The Influence And The Mechanism Of Long Noncoding RNA Associated With H.pylori Infection On The Biological Characteristics Of Gastric Cancer Cells

Part One The screening and verification of lncRNA NR0 and the expression of lncRNA NR0 in gastric cancer tissues and cells and its effect on biological characteristicsObjective:To investigate the expression of long noncoding RNA?NR0?in gastric cancer tissues and cells,and its effect in vitro on the proliferation,migration,invasion and clone formation of gastric cancer cells,and its effect in vivo on proliferation in nude mice.Methods:1 Infected the GES-1 with cultured H.pylori to construct the inflammatory model and to verify the microarray results by Realtime-PCR.2 Expression levels of lncRNA NR0 in gastric cancer tissues,adjacent tissues and cells were detected by Realtime-PCR.3 Expression,survival and essential features of lncRNA NR0 in database by Bioinformatic analysis.4 Proliferatiom,migration,invasion and clone formation abilities of gastric cancer cells with lncRNA NR0 over-expression or low-expression were detected by RTCA-MP,transwell,matrigel chamber and colony-forming assay,respectively.5 Stably transfected SGC-7901 and MGC-803 with a lentivirus construct containing lncRNA NR0 low-expression,and detected the proliferation and clone formation abilities.6 SGC-7901 cells stably low-expression lncRNA NR0 were injected subcutaneously into nude mice and observe tumors proliferation rate.Results:1.Realtime-PCR demonstrated that the inflammatory factors IL-8,TNF-?and IL-1?elevated after added H.pylori 72h,and lncRNA NR0 was higher detected simultaneously.2.Realtime-PCR demonstrated that expression levels of lncRNA NR0 in gastric cancer tissues were higher than those in the adjacent normal gastric tissues?P<0.05?.The expression levels of lncRNA NR0 in gastric cancer cell lines were higher than those in normal gastric epithelial cells?P<0.05?.3.In TCGA database,the expression levels of lncRNA NR0 in gastric cancer tissues were higher than noncancer tissues.Kaplian-Meier survival curve demonstrated that high-expression lncRNA NR0 were related to low-survive.lncRNA NR0 had conflict secondary structure and didn't have the ability to encode protein.4.Low-expression of lncRNA NR0 inhibited proliferation abilities of SGC-7901 and MGC-803?P<0.05?,while over-expression of lncRNA NR0promoted their proliferation abilities?P<0.05?.Transwell migration assays demonstrated that low-expression of lncRNA NR0 inhibited migration and invasionabilitiesofSGC-7901andMGC-803?P<0.05?,while over-expression of lncRNA NR0 promoted their migration and invasion abilities?P<0.05?.Colony formation assay demonstrated that low-expression of lncRNA NR0 inhibited clone formation of SGC-7901 and MGC-803?P<0.05?,while over-expression of lnc RNA NR0 promoted their clone formation abilities?P<0.05?.5.The SGC-7901 and MGC-803 stably low-expression lncRNA NR0demonstrated decreased proliferation and clone formation abilities?P<0.05?.6.The growth rate of nude mice transplanted tumors in sh-NR0 group was significantly lower than that in control group?P<0.05?.Part Two Study on the mechanism of lncRNA NR0 in gastric cancer cellsObjective:To study the mechanism of interaction between lncRNA NR0and proteins in order to investigate the possible role of lncRNA NR0 in promoting cancer.Methods:1 Flow cytometry assay detected the effect of low-expression lncRNA NR0 on cell cycle and apoptosis of SGC-7901and MGC-803 cells.Western blot detected the cell cycle and apoptosis associated protein.2 PARIS kit detected the distribution of lncRNA NR0 in cytoplasm and nucleus.3 RNA-pulldown combining with mass spectrum were performed to detect lncRNA NR0 binding proteins.4 lncRNA NR0 binded to NF45/NF90 was verified by RIP and unlabelled lncRNA NR0 competive binding assay,and the specific binding site was detected by delete mapping assay.5 Western blot detected the effect of low-expression and over-expression lncRNA NR0 on the expression of NF45/NF90.6 Western blot and real-time PCR detected the effect of low-expression NF90 on expression of lncRNA NR0.7 IP detected the effect of lncRNA NR0 on NF45/NF90 compound.8 CHIP and real-time PCR detected the effect of low-expression lncRNA NR0 on the binding of NF45/NF90 and the promoter of IL-2 geen.Results:1.Flow cytometry analysis revealed that compared with the control group,the apoptosis of low-expression lncRNA NR0 were significantly increased in SGC-7901 and MGC-803 cell?P<0.05?.In addition,compared with the control group,there was an increased relative proportion of low-expression lncRNA NR0 SGC-7901 and MGC-803 cells in G₂/M phase?P<0.05?.2.PARIS kit demonstrated that lncRNA NR0 was distributed in cytoplasm and nucleus equably.3.RNA-pulldown combining with mass spectrum demonstrated that lncRNA NR0 could bind NF45/NF90 protein.4.RIP and unlabelled lncRNA NR0 competive binding assay further confirmed that lncRNA NR0 could bind NF45/NF90 protein.Delete mapping assay verified the specific binding site.5.Low-expression and over-expression lncRNA NR0 could not affect the expression of NF45/NF90.6.In contrast with the control group,low-expression NF90 would decrease the expression of lncRNA NR0?P<0.05?.7.IP assay demonstrated that low-expression lncRNA NR0 would destroy NF45/NF90 compound?P<0.05?.8.CHIP assay demonstrate that low-expression lncRNA NR0 would decrease the binding of NF45/NF90 with the promoter of IL-2?P<0.05?.Conclusion:Infected with H.pylori elevated the expression of lncRNA NR0.Simultaneously,the levels of lncRNA NR0 in gastric cancer tissues and cells were up-regulation.And in vitro and in vivo can promote the proliferation of gastric cancer cells.In vivo can also promote the migration and invasion of gastric cancer cells.In addition,lncRNA NR0 can bind to NF45/NF90 to induce the development of gastric cancer.