Correlation Study Of Predicting Target Gene In Gastric Cancer Based On Bioinformatics Analysis

Research background and purposeGastric cancer is one of the most malignant tumors in the world.Its prognosis is relatively poor,which seriously threatens human health and increases the economic burden of patients and society.According to the statistics of the International Agency for Research on Cancer,the number of new cases of gastric cancer in the world in 2012 was about 951000,and the number of deaths from gastric cancer was about 723000,ranking fifth in the incidence of malignant tumors,respectively.The death rate of malignant tumor was the third place.The incidence of gastric cancer in China is very high.The death and incidence of gastric cancer in China account for almost 50%of the world,and the burden of disease is serious,which is the focus of cancer prevention and treatment.According to the latest data from the National Cancer Registry In 2015,there were about 679000 new cases of gastric cancer and 498000 death cases of gastric cancer in China.The burden of disease is very serious and it is a major disease that seriously endangers the health of Chinese residents.The 5-year relative survival rate for gastric cancer in China from 2003 to 2005 was 27.4,while in South Korea from 2005 to 2009,according to the CONCORD-2 report,The 5-year net survival rate of gastric cancer population in Japan was 57.9%and 54.0%respectively,and the survival rate of gastric cancer in China was significantly lower than that in developed countries,which may be due to the early development of gastric cancer in China.Stage screening work is relatively backward,people's awareness of health care is relatively weak.There were no specific symptoms and signs in the early stage,and a high proportion of patients were diagnosed at the late stage.The prognosis of gastric cancer is closely related to the period of diagnosis and treatment.The majority of early gastric cancer can be treated by endoscope,and the 5-year survival rate exceeds 90%.Therefore,attention should be paid to the screening of gastric cancer,the timely detection of diseases in the early stage of gastric cancer and the treatment of symptoms,so as to prolong the survival period of patients,improve the quality of life,and alleviate the financial burden of the patients,their families and society.At present,the diagnosis of gastric cancer mainly depends on gastroscopy,pathology,tumor marker(CEA,CA72-4),enhanced CT,ultrasound endoscopy and so on.It is of limited value in the diagnosis of early gastric cancer.High efficiency,accuracy,false negative and low false positive rate diagnostic methods are urgently needed,indicating the prognosis of thePatients.Shandong is also a high incidence of gastric cancer provinces,gastric cancer prevention and treatment task is arduous,pressure is enormous.Early diagnosis and treatment of gastric cancer should be strengthened.The low 5-year survival rate of gastric cancer was closely related to the following factors:1.Lack of early diagnostic markers.2.The prognostic value of histological index was low.3.At present,the therapeutic effect on advanced gastric cancer is limited.4.Lack of effective molecular markers for targeted therapy.It is important to elucidate the molecular mechanism of the development of gastric cancer and to identify the key biomarkers and effective therapeutic targets.With the development of science and technology,the Human Genome Project shows that only 1.2%of the genome-encoded proteins.Thereare a wide variety of RNAs,including known miRNAs and recently discovered lncRNAs,cirRNA and pseudogenes,which have been thoroughly studied.LncRNAs are still a new,potential and still need to be studied.More and more data show that a large number of non-coding RNAS play an important role in the occurrence,development and metastasis of malignant tumors,and play an important role in the diagnosis and treatment of gastric cancer.The work provides a new perspective and research direction.The hypothesis of competitive endogenous RNA proposed by Poliseno et al has been confirmed by a great deal of evidence.That is,miRNA can cause target gene silencing by binding to mRNA,while ceRNA can regulate gene expression by competitively binding to miRNA.CeRNA can affect gene silencing induced by microRNA by combining microRNA with(microRNA response elements)MREs,a small RNA response element.This reveals the existence of a RNA?microRNA regulatory pathway with significant biological significance.ceRNA is not a newly discovered RNA molecule,but a complex regulatory mechanism.In recent studies,it has been clarified that there are many forms of gene action in transcriptional regulation.MicroRNAs,as one of the important regulatory factors,are the way in which short-stranded RNA,which is about the length of 22nt,inhibits the translation of the target gene or degrades the target gene.Thus reverse regulation of the expression of the target gene.In the actual regulation process,there are not only simple microRNA-mRNA silencing mechanisms,but also more complex network regulation.Some non-coded RNA also exist with the binding site of microRNA acts as a miRNA sponge(miRNA sponge)in the cell,and then releases the inhibition of the target gene by miRNA,which makes the target gene overexpression,and thus the construction of the target gene is more complicated.The large ceRNA network(ceRNETs),mechanism is called competitive endogenous RNA(ceRNA)mechanism.It is very important to study the regulatory mechanism of ceRNA.The analysis of molecular mechanism of cancer and other diseases is of great theoretical and practical significance to the later diagnosis and treatment of diseases and the development of new drugs.In this paper,TCGA database was used for differential LncRNA analysis of gastric cancer by bioinformatics analysis.To establish the total ceRNA regulatory network of LncRNA-microRNA-mRNA;to further analyze the difference between microRNA and mRNA.Find candidate difference LncRNA-microRNA-mRNA(SNHG12-miR320a-TAB3)to prepare for further experiments.To investigate the expression and mechanism of related genes in gastric cancer cell line.Research methodsPart ONE:We take the method of bioinformatics analysis.1.Using TCGA database to find out the difference of gastric cancer by LncRNA analysis.2.To establish the total ceRNA regulatory network of LncRNA-mi croRNA-mRNA;to further analyze the difference between microRNA and mRNA.3.A ceRNA network such as up-regulated LncRNA and down-regulated microRNA then up-regulated target mRNA(encoding gene,PCGs)(or vice versa)was screened.4.Through LncRNA's sub-pathway analysis obtained the connection pathway of LncRNA,and found the connection point'between LncRNA and classical pathway.5.The potential prognostic value of LncRNA,miRNA or PCGs molecules at multiple target sites was studied by COX-related survival analysis.Part TWO:Six cases of gastric cancer and 5 cases of paracancerous tissues were selected.The clinical and epidemiological data including follow-up data were collected to detect the expression of TAB3 in cancer tissues and paracancerous tissues by immunohistochemistry.To investigate the expression of TAB3 in cancer tissues and adjacent tissues and its relationship with clinical data,the expression of SNHG12,miR-320a,TAB3 in gastric cancer cells of AGS was detected by using statistical analysis software.Inhibition of miR-320a expression and detection of SNHG12,TAB3 expression in AGS gastric cancer cell lines by qPCR The proliferation and invasion of AGS cells were detected by CCK-8 and Transwell assays.We used the methods of Western blot to detect the expression of TAB3 protein.Part THREE:SNHG12 overexpression vector was constructed to compare the following functional differences in AGS cells:(1)The change of SNHG12,miR-320a,TAB3 expression in AGS cells.(2)The CCK8 assay was used to detect The proliferation of gastric cancer cells.(3)The invasion of gastric cancer cells was detected by Transwell assay.(4)The changes of TAB3 protein level in AGS gastric cancer cells were detected by western blot.ResultsPart ONE:1.The expression profile data(package/RTCGA 'version 1.8.0)were collected from the normal tissues adjacent to cancer(35 samples)and named normal),cancer tissues(412 samples,named cancer),and a total of 447 samples were selected for analysis,2.Get LncRNA-miRNA-mRNA interaction relation data2.1 Get the ceRNA relationship(LncRNA-miRNA-mRNA)By using starBase v2.0 database,we got 222 lncRNA,8466 mRNA and 43859 interaction pairs in the candidate lncRNA-mRNA intersections,a total of 271 miRNA.2.2 ceRNA in gastric cancerThe TCGA expression profile of cancer group samples containing 22965 genes contains 507 miRNA.The gene in the expression profile was crossed with mRNA and lncRNA in the above lncRNA-mRNA,and the gene expression profile containing 8241 mRNA and the long non-coding RNA expression profile of 38 lncRNA were obtained.The genes in the miRNA expression profile were intersected with the common miRNA,and 18 miRNA expression profiles were obtained.3.Construction of lncRNA coexpression Network(Construct the lncRNA similarity network)CeRNA theory was used to construct the ceRNA network of lncRNA-miRNA-mRNA by using Cytoscape 3.6 software.The coexpression network of lncRNA-miRNA-mRNA ceRNA network containing 19 lncRNA,1,201 mRNA,17 miRNA and 2046 interactions was obtained.4.Construction of subpathway and significance analysis of attractor subpathwayThe pathway database selected by this analysis was KEGG,to identify the difference of gene enrichment pathway in mRNA gene expression profile by Fisher test.After correction,the pathway enriched with pvalue less than 0.05 is a candidate differential pathway.The number of nodes in the molecular set of the pathway is calculated,and if the node number is 8 or so,it is considered to be a candidate subpathway regulated by LncRNAs competition.The(attractor)method of attractor<0.05 is the differential subchannel,which consists of 3 different subchannels:05216-1,04621-1,04310-2.5.Differential subpathway lncRNA evaluationBased on the results of subpathway evaluation,Cytoscape 3.6 software was used to construct transduction pathway-lncRNA-mRNA network,torecognize pathway lncRNA,and analyze its network topology,and 12subpathway IncRNA were obtained.According to different samples(control group and experimental group),the difference of gene expression was calculated by linear expression analysis model(LIMMA),and the difference of gene expression between normal group and disease group was judged by improved t test.There were 10 differentially expressed genes(p<0.05).6.Survival analysis(package survival 'version 2.42-3)There were 48 differentially expressed lncRNA and ceRNApairs genes in 10 pathways.(1).Cox survival curveThe difference of total survival time and relapse free survival time was analyzed.The significant difference was found among them(p<0.01):1 MIR17HG,NEAT1,KCNQ10T1 SNHG12/PLCB1/SMAD3/TAB3/has-miR-133b.According to the results of ceRNApairs,SNHG12-miR-320a-TAB3 was selected as candidate ceRNApairs,for further experiments.Part TWO:1.Inhibition of miR-320a expression can reduce migration andproliferation of AGS cells2.Inhibition of miR-320a expression in AGS cells can reduce the expression of SNHG12.3.The expression of TAB3 in AGS cells is not regulated by miR-320a.Part THREE:1.Overexpression of SNHG12 can cause high expression of miR-320a in AGS cells,and there is a positive correlation between them.2.Overexpression of SNHG12 can enhance the migration ability of AGS gastric cancer cells.3.The overexpression of SNHG12 has no obvious effect on theproliferation ability of AGS gastric cancer cells.4.Overexpression of SNHG12 can decrease the expression of TAB3 protein in AGS gastric cancer cells,but it does not affect the expression of TAB3 mRNA.Conclusion:1.The analysis of TCGA,starBase v2.0 and KEGG database shows that there is a certain correlation between SNHG12,miR-320a,TAB3'in gastric cancer,and SNHG12,TAB3 is closely related to prognosis.2.In AGS cells,SNHG12 regulates miR-320a and then affects the expression of TAB3 protein.SNHG12 does not regulate miR-320a asceRNA,positive correlation between them.3.In AGS cells,SNHG12 and miR-320a can promote the progression of the tumor cells.4.SNHG12 is a potentially effective target gene related to diagnosis,treatment and prognosis of gastric cancer.