Establishment Of H.pylori Infected Gastric Epithelial Cell Model And Study On The Relationship Between LncRNA NR₀26827 And Gastric Cancer

Objective:Helicobacter pylorus（H. pylori） has been shown to play a key role in gastric cancer. However, the molecular mechanisms of gastric cancer infected by H. pylori remain largely unknown. Furthermore, few nucleic biomarkers can be used for the gastric cancer diagnose until now. Long non-coding RNAs（lnc RNAs） are longer than 200 nucleotides in the length, and cannot be translated into the proteins. As lnc RNAs can regulate gene expression in the different levels, and then participate in regulating tumor cells apoptosis and tumor metastasis, thus they are considered as the potential biomarkers to study the mechanisms of tumor genesis and for the diagnosis of tumor. However, the reports associated with lnc RNAs used for the diagnosis and researching of gastric cancer are quite few. The aim of this study was to establish H. pylori infected gastric epithelial cell model firstly, and then to screen the high specific lnc RNA biomarkers for the diagnosis of gastric cancer. In addition, the relationship between lnc RNA and gastric cancer was studied preliminary.Methods:（1） Establishment of H. pylori infected gastric epithelial cell model. H.PyloriNCTC11637 standard strains were cultured by conventional micro aerobic method, and the human cell line U937 was routinely maintained in RPMI 1640. Then, H. pylori with the different concentrations of（100:1 MOI and 300:1 MOI） were used to infect U937 cells for 8, 24, 48 hours, respectively. Meanwhile, equal amount of PBS instead of H.pylori was used as control. The cell morphologic changes of U937 were observed by microscope. Then, the concentrations of IL-2, IL-4, IL-6, IL-10, IL-17 A, IFN-γ, and TNF were detected by Cytometric Beads Array（CBA） technology in normal U937 cell and H.pylori infected U937 cell. In additional, the cell apoptosis was analyzed and confirmed by flow cytometry.（2） Analysis of the expression profile of lnc RNAs in H. pylori infected cell.Human Lnc RNA Array v3.0（8×60K） chips of Arraystar Company were used to profile the lnc RNA expression in normal U937 cell and H.pylori infected U937 cell. The expression profile of lnc RNAs were obtained by data collecting, data standardization, cluster analysis and correlation analysis. Differences of the lnc RNA expression levels >2.0 was defined as up-regulated genes, or else as down-regulated genes.（3） Investigation of the expression of lnc RNA NR₀26827 in clinical samples and preliminary study on the relationship between lnc RNA NR₀26827 and gastric cancer. q RT-PCR was employed to compare the expression differences of lnc RNA NR₀26827 between H.pylori infected epithelial cells and normal cells. Furthermore, expression of lnc RNA NR₀26827 was also examined in 40 paired gastric cancer samples and adjacent non-tumor tissues by q RT-PCR. Based on the above data, we studied the expression levels of lnc RNA NR₀26827 in gastric cancer among different stages.Results:（1） H. pylori infected gastric epithelial cell model has been established. The results of cellular morphology showed that the infected time was no longer than 24 hours and the amount of H. pylori was no more than 100:1 MOI. Otherwise, cell morphology of infected cells would be observed for too long infected time and too much amount of H. pylori. Then, the concentration of cell factors showed that the cell factors IL-6 was significantly higher in H.pylori infected U937 cell than in normal U937 cell. And the concentration of IL-6 was highest when the amount of H. pylori was 100:1 MOI. So normal cellular morphology and significantly changes of IL-6 concentration can be obtained when cells were infected by H. pylori at 100:1 MOI for 24 hours. Moreover, the results of was flow cytometry showed that cellular apoptosis was no significantly changed after H. pylori infection. According to the data of these experiments, cells infected by H. pylori at 100:1 MOI for 24 hours can be used to profile the lnc RNA expression levels by microarray assay.（2） The lnc RNA expression levels between H. pylori infected cell and normal cells were detected by microarray assay. The expression differences of the 79 up-regulated and 52 down-regulated lnc RNAs are more than two folds, while the expression differences of 9 up-regulated and 10 down-regulated are more than five folds. Among these 19 lnc RNAs, the hybridizing signals of 18 lnc RNAs were very weak, indicating the expression levels of the 18 lnc RNAs were very low. But the hybridizing signals of the lnc RNA NR₀26827（Down-regulated with 4841.34 times） is as high as 315135.25.（3） Confirmation of the expression of lnc RNA NR₀26827 in clinical samples and the relationship between lnc RNA NR₀26827 and gastric cancer. The results of q RT-PCR showed that the expression of lnc RNA NR₀26827 in H. pylori infected gastric epithelial cells is lower than that of non-infected cells, which is consistent with the results detected by microarray assay. Lnc RNA NR₀26827 can be detected in gastric cancer tumor tissues, and the expression level was also greatly reduced compared with adjacent non-tumor tissues, which suggest that lnc RNA NR₀26827 is a potential biomarker for diagnosis of gastric cancer. In correlation analysis of the expression levels of lnc RNA NR₀26827 among different stages of gastric cancer, no obvious expression differences of lnc RNA NR₀26827 was observed in different stages of gastric cancer.Conclusions:（1） The model of H. pylori infected gastric epithelial cell was successfully established. Normal cellular morphology and significantly changes of IL-6 concentration can be obtained when cells were infected by H. pylori at 100:1 MOI for 24 hours.（2） The expression of lnc RNAs in H. pylori infected cell were successfully profiled. Lnc RNA NR₀26827, which may be important for gastric tumor genesis and diagnosis, was screened out.（3） The expression level of lnc RNA NR₀26827 was greatly reduced in gastric cancer tumor tissues compared with adjacent non-tumor tissues and the expression level has no obvious differences among different stages of gastric cancer, suggesting that lnc RNA NR₀26827 may be a potential lnc RNA biomarker for gastric cancer diagnosis and lnc RNA NR₀26827 may participate in the whole process of gastric cancer.