CagA Of H Pylori Promotes Gastric Mucosal Epithelial Cell Malignant Transformation Via MiR-155 Induced KLF4 Down Expression

【BACKGROUNDS】Gastric cancer is one of the common types of cancer worldwide,accounting for8% of the cancer cases and the second leading cause of cancer-related death worldwide.Moreover,>70% of gastric cancer related deaths occur in developing countries,especially in Eastern Asia.In china,The 5 leading causes of cancer death among both men and women are cancers of the lung and breast cancer,stomach,liver and colorectum,accounting for most of all cancer deaths that cause serious harm to human health.Gastric cancer arising as the result of multiple risk factors,such as host genetic polymorphisms,smoking or poor diet ect environment and epigenetic factors dysregulations.Persistent infection with Helicobacter pylori is the most important risk factor for the development of gastric malignancies by far,among which gastric adenocarcinoma is the most common type.But little is known about the pathogenesis,particularly how HP interacts with host gene to affect the development and progression of gastric cancer.Recent studies have indicated that Cag A positive and Vac A positive HP strains are considered to more virulent and associated with higher grades of gastric disease.Intracellular Cag A aberrantly activates number signaling pathways including NF-κB,resulting in many cellular responses,such as hyperproliferation and proinflammatory cytokine production,and induction of pathology.Although one half of individuals infected with HP,among which 3% progresses to gastric cancer.This suggests that the occurrence of gastric cancer is the result of the interaction between the infection of Helicobacter pylori and activation of oncogene or the inactivation of tumor suppressor gene in the host cell.Inactivetion of tumor suppressor genes and epigenetic alterations including point mutations,deletions,duplications,recombinations,and methylation of various tumor-related genes are the important mechanisms in inflammation induced carcinogenesis.MicroRNA,a class of small non-coding RNAs that negatively regulate gene expression by directly binding to the 3′untranslated region,involved in global posttranscriptional regulation.MiR-155,have recently emerged as crucial regulators of innate immunity and inflammatory responses.Meanwhile,recent reports show that during HP infection induced gastric disease,miR-155 plays an highlighted regulatory role.Recent studies have revealed that miRNAs have important roles in cancer development as either oncogenes or tumor-suppressor genes by regulating various cancer-related proteins or m RNA expressions.Accordingly,we conducted a preliminary study on the gene KLF4,and achieved some results.Kru¨ppel-like factor 4(KLF4)is a zinc-finger transcription factor.our result showed that Kruppel-like factor 4 expression in human gastric cancer cell and gastric tumor tissue was decreased.Significantly decreased apoptosis was detected in tumor tissues with decreased KLF4 expression.Overexpress Kru¨ppel-like factor 4 suppressed human gastric cancer growth and metastasis in vivo.Our data provide the evidence that the tumor suppressor gene KLF4 plays a critical role in gastric cancer development and progression.Interesting,KLF4 was predicted as targets for miR-155 by using Targetscan or NCBI base.We hypothesis that whether HP infection leads to the loss of KLF4 expression in gastric epithelial/cancer cells? how Cag A interacts with host genes KLF4 to affect the expression of KLF4? Is this process due to the involvement of miR-155 in posttranscriptional regulation?In order to explore the molecular mechanism of the expression of KLF4 in gastric cancer,this study was validated at the cellular level in combination with clinical cases.【Research methods】1.Detection of miR-155 in the gastric musco epithelial cell and gastric cancer cells.In order to evaluate the effect of H.pylori Cag A on KLF4,GES-1 cell were transfected with WT-Cag A or Alpha SR in defferernt time or dose,respectively.2.Gastric cancer or para-carcinoma sections were stained for KLF4 antibody to identify the expression of KLF4;3.Cag A plasmid transfection of GES-1 cell were carried out and the effects of Cag A on the GES-1 cell growth or migration were detected4.Measuring the level of miR-155 in 33 gastric cancer patients’ samples and 33 healthy controls by q RT-PCR.The expression of miR-155 in gastric carcinoma and adjacent tissues was detected by in situ hybridization.5.Expression in GES-1,AGS,MGC-803,SGC-7901,SK-GT5 cell lines was detected.The effect of Cag A on miR-155 expression was observed by agarose gel electrophoresis using ordinary PCR.6.The expression of KLF4 protein and m RNA was affected by miR-155 overexpression after transfected miR-155-3p minic/negative and miR-155-5p minic/negative.The vector containing wildtype(WT)or mutant 3′UTR of KLF4 was cotransfected into HEK293 cells with miR-155 mimics or NC.Fortyeight hours after transfection,luciferase activities were detected through the dual-luciferase analysis,and Renilla luciferase activity was used as internal control.7.we constructed gastric mucosa epithelial cell(GES-1)and gastric cancer cell line(AGS)stably expression of miR-155-5p minic/negative gene.Meanwhile,gastric cancer cell line SGC-7901 stably expression of miR-155inhibitor/negative was established using lentivirus vector.Analysis the effect of miR-155 on KLF4 expression by western bloting.【Experiment Result】1.Cag A inhibits KLF4 protein expression in time-/dose-dependent manner2.Decrease Kruppel-like factor 4 expression in gastric tumor tissue and human gastric cancer cell3.miR-155 is up-regulate in gastric cacner serum samples and gastric cancer tissues4.Cag A increase the proliferation of GES-1 gastric epithelial5.miR-155 is up-regulate in gastric cancer cell lines,High expression of Cag A induced miR-155 expression6.KLF4 is a direct target of miR?155 in GC cells7.The gastric mucosal epithelial cell line GES-1 and the gastric cancer cell line AGS stably expressing miR-155-5p-negative control & miR-155-5p minic were successfully constructed.A gastric cancer cell line SGC-7901 with stable expression of miR-155-5pnegative & miR-155-5p-inhibitor was established.MiR-155 was negatively correlated with KLF4 expression【Conclusion】CagA may be induced by miR-155 expression,targeted interference KLF4 transcriptional translation,inhibition of KLF4 expression,thereby promoting malignant transformation of gastric mucosal epithelium.