-128a in HCC cells. The RNA from the cells was isolated by the one-step TRIzol method and then subjected to quantitative Real Time PCR (qRT-PCR) for detecting the miR-128a expression. MTT assay was engineered to detect the effect of miR-128a on cell proliferation.Then, three publicly available algorithms:PicTar, TargetScan Human 6.2 and miRanda were used to predicte and analyz the potential miRNA targets, and the target genes predicted by at least two programs were taken into consideration. As a result, RND3 was selected for Dual-Luciferase report assay, accounting for the fact of both miR-128a and RND3 have effect on cell proliferation. The expression of RND3 and apoptosis related protein was examined in HCC cells with miR-128a overexpression or underexpression by western blot and qRT-PCR.3. The role of miR-128a in HCC.We use qRT-PCR to detect the expression of miR-128a in 62 cases of HCC tissues in the paraffin sections. Kaplan-Meier method was used to analyse the influence of miR-128a on prognosis of HCC. The relationship between the miR-128a expression and clinical pathological parameters was analysed by Spearman rank correlation method. And we use COX proportional hazards regression model to do univarible and multivariable factor analysis on the relationship between the clinical pathological parameters and the prognosis of primary HCC to make clear miR-128a whether can be an independent risk factor for the prognosis of HCC.Results1. The expression of miR-128a in HCC.QRT-PCR was used to detect the miR-128a expression in HCC tissues and adjacent tissues in 19 patients. The result have shown that miR-128a was overexpressed in HCC tissues comparing in adjacent tissues,and there were significant difference about the expression of miR-128a between tumor and normal tissue samples (P<0.05).2. The biological role of miR-128a in HCC cells and the mechanism of proliferation of HCC.By transfecting HCC cells with inhibitor or mimic of miR-128a,the results determined by qRT-PCR showed that miR-128a expression is succesfully decreased or incresed in the cells correspondingly(P<0.01). And by MTT, we found that cells transfected with the miR-128a inhibitor had lower levels of cell proliferation than the NC cells,while cells transfected with the miR-128a mimic had higher levels of cell proliferation than the NC cells (P<0.001).Then,TargetScan Human 6.2, miRanda and Pic TarThe were used for target gene prediction of miR-128a. As a result, RND3 was predicted to be a potential target of miR-128a by computer-based sequence analysis by both TargetScan Human 6.2 and miRanda. Through a luciferase reporter assay that could verify the direct interaction betweenmicroRNA and its target gene,we found that overexpressing miR-128a led to a reduction of luciferase activity when the reporter construct contained the RND3 3’UTR (P<0.01). In contrast, mutation of the conserved miR-128a binding motif abrogated the reduced luciferase expression. Besides, overexpression of miR-128a in HCC cells led to reduced RND3 mRNA and protein expression. Consistently, inhibition of miR-128a led to an increased activity of luciferasre and expression of RND3 contents (P<0.01), further indicating that RND3 is a target of miR-128a in HCC.Futhermore, transfection with miR-128a inhibitor reduced the expression of apoptosis related protein of cyclin D1, cyclin B1 and CDK4 in HCC cells. These results suggested that miR-128a can promote HCC cells proliferation likely through inhibiting cell-cycle progression due to targeting RND3.3. Analysis of miR-128a expression in liver cancer.The miR-128a expression in 62 cases of HCC paraffin specimen was tested by qRT-PCR. Spearman rank correlation was used to analyze the relationship between the expression of mir-128a and HCC clinicopathological parameters. And we found that the expression was only significantly relate to AST (P=0.018).However, there was no significant relationship between the miR-128a expression and the number of the tumors, BCLC staging, portal vein tumor thrombus, distant metastasis, age, gender, pathological grade, tumor size, recurrence.We performed the Kaplan-Meier to analyze miR-128a and HCC prognosis, but the result was found that there was no significant relationship between the miR-128a expression and HCC prognosis disregarding the status of recurrence. By using COX proportional hazards regression model single factor and multi-factor to analyze the relationship between various clinicopathological parameters and HCC prognosis, it was only found that BCLC staging and AST were both the risk factors of HCC prognosis.Conclusions1. miR-128a was upregulated in HCC and it can act as a valuable molecular diagnostic marker.2. miR-128a could promote the proliferation of HCC by targeting RND3.3. Though miR-128a was upregulated in HCC, there was no significant relationship between the miR-128a expression and HCC prognosis and clinicopathological parameters until now. |
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