Hsa＿circ＿0006135 Regulates MiR-1236-3p/MTA2 Axis Promote Progression Of Gastric Cancer

Background and Objective :Gastric cancer is a common malignant tumor of the gastrointestinal tract.In recent years,some progress has been made in the diagnosis and treatment of gastric cancer.However,the prognosis of gastric cancer is still poor because of tumor metastasis and invasion.Many studies have found that circular RNAs(circRNAss)are abnormally expressed in gastric cancer tissues or cell lines,which is an important molecular mechanism for the occurrence and development of gastric cancer,and may also be an important biomarker for early diagnosis,prognosis and targeted therapy of gastric cancer in the future.The purpose of this study was to explore the role of hsa＿circ＿0006135 in GC proliferation,migration,invasion and epithelial-mesenchymal transition(EMT),and its specific mechanism affecting GC progression.Methods :1.Screening and clinical correlation analysis of hsa＿circ＿0006135(1)High-throughput sequencing was used to detect differentially expressed circRNAss in GC tissues and corresponding normal gastric mucosa tissues;(2)Real-time quantitative PCR(qRT-PCR)was used to detect the expression of hsa＿circ＿0006135 in GC tumor tissues and corresponding normal gastric mucosal tissues,GC cell lines and normal gastric mucosal epithelial cells.The relationship between the expression level of hsa＿circ＿0006135 in GC tissues and the clinical characteristics(age,gender,tumor size,lymph node metastasis,TNM,vascular invasion)of GC patients was analyzed;(3)RNase R digestion assay was used to identify the hsa＿circ＿0006135 circular structure.2.In vitro experiments were used to explore the effect of hsa＿circ＿0006135 on the malignant behavior of GC cells.(1)The experiment was divided into two groups : si-circ＿0006135 group and si-NC group.(2)Tumor cell functional experiments were used to evaluate the effects of the above interventions on the proliferation,migration,and invasion of gastric cancer cells;WB experiments were used to detect the expression of EMT-related proteins after the above group intervenes.3.The mechanism of hsa＿circ＿0006135 regulating the malignant behavior of GC cells was explored in vitro.(1)Experimental group design :(1)In order to prove that hsa＿circ＿0006135 regulates GC progression as an competitive endogenous RNA(ce RNA)of miR-1236-3p,the groups were as follows : circ＿0006135-WT +miR-1236-3p group,circ＿0006135-WT + miR-NC group,circ＿0006135-MUT + miR-1236-3p group,circ＿0006135-MUT + miR-NC group,si-circ＿0006135 group,si-NC group,miR-1236-3p group,anti-miR-1236-3p group,anti-NC group,si＿circ＿0006135 + anti-miR-1236-3p group and si＿circ＿0006135 + anti-NC group.(2)To prove that hsa＿circ＿0006135 regulates GC development through the miR-1236-3p /MTA2 axis,the groups were as follows : MTA2 3’UTR-WT + miR-1236-3p group,MTA23’UTR-WT + miR-NC group,MTA2 3’UTR-MUT + miR-1236-3p group,MTA2 3’UTR-MUT +miR-NC group,miR-1236-3p group,miR-NC group,pcDNA group,pcDNA-MTA2 group.pcDNA-MTA2 + miR-1236-3p group and pcDNA + miR-1236-3p group.(2)The targeting relationship of hsa＿circ＿0006135 to miR-1236-3p and miR-1236-3p to MTA2 mRNA was proved by dual luciferase gene reporting experiment.The expression of hsa＿circ＿0006135 and miR-1236-3p in GC cell lines was detected by qRT-PCR.The effects of the above interventions on the proliferation,migration and invasion of GC cells were evaluated by tumor cell functional experiments.WB experiment can be used to detect the effect of the above intervention on the expression of EMT-related proteins and MTA2 protein.4.In vivo experiments to explore the effect and mechanism of hsa＿circ＿0006135 on GC tumor formation.The GC cell line HGC-27,which was successfully transfected with si-circ＿0006135 and si-NC,was used to establish a mouse xenograft subcutaneous tumor model.The tumor volume and weight were monitored.QRT-PCR was used to detect the expression of hsa＿circ＿0006135and miR-1236-3p in subcutaneous tumors.WB experiments can be used to detect MTA2 protein expression.Results :1.Hsa＿circ＿0006135 was highly expressed in GC tumor tissues and GC cell lines.The high expression of hsa＿circ＿0006135 was related to tumor size,lymph node metastasis and TNM stage of GC patients,but not to gender,age,distant metastasis and vascular invasion.2.The knockdown of hsa＿circ＿0006135 inhibited the proliferation,migration and invasion of GC cells,promoted the expression of E-cadherin,and hindered the expression of Vimentin and N-cadherin.3.Mi R-1236-3p mimic significantly reduced the luciferase activity of hsa＿circ＿0006135-WT vector,but had no effect on the luciferase activity of hsa＿circ＿0006135-MUT vector.The expression of miR-1236-3p was low in GC cells,and the knockdown of hsa＿circ＿0006135increased the expression of miR-1236-3p.4.Mi R-1236-3p inhibitor reversed the effects of hsa＿circ＿0006135 knockdown on the proliferation,migration and invasion of GC cells,reversed the inhibition of Vimentin and Ncadherin protein expression,and reversed the promotion of E-cadherin protein expression.5.Mi R-1236-3p mimic significantly reduced the luciferase activity of MTA2 3’UTR-WT vector,but had no effect on the luciferase activity of MTA2 3’UTR-MUT vector.The expression of MTA2 protein was significantly higher than that of adjacent normal tissues and GC cells.Inhibition of miR-1236-3p can promote the expression of MTA2 protein,while overexpression of miR-1236-3p can inhibit the expression of MTA2 protein.6.Overexpression of miR-1236-3p inhibited the proliferation,migration and invasion of GC cells,and these phenomena were reversed after overexpression of MTA2.Overexpression of miR-1236-3p increased the expression of E-cadherin and decreased the expression of Vimentin and N-cadherin in GC cells,which could also be reversed by MTA2 overexpression.7.The silencing of hsa＿circ＿0006135 can significantly inhibit the expression of MTA2 protein in GC cells,which can be reversed by miR-1236-3p inhibitor.8.After knocking down hsa＿circ＿0006135,the volume and weight of subcutaneous tumors in mice decreased,the protein levels of hsa＿circ＿0006135 and MTA2 in subcutaneous tumors decreased,and the level of miR-1236-3p increased.Conclusion :Hsa＿circ＿0006135 acts as a sponge for miR-1236-3p and positively regulates MTA2,thereby promoting the proliferation,migration,invasion and EMT process of GC.Hsa＿circ＿0006135 may become a therapeutic target for GC.