Correlation Between SST Gene Promoter Methylation And Expression And Its Regulatory Mechanism In Gastric Cancer

Backgrounds and Purpose:Gastric cancer is the fifth most common malignant tumor in the world,and the mortality rate of gastric cancer patients is high,ranking fourth in the global malignant tumor mortality rate.Due to the insidious onset of gastric cancer,most patients have found that they are in the advanced stage,and the overall 5-year survival rate is less than 30%.Gastric cancer is a multigene,multi-step,progressive disease malignant process involving both genetic and environmental factors.Among them,intestinal gastric cancer evolves from normal mucosa/superficial gastritis?atrophic gastritis?intestinal metaplasia?dysplasia,and then evolves gastric cancer.The pathogenesis of gastric cancer has not been fully elucidated,its occurrence and development are directly related to the activation of proto-oncogenes and/or the inactivation of tumor suppressor genes.The changes in the function of these genes are not only related to the genetic changes of simple gene sequence changes.At present,it is believed that epigenetic changes also play an important role in the regulation of gene expression and are closely related to the formation of gastric cancer.Epigenetics is defined as a regulatory mechanism that changes gene expression independently of changes in gene sequence and can be inherited.The main regulatory mechanisms of epigenetics are:DNA methylation,histone modification,chromatin remodeling,and non-coding RNA.DNA methylation is one of the important mechanisms of gene silencing.DNA methylation mainly occurs in the GC base-rich region(Cp G island)of the gene,and the Cp G island is often located in the promoter region of the gene.The Cp G island located in the promoter region of the healthy human genome is unmethylated,and some tumor suppressor genes are highly methylated,resulting in silencing of gene expression and eventually inducing tumors.Therefore,analysis of methylation in gene promoter region can be an important marker for early tumor monitoring and prevention.DNA methylation is a relatively reversible process.Application of methylase inhibitors can reverse the abnormal methylation status of some tumor suppressor genes,restore their expression and tumor suppressor function,which provides a new strategy for tumor therapy.In the early stage of this research group,445 genes with hypermethylation and low expression in gastric cancer were screened out through bioinformatics analysis.The protein interaction network was further used to find the core genes,and six core genes were found after verification by the TCGA database,including SST,AR,CASR,CXCL12,GNAS,and PRKACB.Further verification in histology revealed that the m RNA expression level of SST gene in gastric cancer tissues was significantly reduced,and was negatively correlated with promoter methylation.Somatostatin(SST)is a cyclic peptide hormone with extensive biological activities,which is widely found in the central nervous system,gastrointestinal tract and pancreas.It has an important regulatory effect on the secretion of various hormones in human body.D cells of human gastric antrum have the ability to secrete and synthesize SST,and the SST released by it mainly affects the function of surrounding cells through paracrine.The biological function of SST is mainly produced by binding to somatostatin receptor(SSTRs)on the cell surface and then activating the signal transduction pathway after receptor activation.At present,there are few studies on the methylation and expression of SST gene in gastric cancer,and the molecular mechanism of methylation regulating expression has not been studied,which is worth further discussion.According to the preliminary bioinformatics analysis results of this study,SST is hypermethylation and low expression in gastric cancer.We speculate that SST may be a potential tumor suppressor gene.Due to hypermethylation of the promoter,the SST gene expression is silenced,which in turn causes changes in cell biological behavior and participates in the development of gastric cancer.Therefore,this study intends to compare the differential expression of SST at the protein level in different gastric disease tissues that are closely related to the occurrence and development of gastric cancer,and to analyze the correlation between protein expression and promoter methylation.At the same time,we also want to prove the correlation between promoter methylation and expression of SST gene in vitro,and the effect of SST on the biological behavior of gastric cancer,and further explore the molecular mechanism of methylation regulating expression.The purpose of this study is to elucidate the correlation between promoter methylation and expression of SST gene and its regulatory mechanism during the development of gastric cancer,and to provide experimental evidence and theoretical references for finding new methylated tumor markers or potential therapeutic targets for gastric cancer.Methods:Part I:Correlation between promoter methylation and expression of SST gene in different gastric diseases1 Gastric antrum biopsies and ESD specimens from 186 patients who underwent gastroscopy from August 2014 to June 2017 in the Digestive Endoscopy Center of the First Affiliated Hospital of China Medical University were collected,including 49 cases of superficial gastritis,47 cases of atrophic gastritis,44 cases of dysplasia and 46 cases of early gastric cancer.2 Detection of SST protein expression by immunohistochemistry.3 The methylation level of SST gene promoter in different gastric tissues were detected by BGS method.4 Mann-Whitney U nonparametric test was used to analyze the differences of SST expression and promoter methylation level in different gastric disease groups.Spearman rank correlation coefficient was used to analyze the correlation between the methylation level of SST gene promoter and protein expression.Graphpad Prism 8 was used for statistical analysis and graph processing.Part II:Effects of SST overexpression/silencing on biological behavior of gastric cancer cells with different methylation status1 Construction of overexpression and RNAi plasmid:pc DNA3.1(+)was selected as a vector to construct the SST overexpressed plasmid-1,GV230 was selected as a vector to construct the SST overexpressed plasmid-2 with GFP fluorescence.GV248 was selected as a vector to construct three sh RNA coding clones for SST.2 Identification of gastric cancer cell lines with hypermethylation and low expression of SST and hypomethylation and high expression of SST:The expression of SST in MGC803,HGC27,SGC7901,BGC823,AGS and MKN45 gastric cancer cell lines was detected by Real-time PCR.The methylation level of SST gene promoter in the above gastric cancer cell lines was detected by BGS method.3 Overexpressing SST plasmid transfection:The constructed SST overexpression plasmid was transfected into the selected hypermethylated and low expression SST gastric cancer cell,and the overexpression efficiency of SST was detected by Real-time PCR and Western Blot.After 24 hours of plasmid transfection,stably transfected gastric cancer cell with SST overexpression was constructed by G418 screening and identified by Real-time PCR and Western Blot.4 Silencing SST plasmid transfection:The constructed SST silencing plasmid was transfected into hypomethylated and high expression SST gastric cancer cell and stably transfected gastric cancer cell with SST overexpression,respectively.The silencing efficiency of SST was detected by Real-time PCR.5 Detection of proliferation of SST overexpression/silencing cells:The effect of SST overexpression/silent on proliferation of gastric cancer cells with different methylation status was detected by CCK-8 assay.6 Detection of apoptosis of SST overexpression/silencing cells:Flow cytometry was used to detect the effect of SST overexpression/silencing on apoptosis of gastric cancer cells with different methylation status,and Western Blot method was used to detect the effect of SST overexpression/silencing on the expression of apoptotic proteins PARP and Caspase 3 in gastric cancer cells with different methylation status.7 Detection of invasion and metastasis ability of SST overexpression cells:Transwell method was used to detect the effect of SST overexpression on invasion and metastasis of hypermethylated gastric cancer cells.8 Functional analysis of SST:Bioinformatics methods were used to analyze the function of SST.9 Statistical analysis:Graphpad Prism 8 was used for statistical analysis and graph processing.Paired sample T test was used to compare the expression differences between the two groups.Spearman rank correlation coefficient was used to analyze the correlation between average methylation rate of SST gene promoter and m RNA expression.Part III:Regulation mechanism of SST gene promoter methylation affecting expression1 Effect of SST methylation on expression:5-Aza-d C demethylation was used to treat hypermethylated AGS gastric cancer cells and SAM methylation was used to treat hypomethylated BGC823 cells.After 72 hours of treatment,the methylation level of SST promoter was detected by BGS method,and the m RNA expression level of SST was detected by real-time PCR.2 Detection of promoter activity:The effect of methylation of SST promoter on the activity of SST promoter was detected by dual-luciferase reporter gene system.3 Prediction of transcription factors targeted to differential methylation sites of SST promoter:Using JASPAR database to predict and screen transcription factors binding to differential methylation sites of SST promoter.4 Detection of binding ability between promoter and transcription factors:EMSA assay was used to detect the binding ability of specific methylation sites of SST promoter to transcription factors in gastric cancer cells under different methylation states.5 Effect of specific methylation site of SST promoter combined with transcription factors on activity of SST promoter:Dual luciferase reporter gene system was used to detect the effect of specific methylation site of SST promoter combined with transcription factors on the activity of SST promoter.6 Statistical analysis:Graphpad Prism 8 was used for statistical analysis and graph processing.The expression differences between the two groups were compared by paired sample T test.Results:Part I:Correlation between promoter methylation and expression of SST gene in different gastric diseases1 Immunohistochemical methods confirmed that the expression of SST decreased gradually during the gradual evolution of superficial gastritis-atrophic gastritis-dysplasia-early gastric cancer,with the positive expression rates of 15.02%,3.68%,1.74%,and 0.05%,respectively.The differences in expression between any two groups were statistically significant(P<0.05).2 The BGS method confirmed that compared with the non-gastric group(60.2%±6.69%),the average methylation rate of SST gene promoter in the gastric cancer group was increased(63.16%±4.94%)(P<0.05),and the methylation rate at sites 18,20,21 and 22(+100bp,+127bp,+129bp,+138bp)was significantly increased(P<0.05).3 The average methylation rate of SST promoter was negatively correlated with its protein expression(r=-0.156,P=0.039).Among the 4 differential methylation sites,the methylation rate at sites 20 and 21(+127bp,+129bp)was negatively correlated with protein expression(r=-0.156,-0.165,P=0.039,0.029).Part II:Effects of SST overexpression/silencing on biological behavior in gastric cancer cells with different methylation status1 Cell identification:Using Real-time PCR and BGS methods,the AGS gastric cancer cell line with relatively hypermethylation and low expression of SST and the BGC823gastric cancer cell line with relatively hypomethylation and high expression of SST were identified.Spearman rank correlation coefficient analysis showed that the methylation level of SST promoter was negatively correlated with its m RNA expression(r=-0.371,P=0.50).2 Effect of SST overexpression on cell proliferation:The CCK-8 assay results showed that compared with the control group,the SST overexpression group inhibited the proliferation of hypermethylated AGS cells.Similarly,the proliferation of stably transfected AGS cells with SST overexpression was also reduced.3 Effect of SST silencing on cell proliferation:SST was silenced in hyomethylated BGC823 cells and stably transfected AGS cells with SST overexpression respectively,and the results of CCK-8 assay all showed that compared with the control group,the proliferation of gastric cancer cells was enhanced after SST silencing.4 Effect of SST overexpression/silencing on apoptosis:The results of flow cytometry showed that the apoptosis rate of hypermethylated AGS gastric cancer cells overexpressing SST was higher than that of the control group(P<0.05),and the apoptosis rate of hypomethylated BGC823 gastric cancer cells after SST silencing was lower than that of the control group(P<0.05).Western Blot experiment results showed that the expressions of apoptotic proteins PARP and Caspase 3 in hypermethylated AGS gastric cancer cells overexpressing SST were not significantly different from those of the control group,and in SST-silenced hypomethylated BGC823 gastric cancer cells,the expressions of apoptotic proteins PARP and Caspase 3 were not significantly different from those of the control group.5 Effect of SST overexpression on cell invasion and metastasis:Transwell experiment results showed that there was no significant difference in the number of invaded cells and metastatic cells between the hypermethylated AGS cells overexpressing SST and the control group(P>0.05).6 Functional analysis of SST:Using TCGA-STAD dataset for GSEA enrichment analysis,the KEGG enrichment results showed that PI3K-Akt signaling pathway and MAPK signaling pathway regulated cell proliferation.Similarly,KEGG function enrichment analysis was performed using the gastric cancer cell dataset from CCLE database,and PI3K-Akt signaling pathway was also found in the enrichment results.Part III:Regulation mechanism of SST gene promoter methylation affecting expression1 Results of demethylation/methylation on gastric cancer cells:Using 5-Aza-d C to demethylate hypermethylated AGS gastric cancer cells,the results showed that compared with the control group,the average methylation rate of SST promoter in the 5-Aza-d C treatment group decreased by 14.25%(90.73%vs 76.48%)(P<0.05),and the m RNA expression level of SST was up-regulated by 4 times(1.0×10^-7vs 4.22×10^-7)(P<0.05).Using SAM to methylate hypomethylated BGC823 cells,the results showed that compared with the control group,the average methylation rate of SST promoter in the SAM treatment group increased by 9.07%(84.57%vs 93.64%)(P<0.05),the m RNA expression level of SST was down-regulated by 53.52%(2.84×10^-6vs 1.32×10^-6)(P<0.05).2 Results of dual luciferase assay:Compared with the normal SST promoter,the luciferase activity of the methylated promoter was significantly decreased(P<0.05).3 Prediction results of transcription factors:The JASPAR database was used to predict the transcription factors binding to differential methylation sites 20 and 21(+127bp,+129bp)of SST promoter,from which two transcription factors CTCFL and SOX18 with high scores were selected for follow-up experiments.4 Results of EMSA experiment:Compared with unmethylation,the binding of sites 20and 21(+127bp,+129bp)of SST promoter to the transcription factors CTCFL and SOX18 was decreased under methylation.5 Dual-luciferase assay:After overexpression of the transcription factor CTCFL,the activities of the full-length wild type,Cp G island truncated wild type,and Cp G island truncated methylation sites 20 and 21 mutants of SST promoter were all enhanced(P<0.05),and compared with the methylation sites 20 and 21 mutants,the enhanced activity of the SST wild-type promoter was more obvious.After overexpression of the transcription factor SOX18,the activities of the full-length wild type,Cp G island truncated wild type,and Cp G island truncated methylation sites 20 and 21 mutants of SST promoter were all reduced(P<0.05).Conclusion:1.The expression of SST protein was negatively correlated with the occurrence and development of gastric cancer;the expression of SST was negatively correlated with the proliferation of gastric cancer cells,overexpression of SST inhibited the proliferation of hypermethylated gastric cancer cells,while silencing SST promoted the proliferation of hypomethylated gastric cancer cells;the expression of SST may be related to the apoptosis of gastric cancer cells.2.The average methylation rate of SST promoter in gastric cancer tissues was higher than that in non-gastric cancer tissues,and the methylation rates at sites 18,20,21 and 22(+100bp,+127bp,+129bp,+138bp)of SST promoter were significantly increased.3.Methylation of SST promoter can affect its expression.Methylation at sites 20 and 21(+127bp,+129bp)of SST promoter can inhibit its binding to the transcription factor CTCFL,thus inhibiting the activity of SST promoter and silencing the expression of SST gene.