S100A8 Methylation In Primary Hepatocellular Carcinoma (HCC) And Function Research

OBJECTIVE Researching the expression of S100A8 gene in the population,and observe the effect of S100A8 on the proliferation,migration,invasion and tumorigenic ability of primary hepatocellular carcinoma(HCC)at the cellular level and animal level.So as we can further understand the effect of S100A8 on the biological behavior of HCC cells and explore its mechanism of action,and find a new target for the diagnosis and prognosis of primary hepatocellular carcinoma.Method 1.We used the public databases Gene Expression Omnibus(GEO)and The Cancer Genome Atlas(TCGA,https: //tcga-data.nci.nih.gov/tcga/)to retrieve,and proceed follow-up meta-analysis and survival analysis according to the search terms,strict data inclusion criteria and exclusion criteria.2.The TCGA data in the database through strict screening,after its degree of methylation grouping for survival analysis(Kaplan–Meier).3.The S100A8 methylation expression of Cp G sites in HCC tissues and adjacent tumor tissues were tested by the pyrosequencing technique.Furthermore,through the receiver operating characteristic curve explore the loci of cg20070090 clinical values in the diagnosis of HCC.4.Construction of S100A8 lentivirus expression vector,the virus solution was synthesized by human embryonic kidney cell 293 T and lentivirus packaging system.After virus concentration and titer detection,the optimal titer of the virus was used to infect the target hepatoma cells Huh7,MHCC-97 H and Hep G2.5.After the formation of stable cell lines,the expression of S100A8 Mrna and protein was detected by Western blot and Real time PCR(RT-PCR).6.The effect of S100A8 on proliferation,migration,invasion and apoptosis of hepatocarcinoma cells was investigated in vitro by cell function experiment.7.The subcutaneous tumorigenesis in nude mice was investigated to study the effect of S100A8 on the pathogenicity of hepatocarcinoma cells.RESULTS 1.More than two members in the team performed the search on Totally,X articles related to Methylation microarrays were collected from public database GEO and TCGA by more than two members in the group;Strictly follow the inclusive criteria;The result of meta-analysis reveal that: The methylation level of S100A8 was correlated with the occurrence of hepatocellular carcinoma.(SMD=-2.08,95%CI:-2.54--1.52)The P value in Publication bias was less than 0.05 which means Publication bias doesn't exist and the credibility of our result is relative high.The result of meta-analysis was the same after applied the Sensitivity Analysis,which proved the result of our study is very stable.2.The data of 345 HCC patients were extracted from TCGA database after strict criteria and were applied to assess the association of the S100A8 methylation level with survival outcomes.(Kaplan–Meier analysis).The result showed that OS and PFS were both significantly different among the four groups divided by methylation level.(Estimate Median OS: Q1:2.753;Q2:4.274;Q3:5.074;Q4:8.926,log-rank p<0.05.Estimate Median PFS:Q1:2.266;Q2:3.096;Q3:3.981;Q4:8.926.log-rank,p<0.05).The OS and PFS of Low-methylation-level group(Q1)were significantly higher than the‘high-methylation-level-group'(Estimate Median OS time: Q1:2.753;Q2-4:4.907,log-rank p<0.05.Estimate Median PFS time Q1:2.266;Q2-4:3.367,log-rank p<0.05)? 3.In this study,S100A8 overexpression and silencing lentivirus were successfully packaged.After the lentivirus concentration and titration detection,the appropriate dosage of the virus solution was used to infect Huh7,MHCC-97 H and Hep G2.Formation of stable cell lines,to provide a study for the next functional experiments.4.The stable cell lines Huh7-S100A8 and MHCC-97H-S100A8 were successfully screened by antibiotics,and Hep G2-S100A8 failed to screen.Western blot and RT-PCR showed that the expression of Huh7-S100A8,MHCC-97H-S100A8 protein and m RNA were higher than those of blank control group(Control).5.The proliferation of Huh7-S100A8 and MHCC-97H-S100A8 was higher than that of the corresponding Control group(P <0.05).The migration and invasion ability of Huh7-S100A8 and MHCC-97H-S100A8 were significantly higher than those of the Control group after transfection with S100 A 8 overexpressing vector.Apoptosis detection showed no obvious trend.On the experimental part of nude mice subcutaneous tumor,the tumor volume of Huh7-S100A8,MHCC-97H-S100A8 group was greater than Control group.(P<0.05)CONCLUSIONS 1.The result of meta-analysis reveal that: The methylation level of S100A8 was correlated with the occurrence of hepatocellular carcinoma.2.In survival analysis,the OS and PFS of Low-methylation-level group(Q1)were significantly higher than the ‘high-methylation-level-group'.3.In our study,S100A8 overexpression and silencing lentivirus were successfully packaged.Established the cell lines of Huh7-S100A8?MHCC-97H-S100A8.4. Using stable cell lines analyzed the biological function,and S100A8 promoted the human hepatoma cells proliferation,metastasis and invasion.5.Our research suggests that S100A8 can promote the growth of nude mice subcutaneous tumor.