| Objective: Gastric cancer is one of the most common malignancies in digestive tract diseases and the third deadliest malignant tumor worldwide.gastric cancer in early stage often lacks specific clinical manifestations,which delays the best treatment time,leading to the development of advanced gastric cancer,which greatly reduces the fiveyear survival rate of patients.Although the diagnosis and treatment level of gastric cancer has been improved after a long period of technical development and experience accumulation,the 5-year survival rate after radical gastrectomy for advanced gastric cancer is still hovering around 30%-50%.Therefore,it is an urgent problem in the field of the diagnosis and treatment of gastric cancer to explore the molecular mechanism of the gastric cancer to search for molecular markers with higher diagnostic efficacy,and provide new targets for the clinical treatment of gastric cancer.CircRNA is a class of endogenous RNA molecules without 5' cap and 3' poly(A)tail.They are non-coding RNA molecules with complete closed-loop structure formed by "reverse shear" mechanism,and are stable,abundant,and conserved RNA molecules.CircRNA has been confirmed to have an endogenous competitive RNA mechanism with mi RNAs.In addition,circRNA can also play a role by translating proteins or regulating transcription.More and more studies have found that circRNA molecules are widely involved in the regulation of tumor development and progression.Based on our previous circRNA chip data,we investigated the expression of circ?0049447 in gastric tumor tissues and paired adjacent non-tumorous tissues,and further investigated the relationship between the expression level of circ?0049447 and the clinicopathological characteristics and prognosis of gastric cancer patients.The effects of circ?0049447 on the proliferation,migration and invasion of gastric cancer cells and the mechanism of circ?0049447 in gastric cancer were also investigated.Methods: Tissue samples were collected from 80 patients who underwent radical gastrectomy in the First Affiliated Hospital of China Medical University between 2016 and 2017.All the patients were confirmed as gastric cancer by postoperative pathology.None of the patients received neoadjuvant chemotherapy or radiotherapy before surgery.During the operation,we collected tumor tissue samples and matched adjacent nontumorous tissues,which must be more than 5cm away from the tumor.We designed and synthesized specific primers for circ?0049447 using circBASE,NCBI databases and Primer Premier5.0 software across reverse shear sites.Real-time PCR and geNorm software were used to select the optimal internal reference,and the stability of the selected internal reference was verified.RNA in the tissues was isolated by TRIzol and further transcribed into cDNA.With sample of matching the carcinoma tissue adjacent to carcinoma as reference,real-time PCR was applied to detect the expression level of circ?0049447 in 80 patients with gastric cancer tissue samples.Wilcoxon rank test was used to compare the relative circ?0049447 expression level in two different organizations.Nonparametric test method was applied to analyze the correlation between circ?0049447 relative expression level and clinical pathological features.Manner-Whitney U test was used to compare age,sex,tumor factors.Kruskal-Wallis H test was used to compare the tumor site,pT grade,pN grade,pTNM stage and other pathological features.Kaplan-Meier function was used to draw the survival curve of the patients,and the correlation between the relative expression level of circ?0049447 and the overall survival rate was analyzed.ROC curve was drawn to evaluate the diagnostic value of circ?0049447.CircRNA overexpression vector was constructed and transfected into gastric cancer cell lines.The effects of circ?0049447 on the proliferation,migration and invasion ability of gastric cancer cells were detected by CCK-8 assay and Transwell assay.Results: The real-time quantitative PCR products of circ?0049447 were sequenced and the results were accurate.Of all the commonly used reference genes screened for optimal internal parameters,ACTB has the lowest stability M value,which is the optimal reference under current conditions,and the expression of ACTB remains stable after being placed at room temperature and repeated freeze-thaw treatments.The results of real-time PCR showed that the expression of circ?0049447 in gastric cancer tissues was significantly decreased(P < 0.001),the area under the ROC curve(AUC)=0.838,the sensitivity =0.823,and the specificity =0.772,compared with the paired noncancerous tissues.The expression level of circ?0049447 was related to N stage(P=0.029),and did not show any significantly correlation with other pathological factors.Transwell chamber experiment results showed that circ?0049447 could inhibit the migration and invasion ability of cells.The migration ability of gastric cancer cells after overexpressing circ?0049447 was weaker than that of the negative control group(P < 0.05),and the invasion ability of gastric cancer cells after overexpressing circ?0049447 was weaker than that of the negative control group(P < 0.05).CCK8 experiment results showed that circ?0049447 inhibited the proliferation of gastric cancer cells,and the proliferation of gastric cancer cells after overexpressed circ?0049447 was weaker than that of the negative control group(P<0.05).Conclusion: circ?0049447 was significantly down-regulated in gastric cancer tissues,showing high accuracy in the diagnosis of gastric cancer,and may be a potential new biomarker.The overexpression of circ?0049447 can significantly inhibit the proliferation,migration and invasion of gastric cancer cells,which may be a new target for the treatment of gastric cancer. |
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