| Background and PurposeFor a long time,cancer has been a serious threat to human beings because of its high morbidity and mortality.Among them,gastric cancer is a major category that cannot be ignored.Among the new cancers in human,gastric cancer ranks fifth.Gastric cancer is the third leading cause of human death.Non-coding RNA is a type of RNA that does not encode protein,mainly including LncRNA,mi RNA,Circ RNA,etc,but they still have biological functions and play an important role in many diseases by regulating transcription factors,silencing m RNA,and regulating genes.In recent years,there have been many reports on the research of non-coding RNA in gastric cancer.Among them,LncRNA ADAMTS9-AS2(ADAMTS9-AS2)and mi R-223-3p have been reported to regulate the occurrence and development of gastric cancer,but we don’t know its definite mechan-made clearly.Therefore,we aim to research into the regulatory effects of ADAMTS9-AS2 and mi R-223-3p on gastric cancer,and whether there is a regulatory relationship axis between them.MethodsFrom 2012 to 2014,we gathered tissues which located in gastric cancer and healthy stomach,from 45 gastric cancer patients,in the First Affiliated Hospital of Harbin Medical University.Cultured human gastric epithelial cell line(GES-1)and gastric cancer cell lines(GC)in vitro(SGC7901,MKN74,NUGC-4,HGC-27,BGC-823).Real-time quantitative PCR(q RT-PCR)was applyed to monitored the nucleic acid expression of ADAMTS9-AS2 and mi R-223-3p,then Pearson correlation analysis and bioinformatics were used to predict the correlation between ADAMTS9-AS2 and mi R-223-3p.Kaplan-Meier survival analysis was used to evaluate the prognosis of GC patients with differential expression of ADAMTS9-AS2 and mi R-223-3p.Use online star Base software to predict the binding sites of ADAMTS9-AS2 and mi R-223-3p,then take advantage of dual luciferase reporter gene system and pull-down assay to authenticating the binding connection between ADAMTS9-AS2 and mi R-223-3p.SGC7901 and BGC-823 cell lines were selected,transfected with overexpression plasmids,grouped into Control,OELnc,OELnc+OEmi R,CCK8,trypan blue staining and transwell experiments monitored cell proliferation and migration.Western blotting monitored the expression of EMT-related proteins mainly including Vimentin(Vim),N-cadherin(N-cad),E-cadherin(E-cad).ResultsCompared with normal tissues,the results of q RT-PCR showed that,ADAMTS9-AS2 was decrased,while mi R-223-3p was incrased in GC tissues,we also got the identical results in vitro.In addition,Pearson and pan-cancer predicted ADAMTS9-AS2 may negatively regulated mi R-223-3p.Kaplan Meiier survival analysis had a manifestation of that patients with increased expression of ADAMTS9-AS2 and decreased expression of mi R-223-3p had a better prognosis and longer survival time.The results of dual luciferase reporter gene system and pull-down experiments showed that ADAMTS9-AS2 and mi R-223-3p have binding sites,ADAMTS9-AS2 can negatively regulate the expression of mi R-223-3p.CCK8,trypan blue staining and Transwell experiment results show that overexpression of ADAMTS9-AS2 inhibited the proliferation,survival,and migration ability of GC cell lines,while overexpression of mi R-223-3p can reverse this phenomenon.The results of Western Blot experiments showed that ADAMTS9-AS2 inhibited the N-cad and Vim and promoted E-cad in GC cells,and these expressions were reversed by mi R-223-3p.Repeat each experiment at least 3 times.*P<0.05,* * P<0.01.ConclusionsLncRNA ADAMTS9-AS2 was low expressed in gastric cancer tissues and cells,and it was negatively correlated with the expression of mi R-223-3p;The prognosis of GC patients with high expression of LncRNA ADAMTS9-AS2 and low expression of mi R-223-3p is better;LncRNA ADAMTS9-AS2 bound and negatively regulated mi R-223-3p expression;LncRNA ADAMTS9-AS2 and mi R-223-3p regulate the expression of EMT-related proteins in gastric cancer cells,they affects cell migration and invasion,ultimately participates in the occurrence and development of gastric cancer. |
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