The Regulation Of CK7 And CK19 Expression And Its Biology Implications In Hepatitis Associated Hepatocellular Carcinoma In Guangxi Population

Research content: This study is divided into four parts: 1.Clinical pathological features and prognostic analysis of CK7 and CK19 under different expression in patients with hepatitis surface antigen positive primary hepatocellular carcinoma.2.Based on the genome-wide expression profile data of hepatocellular carcinoma from the dataset of GSE14520 in the GEO database,clinical pathological features,prognosis,expression regulation and differential gene analysis of CK7 and CK19.3.According to the expression of CK7 and CK19 immunohistochemistry,the genetic variation of CK7 and CK19 was analyzed based on the whole exon analysis strategy.4.After the silencing of CK7 and CK19 genes in hepatoma cell lines by siRNA,the biological behavior of hepatoma cells was analyzed.PART 1:CLINICOPATHOLOGICAL FEATURES AND PROGNOSIS ANALYSIS OF CK7 AND CK19 EXPRESSION IN HEPATITIS B ASSOCIATED HEPATOCELLULAR CARCINOMA IN GUANGXIObjective: To investigate the clinicopathological features and prognostic analysis of cytokeratin 7,CK7/KRT7 and cytokeratin 19(CK19/KRT19)in hepatitis B associated hepatocellular carcinoma.Materials and Methods: A total of 394 cases of hepatic tumor resection were performed in the Department of Hepatobiliary Surgery,the First Affiliated Hospital of Guangxi Medical University from 2001 to 2013.CK7/CK19 immunohistochemical staining was performed on the cancer tissues.The effects of clinical factors on their expression were analyzed by total 394 cases,CK7expression(positive-negative 1:3 match),CK19 expression(positive-negative1:3 match),and CK7/CK19 expression(positive-positive 1:3 match).The combination of CK7 and CK19 expression and follow-up data was used to analyze the effect of CK7 and CK19 on the prognosis in the above 4 groups.RESULTS: Immunohistochemical staining of CK7 in 394 cases of hepatocellular carcinoma was positive in 43 cases and positive in CK19 in 70 cases.In the total 394 cases,the expression of CK7 and CK19 was correlated with the level of AFP(?2=13.971,P<0.001;?2=7.608,P=0.006).The recurrencerate of CK7 positive expression within 2 years was higher than that of negative expression(HR=2.014,95% CI=1.110-3.657),CK7/CK19 double positive expression had worse 2-year,5-year and long-term survival and tumor-free survival compared with double negative expression.In propensity score matching(1:3)analysis of CK7 expression,CK7 expression was correlated with AFP levels(?2=11.251,P = 0.001),and CK7 positive expression was higher than the negative expression rate in 2 years(HR = 2.131,95% CI=1.027-4.422).In propensity score matching(1:3)analysis of CK19 expression,CK19 expression was correlated with AFP levels(?2=5.151,P=0.023).CK19 negative and positive expression in 2 years,5 years,and long-term survival time was no statistically significant difference in tumor survival time.In propensity score matching(1:3)analysis of CK7/CK19 expression,CK7/CK19 double positive expression had worse 2-year,5-year,and long-term survival and tumor-free survival compared with double-negative expression.Conclusion: In hepatitis B-related hepatocellular carcinoma,compared with patients with double-negative CK7/CK19 expression,double-positive patients have a worse prognosis and shorter recurrence time,suggesting that CK7/CK19 may be a potential predictor of clinical prognosis.PART 2:ANALYSIS OF WHOLE GENOME EXPRESSION PROFILES OF HEPATOCELLULAR CARCINOMA BASED ON THE EXPRESSION OF CK7 AND CK19 IN THE GEO DATABASE GSE14520DATASETObjective: To analyze the molecular mechanism related to the regulation of CK7 and CK19 expression in HBV related hepatocellular carcinoma.Materials and Methods: Analysis of expression profile data of 212 cases of hepatocellular carcinoma patients with hepatitis B background data in the GEO database GSE14520 dataset,CK7 and CK19 according to the first part of the positive rate of this study(CK7 positive rate 43/394,CK19 positive rate70/394)(by rate)and the median of the expression profile(by median)were analyzed.We pre-processed the downloaded chip raw data and clustered with the control group to screen for differentially expressed genes among the samples.Gene function enrichment analysis,signal pathway enrichment analysis,construction of gene interaction network,and the combination of CK7,CK19 expression and follow-up data were analyzed in the above two groups for their role in prognosis.Results: According to different groups,we differentially expressed geneswith high and low expression of CK7 and CK19 in the GSE14520 dataset.We found that there are complex interactions between these differential genes and CK7 and CK19 in gene and protein interaction networks..In the CK7 and CK19 expression,according to the first part positive rate(CK7 positive rate 43/394,CK19 positive rate 70/394)(by rate)group analysis,CK19 expression correlated with AFP levels(?2=14.240,P<0.001),CK19 The expression was correlated with BCLC stage(?2=10.372,P=0.016).There was no statistically significant difference in the survival time and survival time of CK7 and CK19 yin and yang expression at 2 years,5 years and long-term.In CK7 and CK19 expression by median group analysis,CK19 expression correlated with AFP levels(?2=16.883,P<0.001),and CK19 expression correlated with BCLC stage(?2=12.791,P=0.005).Factor analysis Patients with high CK19 expression had poor survival time and disease-free survival time relative to those with low expression(Log-rank P=0.012,Log-rank P=0.035,respectively).Multivariate analysis of CK7 and CK19 yin and yang expression showed statistically significant differences in the 2-year disease-free survival time(HR=0.586,95%CI=0.372-0.923;HR=1.633,95% CI=1.020-2.614).Conclusion: We have discovered differentially expressed genes with high and low expression(different grouping)of CK7 and CK19.In gene and protein interaction networks,there are complex interactions and co-expression relationships between these differential genes and CK7 and CK19.PART 3:EXOME-WIDE ASSOCIATION STUDY IDENTIFIED THE GENETIC VARIANTS ON EFFECTING THE CK7 AND CK19 EXPRESSION IN HBV RELATED HCC PATIENTSObjective: To analyze the whole exon polymorphisms of hepatitis B surface antigen-positive hepatocellular carcinoma(HCC)in Guangxi and its effect on clinical prognosis in different status of CK7 and CK19 expression.Materials and Methods: Whole exon single nucleotide polymorphism(SNP)genotyping microarray scanning was performed on 394 cases of hepatitis B surface antigen positive hepatocellular carcinoma patients,according to CK7(positive and negative expressions were matched 1:3),CK19(1:3 match between positive and negative expressions)and CK7/CK19(double positive and double negative expressions were matched 1:3).Genome-wide association analysis was performed.SNPs with MAF>0.1 and P<0.001 were selected for clinical prognostic analysis.At the same time,the gene for these SNPs was analyzed by median grouping in the GSE14520 dataset for clinical prognostic analysis.Results: According to group analysis of CK7(positive and negative expressions were matched 1:3),the rs6930740 polymorphism inequality influenced the expression of CK7 and affected the long-term survival time(log-rank P=0.002).The rs1816392 locus polymorphism may affect long-term disease-free survival in patients(log-rank P=0.022).Analysis of grouping by CK19(positive and negative expressions by 1:3 match)revealed that rs2288419,rs7516332,rs239259,rs3829868,rs2602381,and rs4381746 polymorphisms affected long-term survival of patients(log-rank P=0.006,log-rank P=0.016,log-rank P=0.021,log-rank P=0.040,log-rank P=0.046,log-rank P=0.047,respectively).The rs239259 polymorphism affects long-term disease-free survival in patients(log-rank P=0.001).Analysis of grouping by CK7/CK19(positive and negative expressions according to 1:3 match)showed that the rs3781684 polymorphism affected long-term tumor-free survival of patients(log-rank P=0.020).We analyzed the clinical prognostic analysis of these SNPs loci in the GSE14520 dataset by median grouping: The high expression of the UGT1A10 gene with low expression of the rs2602381 locus has a good survival time(HR=0.589,95%CI= 0.372-0.932).The low and high expression of UGT1A10 gene in the GSE14520 dataset affected the long-term survival of patients.The high-expression group had a longer survival time than the low-expression group(HR=0.589,95% CI=0.372-0.932).Conclusion: We analyzed the data of whole exons and found that different CK7 and CK19 expression states had different single nucleotide polymorphism distribution and gene expression distribution.Although the genetic polymorphisms of the above candidate SNPs were significantly associated with the prognosis of our study population,it is still need further validation.PART 4:STUDY ON BIOLOGICAL BEHAVIOR OF CK7 AND CK19 GENES IN HEPATOMA CELL LINESObjective: To investigate the effects of siRNA silencing CK7 and CK19 genes on gene expression,migration,invasion,cell cycle and apoptosis of hepatocellular carcinoma MHCC-97 H and SMMC-7721 cell lines.Methods: Three siRNA fragments targeting CK7 and CK19 genes were transfected into human hepatocellular carcinoma MHCC-97 H and SMMC-7721 cell lines.Three siRNAs in each cell line were detected by real-time fluorescence quantitative PCR and Western Blot.In the fragment,the best siRNA fragment for CK7 and CK19 was silenced.Then,the siRNA fragment was transfected into the human hepatocellular carcinoma MHCC-97 H and SMMC-7721 cell lines.48 hours later,the expression of CK7 and CK19 genes was detected by real-time fluorescence quantitative PCR.After 72 hours,CK7 and CK19 were detected by Western Blot technique.Protein expression levels,scratch assays were used to detect cell migration,detection of Matrigel invasion assay for detection of cell invasion,and flow cytometry was used to detect changes in cell cycle and apoptosis.In the second part of this study,the differential genes were screened by the median CK19 grouping to analyze the signal pathways of CK19 affecting the biological behavior of MHCC-97 H and SMMC-7721 cell lines.Results: The expressions of CK7 and CK19 in the transfected hepatoma MHCC-97 H and SMMC-7721 cell lines were detected by real-time fluorescence quantitative PCR and Western Blot techniques.The si-CK7-C silencing effect for CK7 gene was the best,and The si-CK19-C silencing effect for CK19 gene was the best.After these two fragments were used for subsequent experiments in the transfected hepatoma MHCC-97 H and SMMC-7721 cell lines.After silencing CK7 gene,the expression of CK7 gene in hepatoma cells decreased,and the migration ability of MHCC-97 H cell line in liver cancer cells weakened.Mitotic arrest in SMMC-7721 liver cancer cell line.After silencing CK19 gene,the expression of CK19 gene in hepatoma cells was decreased,and the migration ability of MHCC-97 H in hepatoma cell lines was weakened.After silencing CK7 and CK19,the invasion of MHCC-97 H and SMMC-7721 cancer cells was weakened.Signaling pathway analysis revealed that the relative expression levels of the 22 genes in the pathway were detected by RT-PCR in the low or in the unexpressed state.Conclusion: SiRNA targeting CK7 and CK19 genes can reduce the expression of CK7 and CK19 genes.After silencing the CK7 gene,cell migration in the MHCC-97 H cell line was weakened and mitosis in the SMMC-7721 hepatoma cell line was arrested;cell migration was suppressed in the MHCC-97 H cell line after silencing the CK19 gene.After silencing CK7 and CK19,the invasion of cancer cells MHCC-97 H and SMMC-7721 cells was weakened.Because these CK7 and CK19 may be a molecular target with potential therapeutic value in hepatocellular carcinoma.It provides experimental basis for the next step to verify expression regulation at the cellular level.Analysis of signal pathways affecting the expression of CK19 in hepatoma SMMC-7721 and MHCC-97 H cell lines revealed that the genes in the pathwaywere detected by RT-PCR in the low or in the unexpressed state..This needs further analysis of the signal pathways that influence the expression of CK19 for us to search a suitable hepatoma cell line.