| Objective:Gastric cancer is a malignant disease with high morbidity and mortality,which seriously threatens human health.Gastric adenocarcinoma is the main classification.H.pylori infection is known as a major risk factor for the development of gastric cancer.H.pylori infection does not always result in cancer,and the primary factor for this is that different H.pylori genotypes have varied harmful consequences.causes.Previous studies have found that patients infected with Hp strains cagA~+have a higher risk of developing gastric cancer and precancerous lesions than patients infected with Hp strains cagA~-.Recent research data have shown that micro RNAs(miRNAs)play a key role in tumor development.At present,there are fewer studies on miR-1224-5p,mainly involving the functional role in the development of malignant tumors such as preventing scarring controlling silicon-induced lung fibrosis,colorectal cancer,hepatocellular liver cancer,osteosarcoma,and melanoma.To date,there have been no studies and reports on miR-1224-5p and cagA~+Hp-associated gastric carcinogenesis have been published.In this paper,we initially explored the effect of miR-1224-5p on cagA~+Hp-associated gastric cancer in gastric cancer.Methods:1.The differential expression of miRNAs in cagA~+Hp human gastric cancer tissues and their paracancerous tissues was compared by pre-collection of clinical samples applying second-generation sequencing.Ten core miRNAs were identified by using│logFC│>1 and P2.Nineteen tissue samples with postoperative pathological diagnosis of gastric adenocarcinoma were collected from the First Affiliated Hospital of Kunming Medical University from May 2021 to November 2021,of which 12 were male and 7were female.,and gastric cancer tissues were classified into cagA~+Hp and cagA-Hp using RTq-PCR,and the differences in expression levels of miR-1224-5p in gastric cancer tissues and paraneoplastic tissue specimens were verified.3.3.To explore the effect of cagA~+Hp on miR-1224-5p expression at the cytological level.The effect of cagA~+Hp on miR-1224-5p expression in GES-1,AGS was analyzed.The ratio of cell number to cagA~+Hp colony concentration was 1:0,1:50,1:100,and cagA~+Hp was co-cultured with GES-1 and AGS for 48 h.The ratio of cell to cagA~+Hp colony concentration was 1:100,and cagA~+Hp was co-cultured with GES-1 and AGS for 0 h,24 h,and 48 h.The ratio of cell number to cagA~+Hp colony concentration was 1:100,and cagA~+Hp was co-cultured with GES-1 and AGS for 0 h,24 h,and 48 h.4.cagA~+Hp plasmid was transfected with GES-1 and AGS cell lines,and the effect of cagA~+Hp on miR-1224-5p expression was analyzed.5.Lentiviral transfected AGS cells were constructed and named as miR-NC group,miR-1224 group,anti-NC group,anti-1224 group,and after sieved into stable transfectants with puro,and co-cultured with cagA~+Hp at 1:100 for 24 h and named as miR-NC+cagA~+Hp group,miR-1224+cagA~+Hp group,respectively The miR-NC+cagA~+Hp group,anti-NC+cagA~+Hp group,anti-1224+cagA~+Hp group.And the transiently transfected AGS cells were constructed and named as NC group,mimics group,NC inhibitor group and inhibitor group respectively6.Western blot detected the expression of autophagy proteins Beclin1,ATG5,LC3B,P62.7.The number of cellular autophagic vesicles in each group was detected by transmission electron microscopy,and the number of microtubule-associated protein light chain 3(GFP-LC3)spots in each group was observed by immunofluorescence microscopy and fluorescence confocal microscopy.8.The effects of miR-1224-5p-NC,mimics,NCinhibitor and inhibitor on the value-added and apoptosis of gastric cancer cells were detected by using CCK8 assay,clone formation assay and flow cytometry.9.All experimental data were analyzed using Graph Pad Prism 6.0 software.p<0.05was statistically significant.Results:1.The results of bioinformatics analysis by pre-collection of clinical samples showed that 10 core miRNAs,including 5 down-regulated miRNAs(miR-1224-5p,miR-876-3p,miR-363-5p,2 miR-129-2-3p,miR-135a-5p)and 5 up-regulated miRNAs(miR-548ar-3p,miR 3690,miR-708-3p,miR-335-3p,miR-584-5p)as miRNAs that are closely associated with cagA~+Hp-associated gastric carcinogenesis.We verified the expression of 10 core miRNAs in cagA~+Hp,cagA~-Hp normal gastric mucosa,chronic non-atrophic gastritis,chronic atrophic gastritis,gastric precancerous lesions(heterogeneous hyperplasia)and gastric cancer tissues.The results showed that compared with the cagA-Hp group only miR-1224-5p was relatively low expression in the cagA~+Hp group.2.RT-PCR results showed that miR-1224-5p expression in cagA~+Hp gastric cancer tissues was lower than that of adjacent tissues.the expression of miR-1224-5p in AGS cells gradually decreased with the prolongation of cagA~+Hp infection time and the increase of infection concentration.The expression of miR-1224-5p in AGS cells was lower than that in GES after transfection of cagA~+plasmid.3.Western blot detected the expression of autophagy proteins Beclin1,ATG5,LC3B,P62 and the result showed that the expression of Beclin1,ATG5,was increased and promoting LC3Ⅰ to change to LC3Ⅱ of LC3B and the expression of P62 was overexpressing in AGS cells about miR-1224-5p.And while knocking down results were opposite.4.The result of transmission electron microscopy results showed that autophagsome increased in autophagic vesicles in the miR-1224-5p overexpression group,thenautophagsome decreased after knockdown and co-culture with cagA~+Hp The same results were obtained by immunofluorescence microscopy and fluorescence confocal microscopy by observing the number of green fluorescent protein-light chain protein 3(GFP-LC3)spots.5.miR-1224-5p inhibitor promotes cell proliferation and inhibits apoptosis and.Promotes the development of gastric cancer.Conclusion:It can be concluded that cagA~+Hp infection leads to down-regulation of miR-1224-5p expression to inhibit autophagy and promote value-added to reduce apoptosis.This promotes the development of gastric cancer. |
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