Study On CircRNA As The Molecular Marker Of Liver Cancer

Part1 Expression level of four kinds of circRNA in hepatic tissues and diagnostic value in liver cancerObjective: To explore the expression of hsa＿circRNA＿404974,hsa＿circRNA＿001387,hsa＿circRNA＿100246 and hsa＿circRNA＿091581 in cancer tissues and adjacent tissues of patients with liver cancer.To analyze the relationship between the expression of circRNA in liver cancer tissues and the basic clinical information of patients with liver cancer.To explore which circRNA can be used as a molecular marker of liver cancer.Methods:1.The expression of hsa＿circRNA＿404974,hsa＿circRNA＿001387,hsa＿circRNA＿100246 and hsa＿circRNA＿091581 in 55 pairs of cancer tissues and their adjacent tissues were analyzed by q RT-PCR.2.To analyze the relationship between the expression circRNA in cancer tissues and the clinical features of patients.3.ROC curve was used to analyze which circRNA can be used as a molecular diagnostic marker of liver cancer.4.SPSS21.0 software was used for statistical analysis of the data in this paper.The expression of circRNA in cancer tissues and matched adjacent tissues of the patients with liver cancer were analyzed by Wilcoxon signed rank test.The relationship between the expression level of circRNA in the cancer tissues and the clinical data were analyzed by Student’s t test or Wilcoxon rank test.The diagnosis test was analyzed by ROC curve.Results: 1.The expression of hsa＿circRNA＿404974 in hepatic tissues was lower than its adjacent tissues,the difference was statistically significant(P<0.001).The expression of hsa＿circRNA＿001387 in hepatic tissues was higher than its matched adjacent tissues,the difference was statistically significant(P<0.001).The expression level of hsa＿circRNA＿100246 in hepatic tissues was lower than its adjacent tissues,the difference was statistically significant(P<0.001).The expression level of hsa＿circRNA＿091581 in hepatic tissues and adjacent tissues had no significant difference(P>0.05).2.The analysis of the expression amount and clinical information of patients with cancer showed that,the expression of hsa＿circRNA＿404974 in cancer tissues had significant differences with the number of tumors(P<0.05)and star lesions of tumor adjacent(P<0.05);the expression of hsa＿circRNA＿001387 in cancer tissues had a significant difference with AFP level(P<0.05);the expression of hsa＿circRNA＿100246 in cancer tissue also had a significant difference with AFP level(P<0.05).3.ROC curve was drawn according to the expression of circRNA in hepatic tissues.The results of hsa＿circRNA＿404974 showed that the area under the curve was 0.896,P<0.001,95% confidence interval was(0.828,0.964),the accuracy was 87.27%.The results of hsa＿circRNA＿001387 showed that the area under the curve was 0.884,P<0.001,95% confidence interval was(0.816,0.952),the accuracy was 85.45%.The results of hsa＿circRNA＿100246 showed that the area under the curve was 0.714,P<0.001,95% confidence interval was(0.619,0.809),the accuracy was 68.18%.The results of combined diagnosis showed that the area under the curve was 0.953,P<0.001,95% confidence interval was(0.912,0.994),sensitivity was 90.91%,specificity was 90.91%.Conclusion:1.Hsa＿circRNA＿404974 was markedly downregulated in HCC tissues,the low expression of hsa＿circRNA＿404974 in liver cancer is related to the number of tumor and its adjacent cancer focus,may be involved in the development of liver cancer.Hsa＿circRNA＿404974 as a molecular marker of liver cancer has a good diagnostic value.2.Hsa＿circRNA＿001387 was markedly upregulated in HCC tissues,the high expression of hsa＿circRNA＿001387 in liver cancer is related to AFP level,may be involved in the development of liver cancer.Hsa＿circRNA＿001387 as a molecular marker of liver cancer has a good diagnostic value.3.Hsa＿circRNA＿100246 was markedly downregulated in HCC tissues,the expression of hsa＿circRNA＿100246 in liver cancer is related to AFP level,may be involved in the development of liver cancer.Hsa＿circRNA＿001387 as a molecular marker of liver cancer has an ordinary diagnostic value.4.The expression level of hsa＿circRNA＿091581 in the cancer tissues and its adjacent tissues had no statistical significance,which may not be involved in the occurrence and development of HCC.Part2 Effect of stable overexpression of hsa＿circRNA＿404974 on the function of HCC cell linesObjective: To construct stable overexpression cell lines of hsa＿circRNA＿404974 and explore its effect on the proliferation,migration and invasion of HCC cell lines.Methods:1.Applying monoclonal cell culture technology to construct stable overexpression HCC cell lines of hsa＿circRNA＿404974.2.The CCK8 experiment was carried out to analyze the influence of hsa＿circRNA＿404974 overexpression on the cell proliferation ability of HCC cell lines.3.Clone formation assay was carried out to analyze the influence of hsa＿circRNA＿404974 overexpression on the cell proliferation capacity of HCC cell lines.4.Transwell migration experiment was carried out to analyze the migration effect of hsa＿circRNA＿404974 overexpression on HCC cell lines.5.Transwell invasion experiment was carried out to analyze the invasion effect hsa＿circRNA＿404974 overexpression on HCC cell lines.6.The data were analyzed by spss21.0 software,the measurement data were analyzed by Student’s t test or rank sum test.Results:1.Two clones of hsa＿circRNA＿404974 overexpression and one negative control were screened from QGY-7703 and Huh-7 cell lines,named clone 3,clone 15 and nc of QGY-7703 cell line;clone 5,clone 6 and nc of Huh-7 cell line.Six cell clones were used for further functional experimental study.2.The results of CCK8 assay showed that in QGY-7703,there was no significant difference between nc group and clone3 group,between nc group and clone15 group at 48 hours and 72 hours;in Huh-7,there was no significant difference between nc group and clone5 group,between nc group and clone6 group at 0 hours,24 hours,48 hours and 72 hours.3.The results of cloning formation assay showed that in QGY-7703,between nc and clone3,between nc and clone15,there were no significant difference;in Huh-7,between nc and clone5,between nc and clone6,there were no significant difference.4.The results of Transwell migration showed that in QGY-7703,compared with nc,the number of through the basement membrane in clone3 was less than that in nc,with a significant difference;compared with nc,the number of through the basement membrane in clone15 was less than that in nc;in Huh-7,compared with nc,the number of through the basement membrane in clone5 was less than that in nc,with a significant difference;compared with nc,the number of through the basement membrane in clone6 was less than that in nc.5.The Transwell invasion assay showed that in the QGY-7703,the number of clone3 was less than that in the nc,and that in the clone15 was less than that in the nc;in Huh-7,the number of clone5 was less than that in the nc,and that in the clone6 was less than that in the nc.Conclusion:1.Two over expressed HCC cells were successfully constructed by drug screening.2.The overexpression of hsa＿circRNA＿404974 can inhibit cell migration and invasion,and indicate hsa＿circRNA＿404974 may function in metastasis of HCC.