Molecular Mechanism And Clinical Significance Of MiR-483-5p In Recurrence Of Hepatocellular Carcinoma

Part I.Differentially expressed mi RNA in different interval recurrent hepatocellular carcinomaOBJECTIVE: To screen differentially expressed mi RNA in hepatocellular carcinoma tissues with different recurrence interval.PATIENTS AND METHODS: 38 cases of recurrent hepatocellular carcinoma underwent resection twice in the hospital were collected,microsatellite LOH was detected in the primary-recurrence tissue samples to determine the clonal origin of recurrent hepatocellular carcinoma.The cases was divided into short-term recurrence group(?2 years)and long-term recurrence group(>2 years)according to the recurrence interval.mi RNA array was used to screen differentially expressed mi RNA between two groups,and fluorescent quantitative PCR method was used to verify the expression level of candidate mi RNA.RESULTS: 12 pairs of recurrent HCC were polyclonal and 26 pairs were monoclonal were detected with microsatellite LOH.There were significant differences between the two types of recurrent hepatocellular carcinoma in recurrence time,hepatitis,the size of tumor,the degree of tumor differentiation,and vascular invasion.Monoclonal RHCC was used to screen the differentially expressed mi RNA,5 mi RNAs(hsa-mi R-133 a,hsa-mi R-4251,hsa-mi R-4279,hsa-mi R-483-5p,hsa-mi R-642b-3p)were found between short-term and long-term recurrence groups.The most significant difference mi RNA(mi R-483-5p)was confirmed by fluorescence quantitative PCR.CONCLUSIONS: Monoclonal RHCC has higher invasiveness and worse prognosis.There is differentially expressed mi RNAs between short-term and long-term recurrence groups,and among them,mi R-483-5p expression was the most significant,which may have important biological effects.Part II.Biological function of mi R-483-5p in hepatocellular carcinomaOBJECTIVE: To clear the biological function of mi R-483-5p in hepatocellular carcinoma by cytology and animal experiments.PATIENTS AND METHODS: mi R-483-5p mimics or inhibitor were transfected with into HCC cell lines to simulate the state of over expression or knockdown of mi R-483-5p in HCC cells.To detect the migration and invasion of HCC cells by Transwell,and the proliferation of HCC cells by CCK8.Establishment of stable cell line expressing mi R-483-5p by infecting HCC cells by lentivirus,and detect tumor cell invasion and metastasis by orthotopic implantation in nude mice.RESULTS: The expression level of mi R-483-5p inmimics transient and lentivirus infected HCC cells was significantly increased.mi R-483-5p mimics can significantly promote the migration and invasion of HCC cells,and mi R-483-5p inhibitor can significantly reduce the migration and invasion of HCC cells in vitro.mi R-483-5p mimics and inhibitor had no significant effect on the proliferation of hepatoma cells.The HCC cells over-expressing of mi R-483-5p increased the number of liver metastasis and distant metastasis significantly in nude mice.CONCLUSIONS: mi R-483-5p can promote the migration and invasion of HCC cells in vitro,and the liver and distant metastasis of HCC cells in vivo.Mi R-483-5p is an important molecule in metastasis and recurrence of hepatocellular carcinoma.Part III.Identification,mechanism and clinical significance of downstream target genes of mi R-483-5pObjective: To seek and identify the downstream target genes of mi R-483-5p,and to clarify the mechanism and clinical significance.Methods: Downstream target genes of mi R-483-5p were predicted with bioinformatics software,target gene expressed in reverse with mi R-483-5p was detected with fluorescent quantitative PCR,determined the binding sites between mi R-483-5p and target genes with luciferase reporter vector assay,the relationship between mi R-483-5p and target genes was determined by functional recovery assay,and the relationship between the target gene and the clinical pathological parameters and the influence on the prognosis were detected by immunohistochemistry.Results: Target Scan software selected 55 genes with potential binding sites for mi R-483-5p,and activated leukocyte cell adhesion molecule(ALCAM)was identified as the target gene of mi R-483-5p by fluorescent quantitative PCR.Luciferase reporter vector experiments further confirmed that ALCAM and mi R-483-5p had a binding site,The functional recovery experiment showed that ALCAM was able to restore the migration and invasion of mi R-483-5p after it was introduced.Clinical analysis showed that the expression of ALCAM was significantly correlated with liver cirrhosis,tumor size and vascular invasion,the expression of ALCAM was related to the prognosis of patients,and was an independent risk factor for postoperative recurrence.Conclusion: As the downstream target gene of mi R-483-5p,ALCAM plays an important role in the migration and invasion of HCC cells,the expression is also correlated with clinicopathological parameters and TTR,and is great significance in clinical diagnosis,treatment and prognosis.