IncRNA Promotes Helicobacter Pylori-related Gastric Cancer Through Epigenetic Regulation Of VPS11

IntroductionThere are about 1 million newly diagnosis cases of gastric cancer worldwide every year,of which 47%are found in China,so gastric cancer is a serious threat to human health and quality of life.Helicobacter pylori?H.pylori?is class ? carcinogen and more than half the world's population is infected H.pylori.The prevalence of H.pylori infection in China range from 41.35%to 72.3%.H.pylori infection can lead to chronic inflammation of the gastric mucosa,resulting in atrophic gastritis,intestinal metaplasia or intraepithelial neoplasia,which may develop cancer.Therefore,a comprehensive study of the pathogenesis of H.pylori related gastric cancer has important scientific value and clinical significance.long non-coding RNAs?lncRNAs?are a class of RNA whose molecules is longer than 200 bases without coding proteins,but they have complex biological functions.More and more studies show that mutation and abnormal expression lncRNA can lead to multiple diseases,including cancer,but their role in the pathogenesis of H.pylori is rarely reported.Epigenetic refers to heritable change of gene expression without DNA sequence change.Epigenetic mechanisms have been extensively studied including DNA methylation,non-coding RNA,histone modification,chromatin remodeling.Usually,there is not single epigenetic modification involving in potential mechanism.More and more studies show that in the process of tumor formation,1ncRNA could involve in other epigenetic regulations forming a epigenetic regulatory network.In previous studies we have collected H.pylori+and H.pylori-gastric carcinoma to perform lncRNA microarray and DNA methylation microarray.By cross-analysis if two sets microarray results,we found 14 pairs of IncRNA and abberrant methylation genes in close positions.Among them,lncRNA NR 033122 and VPS 11 are located closest,both in chromosome11,suggesting that lncRNA NR 033122 have a potential role of silence VPS11 by epegentic manner in H.pylori related gastric cancer.Objective1.To screen the abberrant IncRNA expression profile in H.pylori related gastric cancer and to vertify expression of key IncRNA.2.To screen the abberrant methylation gene profile in H.pylori related gastric cancer.To select key genes which have potential links to IncRNAs by cross-analysis and vertify methylation level of key genes.3.To observe the influence of key IncRNA and abberrant methylation gene on phenotype of gastric cancer cells,respectively.4.To study the mechanism underlying the epigenetic regulation of gene by key IncRNA in H.pylori related gastric cancer.Methods1.Human IncRNA Microarray was used to compare the lncRNA expression profile difference between 3 surgical remocal H.pylori+and 3 H.pylori-gastric cancer tissue.2.Infinium Methylation BeadChip was used to compare the DNA methylation profile difference between the same gastric cancer tissue.3.Selection potential lncRNA,which could involve in epigenetic regulation by cross-analysis.4.Real-time PCR was used to detect IncRNA NR₀33122 expression in 60 gastric cancer sample and 60 chronic gastritis sample.Pyrosequencing was used to detect the methylation level of VPS 11 promoter,VPS 11 mRNA and protein was detected by real-time PCR and Western blot,respectively.5.GES-1,SGC-7901,MGC-803,MKN-45 and AGS was stimulated by H.pylori,and were detected as previously mentioned.6.H.pylori infection mice model was constructed and sacrificed at 2m,6m,12m after H.pylori infection.Real-time PCR was used to detect homology IncRNA NM₁33226.2.Pyrosequencing was used to detect the methylation level of VPS 11 promoter,VPS11 mRNA and protein was detected by real-time PCR and Western blot,respectively.7.IncRNA NR₀33122 siRNA and over-expression plasmid was constrcted and transfected into SGC-7901,MGC-803 and AGS cells.Real time PCR and Western Blot were used to detect transfection efficiency.Then CCK-8 was used to evaluate proliferation and Transwell invasion assay was used to evaluate invasion.8.VPS 11 siRNA and over-expression plasmid was constrcted and transfected into SGC-7901,MGC-803 and AGS cells.Real time PCR and Western Blot were used to detect transfection efficiency.Then CCK-8 was used to evaluate proliferation and Transwell invasion assay was used to evaluate invasion.9.IncRNA NR 033122 siRNA and over-expression plasmid was transfected into gastric cancer cells to detect methylation level of VPS 11 promoter,VPS 11 mRNA and protein level and DNMTs protein level as previously mentioned.10.Tumor formation and transfer experiment by tail vein injection in nude mice were used to observe the influence of Inc RNA NR 033122 and VPS 11 on gastric cancer cell proliferation and invasion in vivo.11.To predict target gene of lncRNA NR₀33122 by bioinformation anlysis and be vertifed in vitro.12.RNA pulldown was used to confirm the binding between Inc RNA NR 033122 and NF-?B after lncRNA NR₀33122 over-expression plasmid transfection.13.Co-IP was used to confirm the binding between Inc NF-?B and DNMTs after H.pylori stimulation.Results1.46 up-regulated and 80 down-regulated IncRNA was found in H.pylori related gastric cancer by IncRNA microarray.2.86 hypermethylation and 62 hypomethylation gene was found in H.pylori related gastric cancer by DNA methylation microarray.3.Cross-analysis found 14 pair of IncRNA and aberrant methylation gene in a close position,and we selected IncRNA NR₀33122 and VPS11 to do further research.4.Clinical sample vertification:lncRNA NR₀33122 and VPS 11 were down-regulation in gastric cancer tissue?P<0.01?and both of them could be reduced by H.pylori infection.The methylation level of VPS 11 promoter in gastric cancer tissue was higher than chronic gastritis tissue?P<0.01?,meanwhile mRNA and protein level of VPS 11 were decreased?P<0.01?.There was a negative correlation between IncRNA NR₀33122 expression and VPS 11 methylation level.There was a positive correlation between IncRNA NR₀33122 expression and VPS 11 expression.5.Cell model vertification:expression of IncRNA NR₀33122 in SGC-7901,MGC-803,MKN-45 and AGS was down-regulated after H.pylori stimulation?P<0.01?.The methylation level of VPS 11 promoter was increased in all cell lines?P<0.01?.mRNA and protein level of VPS11 were decreased in all cell lines?P<0.01?.6.Mice model vertification:expression of IncRNA NM₁33226.2 was down-regulated in H.pylori infection mice model?P<0.01?.The methylation level of VPS11 promoter was higher in H.pylori infection group?P<0.01?and VPS11 mRNA was lower?P<0.01?.7.Low-expression of IncRNA NR 033122 promoted proliferation and invasion in gastric cancer cells,however over-expression of IncRNA NR₀33122 inhibited proliferation and invasion.8.Low-expression of VPS 11 promoted proliferation and invasion in gastric cancer cells,however over-expression of VPS 11 inhibited proliferation and invasion.9.Low-expression of IncRNA NR₀33122 induced hypermethylation of VPS 11 promoter and down-regulated expression of VPS 11 mRNA and protein.While over-expression of IncRNA NR₀33122 induced opposite results.10.Low-expression of IncRNA NR₀33122 and VPS 11 promoted proliferation of gastric cancer cells in vivo?P<0.01?,while no siginificant result was observed in cell invasion.11.NF-?B could be potential target gene of Inc RNA NR 033122 according to bioinformation analysis and H.pylori infection could activate KF-KB.12.The binding between IncRNA NR 033122 and NF-?B was confirmed by RNA pulldown after transfection of IncRNA NR 033122 over-expression plasmid.13.The binding between NF-?B and DNMT1,DNMT3a respectively were confirmed by co-IP after H.pylori stimulation.Conclusions1.H.pylori infection decrease IncRNA NR₀33122 expression and IncRNA NR 033122 plays a role of tumor suppressor in gastric cancer.2.H.pylori infection silence VPS 11 by hpyermethylation and VPS 11 plays a role of tumor suppressor in gastric cancer.3.lncRNA NR 033122 and VPS11 have a cloes position and IncRNA NR₀33122 involves in epigenetic regulation of VPS 11.4.H.pylori infection down-regulate lncRNA NR₀33122,which activates NF-?B.NF-?B recrutes DNMT1 and DNMT3a,which induce hypermethylation of VPS 11 promoter in gastric cancer.