Study On The Combination Of NONO And SNHG17 And Its Mechanism Of Action

Gastric carcinoma(GC)is one of the most common cancers in the world.Despite the steady decline of morbidity and mortality in developed countries in the past 50 years,gastric cancer is still a considerable burden worldwide.The incidence of stomach cancer is highest in east Asia,including China,which contributes 40%of new cases of stomach cancer worldwide each year.Helicobacter pylori(HP)is a major risk factor for gastric cancer.About 75%gastric cancer are caused by Helicobacter pylori infection.The International Agency for Research on Cancer(IARC)classifies Helicobacter pyhlori as class I carcinogen causing human gastric cancer in 1994.Helicobacter pylori is a gram-negative bacterium,which is colonized in the mucosal layer of mucous cells and on the surface of the stomach.Helicobacter pylori infection can cause the inflammatory reaction of the host,leading to chronic gastritis and pepticulcer,and eventually developing into gastric cancer.The study found that Helicobacter pylori infection can promote the expression of many proinflammatory cytokines,such as TNF-?,IL-1?,IL-6 and IL-8 and so on.These inflammatory factors may play a key role in the development of GC.In recent years,the function of the long non-coding RNA(lncRNA)has been widely recognized.It can regulate gene expression and cell signaling by adjusting the chromatin remodeling,control gene transcription,mRNA transcription and protein function or position.Studies have found that long non-coding RNA can be used as potential biomarkers of diagnosis and prognosis of GC.Many IncRNAs in HP-related gastric cancer were screened out by chip technology in the early stage of our laboratory.Dr.Han taotao found that lncRNA-SNHG17 can bind to NONO protein through the 716-720 base sequence AGGGA,and proposed that the function of SNHG17 may be related to the regulation of genomic stability.This study aims to further explore the binding and mechanism of SNHG17 and NONO.For detecting the expression of SNHG17,we added fresh Helicobacter pylori(international standard strains and clinical strains isolated from gastric cancer patient)to normal gastric mucosa epithelial cells and gastric cancer cells in vitro.qRT-PCR experiments show that the international standard strains and clinical strains isolated from gastric cancer patient can make the expression of SNHG17 rise.In this study,CRISPR/Cas9 technology was used to establish gastric cancer cell lines with SNHG17 and NONO binding site mutations,and finally two homozygous gastric cancer cell lines with binding site mutations were successfully obtained.qRT-PCR results showed that in gastric cancer cells with SNHG17 mutation,the expression levels of SNHG17 and snoRNA did not change after HP infection.RIP experiment found that HP infected binding site mutation of gastric cancer cells,the combination of NONO and SNHG17 will be suppressed.Comet experiment showed that after the SNHG17 mutant SGC-7901 cells were infected with Helicobacter pylori,the degree of genomic DNA damage was more serious.Immunofluorescence assay showed that,compared with gastric cancer cells in the control group,after HP infected,the formation of y-H2AX foci in SNHG17 mutant cells increased and the number of DNA double strand breaks(DSB)increased.Western Blot results also showed that the expression of y-H2AX increased,and the expression of Rad51 is restrained,and the amount of Ku70 and Ku80 increases.We speculated that the expression of y-H2AX increase might be due to the raise of NONO protein is insufficient,leading to the DSB repair delay and the number of DSB increases.Changes of Rad51 and Ku70/Ku80 may change the DSB repair pathway in host cells.In addition,based on previous reports,this study established a chronic infection model to simulate the status of infection in vivo,and the effect of SNHG17 on genome rearrangement was detected by whole-genome sequencing.We sequenced and found that CNV of gastric cancer cells decreased after SNHG17 knockdown,suggesting that SNHG17 knockdown is conducive to genome stability.Based on the experimental results of this study,we speculate that SNHG17 is closely related to genomic stability,which provides a new idea for the diagnosis and treatment of gastric cancer.