| Hepatocellular carcinoma(HCC)is the fourth most common malignancy in China and it is the second leading cause of death in humans.Patients with HCC are usually diagnosed with advanced disease,and most patients lose the chance of surgery due to the lack of early diagnostic markers and nonspecific clinical symptoms in early stage.Therefore,it is urgent to study the underlying mechanism of the development and invasion of HCC to explore effective prevention and treatment strategis for patients with HCC.The differentiation,development and the growth of cell is dependent on the dynamic balance of cell proliferation and apoptosis.A variety of disease even tumor will appear once the balance broken.Numerous studies have identified that the process of cell proliferation is directly related to the development,invasion and metastasis of tumor.MiRNAs are highly conserved small RNA molecules of 19-25 nucleotides in length that could bind the partial sequence homology or splitting to regulate the expression of target genes,which play a vital role in the development,proliferation,differentiation and apoptosis of cells.A large number of new miRNAs have been identified,and the number of identified human mature miRNA has reached more than 2500,which can regulate over one third of the genes in human.HCC can be caused by the unbalanced regulation of gene transcriptional and abnormal proliferation of local tissues.The development of HCC is a complex process involved in the disordered expression of multiple oncogenes and anti-onco genes.MiRNAs can reduce the activity of anti-oncogenes and further prompt cells malignant transformation by regulating cell differentiation and apoptosis in the development of HCC.It can also be used as an anti-oncogene to reduce the activity of the original oncogene,invasion and metastasis genes,thereby inhibiting the development and metastasis of HCC.In addition,miRNAs widely participate in various cellular activities by regulating gene expression levels,such as proliferation,differentiation,apoptosis,and cell skeleton rearrangement.These findings indicated that miRNAs play an important role in the pathophysiological processes of malignant transformation,invasion and metastasis of liver cells.It has been extensively confirmed that miRNAs are involved in a series of important cell processes,including early development,proliferation,differentiation,apoptosis,death and fat metabolism.Emerging evidences identified that miRNAs plays an important role in various pathophysiological processes including abnormal transformation,invasion and migration of human normal hepatocytes.Recent research has found that half of them are located in cancer-associated sites,indicating that miRNAs may be implicated with the development of tumors.Indeed,multiple miRNAs have been reported to be closely associated with the tumor development,pathologic classification,clinical stage,the drug resistance and prognosis of cancer patients.Several miRNAs can directly participate in the development of tumor(including prostate cancer,pancreatic cancer,lung cancer,colon cancer,etc).Therefore,miRNAs can serve as both oncogenes and anti-oncogenes.Accumulating evidence verified the abnormal expression of some specific miRNAs in HCC,indicating that miRNAs may play an important role in the development of HCC.Recent studies have shown that multiple miRNAs are highly expressed in hepatocellular carcinoma,including mir-181,mir-21,mir-324,mir-186,mir-155 and so on.In addition,some miRNAs were poorly expressed in hepatocellular carcinoma,which were: mir-139,mir-198,mir-199a-3p,mir-199a-5p,mir-195,mir-34 a.Microrna-198 is an important molecule of the miRNAs family.Some data showed that mir-198 was low or not expressed in prostate cancer,colon cancer,pancreatic cancer,lung cancer and other tumors.However,the situation in liver cancer is not clear.Therefore,our research team speculated that mir-198 may have an important influence on the induction and progression of HCC.We preliminarily predicated that miR-198 may play a role in the development of HCC.Therefore,we detected the expression of miR-198 in patients with HCC and HCC cell by RT-PCR.The results demonstrated that the expression level of miR-198 was lower in HCC ell lines and tissues,suggesting that miR-198 played a role in the development of HCC.In this study,we detected the expression of miR-198 was detected in different HCC cell lines.We also analyzed the expression level of miR-198 in the HCC tissues and adjacent normal tissues and the relationship between the expression level of miR-198 and the survival time of HCC patients.The effects of miR-198 on the proliferation and migration of Huh7 cell lines were also detected.The predicted target gene ZEB2 was identified by RT-PCR and Western Blot.The content is divided into three parts.Part One Expression of miR-198 in different hepatocellular carcinoma cell lines and tissues and its relationship with survival of hepatocellular carcinoma Method1 Firstly,real-time PCR was used to determine miR-198 in different cell lines and tissues of hepatocellular carcinoma.2 The correlation between the expression level of miR-198 and the clinical data and prognosis of hepatocellular carcinoma patients was analyzed.Results1 Compared with normal liver cells,miR-198 was down-regulated in HepG2,Huh7,Hep3 B,and MHCC97 H,with the lowest expression in Huh7 cells.2 The expression levels of miR-198 were analyzed in 65 patients with liver cancer tissue samples.The results showed that the relationship between miR-198 expression levels and the tumor differentiation degree(high,medium and low),TNM stage(I II,III IV),the presence of vascular tumor emboli and presence of blood vessels has no significance,while the relationship between miR-198 expression levels and with or without coated invaded and presence of distant metastasis has obvious differences.And compared with the corresponding tissue adjacent to carcinoma,miR-198 expression level is closely related to liver cancer patients survival time length,there was a positive correlation.SummaryMiR-198 was down-regulated in HepG2,Huh7,Hep3 B and MHCC97 H cells as well as in HCC tissues.The expression levels of miR-198 were closely associated with the clinical parameters and positively correlated to the survival time of patients with HCC.These results indicated that miR198 serves a negative regulator in HCC patients,and the expression levels of miR198 were positively related to the survial time in patients with HCC.Part Two The biologic function of miR-198 in Huh7 Methods1 Transfected Huh7 cells with miR-198 minics in vitro.2 Flow cytometry was used to detect the effect of miR-198 on the apoptosis of Huh7 cells.3 CCK8 and soft AGAR cloning were used to detect the effect of miR-198 on the proliferation of Huh7 cells.4 Transwell and Wound Healing assay were used to detect the migration of Huh7 cells after miR-198 mimics treatment.5 In vivo subcutaneous tumorigenesis experiment in nude mice was used to test the effect of miR-198 on Huh7 cells.Results1 The flow cytometry results showed that miR-198 can increase the apoptosis of Huh7 cells.2 Compared with the control group,the proliferation ability of Huh7 cells was significantly lower in miR-198 mimics group.3 The Transwell assay results showed that miR-198 could inhibit the migration of Huh7 cells.4 Compared with the control group,tumorigenic ability in nude mice was significantly inhibited in miR-198 mimics group.SummaryThe results of this part showed that miR-198 could inhibit the proliferation and increase the apoptosis of Huh7 cells.Moreover,miR-198 could inhibit the migration and tumorigenicity of Huh7 cells.These results suggested that miR-198 played an important role in the occurrence and development of HCC.Part Three The miR-198 target gene prediction and preliminaryfunctional identification Methods1 The target genes of miR-198 were predicted by bioinformatics method.2 Dual luciferase positioning was used to verify the target gene.3 The expression of ZEB2 in HCC was detected by immunohistochemistry.Western Blot with RT-PCR were performed the expression of ZEB2 in NC,miR-198 mimic and si-ZEB2 group.4 Slience the expression of ZEB2 by RNA interference,and then the migration ability of Huh7 cells was detected by Transwell assay.Results1 Bioinformatics results showed that ZEB2 was the target gene of miR-198.2 Luciferase assay showed that ZEB2 3'UTR-WT together with miR-198,but not other groups,caused a notable reduction in the luciferase activity,indicating that ZEB2 could be the target gene of miR-198.3 Transwell assay results showed that the migration ability of Huh7 cells was significantly reduced in miR-198 mimic and si-ZEB2 groups.4 Transwell assay results the migration ability of Huh7 cells was significantly reduced in miR-198 mimic and Si-ZEB2 group.5 The expression of MiR-198 and ZEB2 showed a negative correlation in HCC tissues.SummaryThe results of this part showed that ZEB2 is target gene of miR-198.MiR-198 might inhibit the proliferation and migration ability of Huh7 cells through regulating ZEB2 expression.MiR-198 might inhibit the migration ability of Huh7 cells through inhibiting the expression of ZEB2.ConclusionThe results of this study demonstrated that miR-198 was down-regulated in HCC cells.MiR-198 could negatively regulate the proliferation and migration ability of HCC cells,and its low expression was closely related to the occurrence and development of HCC.Moreover,miR-198 could inhibit the expression of its target gene,ZEB2,thus limiting the development of HCC.MiR-198 could be used as a new molecular biomarker and therapeutic target for HCC. |
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