The Role And Mechanism Of Long-chain Non-coding RNA In H.pylori Infection-associated Gastric Cancer

The regulation of gene expression by lncRNA at the epigenetic level,transcriptional and post-transcriptional level has been widely studied.LncRNA are involved in many important pathological processes,such as cell differentiation,apoptosis and individual development.The dysregulated expression of lncRNA was closely associated with the pathogenesis of tumor.Many lncRNA functioned as oncogenes or tumor suppressors have been reported recently,suggested that lncRNA may be as diagnostic,prognostic and therapeutic biomarkers in tumors in the future.Gastric cancer(GC)was one of the most common malignancies and represented the second leading cause of cancer-associated mortality worldwide,which remains a serious threat to the public health.Many factors are implicated in the carcinogenesis and development of gastric cancer,including environmental risk factors,genetic alterations and host itself.Among them,H.pylori was a well known risk factor for GC.In 1994,The International Agency for Research on Cancer(IARC)defined H.pylori as a class I carcinogen,and strongly associated with GC.H.pylori infection can induce chronic inflammation in the gastric mucosa,which induces the normal gastric epithelium to progress through a series of well-defined steps into carcinoma.However,the mechanism of H.pylori infection in gastric cancer progression is unclear.In this study,we used high-throughtput IncRNA microarray to screen dysregulated IncRNA in gastric cancer,and to investigate the biological function fully understood lncRNA.Cultured H.pylori was used to infect the normal gastric epithelial cell line GES-1,to construct the H.pylori infection model.lncRNA microarray was applied for detecting the IncRNA in H.pylori infection model.Meanwhile,we tested the expression of IncRNA in normal gastric mucosa,atrophic gastritis(Hp+),gastric cancer tissues(Hp+)by IncRNA microarray technology.We found that the expression of many mRNA involved in cell cycle,genomic stability had great alterations.Thus,bioinformatics analysis was performed to establish a co-expression network between dysregulated lncRNA and mRNA.From the co-expression network,we picked a IncRNA named SNHG17(small nuclear RNA host gene 17)for further investigation.Then,we examined the expression level of SNHG17 in 128 gastric cancer tissues and the adjacent normal tissues.Results showed that SNHG17 in gastric cancer tissues was significantly upregulated compared with the adjacent tissues.We also examined SNGH17 expression in normal gastric epithelium,atrophic gastritis,intestinal metaplasia,and dysplasia tissues with or without H.pylori infection.According to the qRT-PCR results,SNHG17 expression was gradually upregulated during the H.pylori infection process.RNA fluorescence in situ hybridization assay showed that SNHG17 was predominantly located in the nuclear.To investigate the function of SNHG17,we used siRNA technology to study the effect of SNHG17 on genomic stability.Results showed that knockdown of SNHG17 expression could maintain genomic stability,whereas didn’t influence the proliferation of gastric cancer cells.To exploreing SNHG17 function in vivo,we introduced SNHG17-specific short hairpin RNAs(shRNA)into SGC-7901 cells,named SGC-7901-shl7 cells.We established control or SNHG17-shRNA-expressing SGC-7901 derived xenografts in nude mice and treated with a single dose of 10 Gy IR to each tumor.In the IR-treated groups,the growth of SNHG17-knockdown tumors was significantly attenuated compared with the control group.Our results suggest that suppression of SNHG 17 expression improve genomic stability in vivo,thus sensitizing tumors to IR treatment.To exploring the molecular mechanism of SNHG 17,we performed RNA-pull down assay combined with Mass spectrometry to screen the protein interacted with SNHG17.According to the results,SNHG17 might interact with NONO in Hp infected SGC-7901 cells.RNA IP verified the binding of NONO with SNHG17.Moreover,we found that SNHG 17 could bind with NONO protein with a binding site located in 716-720 nucleotide of SNHG 17 transcript.Then we mutated this binding site in SNHG 17 and found that the capacity of SNHG 17 interacting with NONO was abolished obviously,which suggested that SNHG17 could interact with NONO directly.Also,we found that knock down SNHG17 could not regulate the expression level of NONO protein,and the alteration of NONO expression do not influence SNHG 17 neither.Taken together,our study firstly reported a lncRNA SNHG 17 associated with H.pylori infected gastric cancer by lncRNA microarray.To investigate the biological functions and molecular mechanisms,we found that SNHG 17 was significantly upregulated in H.pylori positive atrophic gastritis and gastric cancer,and involved in gastric cancer migration and genomic stability.Therefore,this study found that SNHG 17 was a IncRNA,affected gastric cancer cells migration and genomic stability,might be a potential biomarker for the early diagnosis and treatment of GC.