Molecularmechannismof CK19 Involved In The Regulation Of Postoperative Recurrence Of HBV-associated Primary Hepatocellular Carcinomain Guangxi

Research content:The study was divided into three parts:1.Correlation analysis of relative expression of CK19 and clinicopathological features and clinical prognosis in primary tumors and recurrent tumor tissues of patients with primary hepatocellular carcinoma associated with HBV in Guangxi;2.Small RNA interference Technique,after silencing CK19 gene,the effects of CK19 on the biological behavior of liver cancer cell lines MHCC-97H and Hep-3B were analyzed.3.High-throughput sequencing technology was used to analyze the differential RNA expression(mRNA/miRNA/lncRNA/circRNA)of CK19 gene affecting the biological behavior of Hep-3B cells and construct a competitive endogenous RNA(ceRNA)network regulation,from the transcriptomics level to explore the potential molecular mechanism of CK19 positive expression of HCC with higher malignant biological behavior.PART1 CORRELATION BETWEEN CK19-POSITIVE EXPRESSION AND CLINICOPATHOLOGICAL FEATURES AND CLINICAL PROGNOSIS IN PATIENTS WITH PRIMARY HEPATOCELLULAR CARCINOMA ASSOCIATED WITH HBV IN GUANGXIObjective:Comparative analysis was made on the relative expression and the correlation between clinical pathological characteristics and clinical prognosis.of cytokeratin 19(CK19/KRT19)between primary and recurrent HBV-associated Hepatocellular Carcinoma(HCC)in Guangxi.Materials and Methods:A total of 24 cases of recurrent HBV-associated primary hepatocellular carcinoma with hepatic tumor resection at the First Affiliated Hospital of Guangxi Medical University from January 2002 to December 2017 were collected.Tumor and recurrent tumor tissues were stained with CK19 immunohistochemical staining,and the relationship between clinicopathological factors and CK19 protein expression in primary tumors and recurrent tumors of 24 patients with HCC and the prognosis of HCC patients were analyzed.Results:In 24 patients with HCC,regardless of primary tumor or recurrent tumor,CK19 was only expressed in cancer tissues,and no expression of CK19 was detected in adjacent tissues.The proportion of CK19 protein positive cells in HCC recurrent tumor tissues was significantly higher than that in primary tumors(X2=8.545,P=0.003),and the degree of differentiation of recurrent tumors was significantly different from that of primary tumors(X2=4.128,P=0.047),suggesting that the proportion of CK 19-positive cells in HCC recurrent tumors is higher,and the degree of differentiation is more obvious than that of primary tumors;HCC cases with positive HCC primary tumor CK19 expression have shorter tumor-free survival time(P=0.05).Conclusion:In the specific subtype of HBV-associated HCC CK19 positive expression in Guangxi,the proportion of CK 19-positive cells in the primary tumor was higher when the tumor recurred,and the tumor-free survival time of HCC cases with positive CK19 expression was shorter.It is suggested that the positive expression of CK 19 may be a risk factor for tumor recurrence.PART 2 EFFECT OF CK19 ON BIOLOGICAL BEHAVIOR OF HEPATOCELLULAR CARCINOMA CELL LINES MHCC-97H AND HEP-3BObjective:To investigate the effects of siRNA silencing CK19 gene on proliferation,migration,invasion,cell cycle and apoptosis of hepatoma cell line MHCC-97H and Hep-3B.Materials and Methods:The siRNA-CK19 fragment was transfected into human hepatoma cell line MHCC-97H and Hep-3B to silence the CK19 gene,and then Real-time PCR,Western Blot,cellular immunohistochemistry,flow cytometry,CCK8,scratch test,Transwell chamber assay was used to detect the effects of silencing CK19 on proliferation,migration,invasion,cell cycle and apoptosis of hepatoma cell lines.Results:After transfected liver cancer cell lines MHCC-97H and Hep-3B were detected by Real-time PCR and Western Blot,we found that the silencing effect of si-RNA3 against CK19 gene was the best.In the hepatoma cell lines MHCC-97H and Hep-3B transfected with si-RNA3,the expression of CK19 gene and protein was decreased after silencing CK19 gene,and its vertical,horizontal migration and invasion in hepatoma cell line MHCC-97H.The ability was weakened,and the percentage of cells in the G2 phase of liver cancer cell lines MHCC-97H and Hep-3B increased,and the percentage of apoptosis increased significantly.Conclusion:siRNA targeting silencing of CK19 gene in hepatocellular carcinoma cell lines MHCC-97H and Hep-3B significantly reduced the expression of CK19 gene and protein,and increased the cell content and percentage of apoptosis in G2 phase,suggesting that HCC after silencing CK19 gene The migration and invasion ability of the cells were significantly weakened,and the cell cycle arrest of the hepatoma cell lines MHCC-97H and Hep-3 B was induced in the G2-M phase to inhibit the proliferation of HCC.PART 3 CONSTRUCTION OF COMPETITIVE ENDOGENOUS RNA REGULATORY NETWORK OF CK19 POSTIVE HEPATOMA CELL LINE HEP-3B BASED ON TRANSCRIPTOME SEQUENCINGObjective:High-throughput sequencing technology was used to analyze the differential RNA expression(mRNA/miRNA/lncRNA/circRNA)of CK19 gene affecting the biological behavior of Hep-3B cells and construct competitive endogenous RNA(ceRNA)network regulation.From the transcriptomics level,the potential molecular mechanism of CK 19-positive HCC with higher malignant biological behavior was explored.Methods:In this study,CK19 gene expression in hepatocellular carcinoma cell line Hep-3B was silenced by transfection of siRNA,and whole transcriptome sequencing was performed using Illumina HiSeq 2500 platform.And use the edgeR,R package clusterProfiler and other software to carry out related bioinformatics function annotation and enrichment analysis.Results:Each sample was compared to a reference genomic sequence,as well as lncRNA,circRNA,miRNA,and gene prediction.A total of 20066 lncRNAs were identified,of which 14704 were new lncRNAs;3704 miRNAs,of which 1806 were known miRNAs,1898 were new miRNAs;25,846 genes,of which 6,199 were new genes;and 1,668 new were predicted CircRNA.Quantification of each RNA and differential screening analysis revealed 1099 lncRNAs(641 up-regulated,498 down-regulated),10 circRNAs(4 up-regulated,6 down-regulated),281 miRNAs(178 up-regulated,103 Down-regulated)and 779 mRNAs(393 up-regulated,386 down-regulated),and functionalized annotation and enrichment analysis of differentially expressed mRNA,RNRNA-derived genes,lncRNA target genes,and miRNA target genes were constructed based on ceRNA The network differs in the relationship between lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA regulation.Conclusion:We performed transcriptome sequencing analysis of CK19-silencing hepatoma cell lines and found that potential CK19 regulates differentially expressed genes related to proliferation,cell cycle and apoptosis of hepatocellular carcinoma cell lines,and constructed corresponding ceRNAs to elucidate the influence of CK19 on HBV-associatedogens.The molecular mechanisms involved in postoperative recurrence of hepatocellular carcinoma provide direction and strategy.