Research Into The Microrna-223Overexpression In H.pylori-associated Gastric Cancer And Into Its Promotion To Cancer Cell Proliferation And Migration

BackgroundsGastric cancer is one of the most common and the second deadly malignanciesworldwide, threatening human health, and leads the morbidity for all malignancies inChina. Because of its asymptoms, it is rarely detectable at its early stage, and has apoor prognosis. There are1/4death cases being caused by the gastric cancer, which isthreatening the health of Chinese in particular. However, the pathogenesis of gastriccancer is not clear, and is ascribed to multiple factors, such as genetics, environmentalpollution and life habits. Studies confirmed that the gastric infection with H. pylorimight be one of the most important environmental factors. The Lipopolysaccharide(LPS), Cytotoxin-associatedgeneA (CagA) and other virulent factors can promotesustained chronic gastritis, gastric erosions or ulcers; can enhance the proliferation ofgastric cancer cells and promote progression of gastric cancer.microRNAs (miRNAs or miRs) are a group of intrinsic single-stranded smallnon-coding RNA molecules with a length of18-24nt, can regulate the mRNAexpression of targeted gene, and thus promote the proliferation, apoptosis,differentiation of cells. microRNAs are associated with various types of diseases,especially closely with tumor occurrence, development and metastasis. Recently,accumulating reports indicate that microRNAs play important regulatory roles in thedevelopment of gastric cancers, and there is significant difference in the microRNAexpression profiling between gastrointestinal cancers and normal gastrointestinaltissues. For example, microRNA-223, which is the first confirmed microRNA inbioinformatics, was overexpressed in Barrett’s esophagus, colony cancers, gastriccancers and other gastrointestinal diseases, and was valuable in the diagnosis ofmalignant tumors of the gastrointestinal tract.Given the high importance of H.pylori’s roles in the tumogenesis of gastriccancers, and in order to reveal the pathogenesis of gastric cancers, we need to explore the regulation by H.pylori infection on the microRNA profile which is closelyassociated with gastric cancers. And in the present study, we compared themicroRNA-223between gastric cancers with H.pylori-positive and those withH.pylori-negative, and investigated the correlation of microRNA-223expression withclinic-pathological items and prognosis. Then we explored the regulation of H.pyloriinfection on microRNA-223expression, and investigated the regulatory role ofmicroRNA-223on the proliferation, migration and other characteristics. The presentstudy reveals the role of microRNA-223in the pathogenesis and moleculardeterminants in gastric cancers. There are three chapters, with detailed objectives,methods, results and conclusions are as following.Chapter I microRNA-223expression in H.pylori-associated gastric cancersI ObjectivesThe present study is to explore the association of H.pylori infection with themicroRNA-223expression in gastric cancers, and to investigate the role ofmicroRNA-223in the pathogenesis of gastric cancers.II MethodsWe determined the expression in both mRNA and protein levels of CagA, whichwas associated with the H.pylori infection with Real-time quantitative PCR andwestern blot assay, and screened the cancer specimens with H.pylori-positive or withH.pylori-negative. Then we quantified the microRNA-223expression levels in bothgroups of specimens. Finally, we analyzed the association of microRNA-223expression with survival time and other clinicopathological characteristics, usingKaplan–Meier and other statistical methods.III Results38gastric cancer specimens with H.pylori-positive and27gastric cancerspecimens with H.pylori-negative were identified; the expression of CagA in bothmRNA and protein levels was significantly higher in H.pylori-positive specimensthan in H.pylori-negative specimens.There was a significantly higher level of microRNA-223expression in theH.pylori-positive specimens than in the H.pylori-negative specimens. ThemicroRNA-223level was2.23folds high in former group than which in the latter group.The microRNA-223expression level closely correlated with such clinicopathological characteristics as tumor size, tumor migration and TNM stage,whereas not with the patient age, cell differentiation and PT stage. And theaverage survival time in the microRNA-223-overexpressed patients was markedlyless than in control patients.IV ConclusionsmicroRNA-223expression was closely associated with the H.pylori infection, andcorrelated with the clinicopathological characteristics of gastric cancers. It implies theimportant role of microRNA-223in the development and progression of gastriccancers. Chapter II H.pylori infection induce microRNA-223expression in vitro in gastriccancer cellsI ObjectivesThis part of study was to investigate the induction by H.pylori infection to themicroRNA-223expression and the expression of microRNA procession-relatedDrosha and Dicer.II MethodsRT-qPCR was utilized to quantify the microRNA-223expression levels in thegastric cancer cell lines, with or without H.pylori infection. RT-qPCR and westernblotting were adopted to examine the promotion of H.pylori infection to the expression ofDrosha and Dicer in both mRNA and protein levels in SGC-7901cancer cells.III ResultsH.pylori infection promoted the microRNA-223expression in AGS, SGC-7901and MKN-4538gastric cancer cells.There was a time-dependence of the microRNA-223expression promotion byH.pylori infection.H.pylori infection promoted the expression of Drosha and Dicer in SGC-7901gastric cancer cells in both protein and mRNA levels.IV ConclusionsH.pylori infection induced the microRNA-223expression in AGS, SGC-7901and MKN-4538gastric cancer cells, and promoted the expression of microRNAprocession-related Drosha and Dicer. Chapter III Influence of microRNA-223on the proliferation and migration ofgastric cancer cellsI ObjectivesTo explore the molecular mechanism of the microRNA-223promotion to thetumorgenesis of gastric cancers, we explored the influence of microRNA-223mimicstransfection on the proliferation and migration of gastric cancer cells.II MethodsMTT assay was performed to examined the influence of the transfection withmicroRNA-223mimics or control microRNA mimics on the cellular viability; CCK-8assay and colony forming assay were performed to investigated the influence ofmicroRNA-223on the proliferation of gastric cancer cells; the scratch assay wasperformed to determined the influence of microRNA-223on the migration of gastriccancer cells; transwell assay and invasion assay were performed to determine theinfluence of microRNA-223on the invidity of gastric cancer cells.III ResultsThe transfection with either microRNA-223mimics or with the controlmicroRNA mimics reduced the viability of gastric cancer cells, and there was nosignificant difference between the two mimics.The transfection with microRNA-223mimics promoted the proliferation and thecolony formation of gastric cancer cells.The transfection with microRNA-223mimics promoted the migration andinvasion of gastric cancer cells.IV ConclusionsThe transfection with microRNA-223mimics promoted the proliferation, colonyformation, migration and invasion of gastric cancer cells.