摘要
目的 探讨circGFRA1对胶质瘤细胞增殖、凋亡、侵袭的影响,分析miR-138-5p/膜联蛋白A2(ANXA2)信号通路在其中发挥的作用.方法 选取2020年1月至2022年1月河南省第二人民医院收治的72例脑胶质瘤患者以及72例行颅脑损伤内减压术的患者,分别取脑胶质瘤组织与脑正常组织,体外培养人神经星形胶质细胞株、人神经胶质瘤细胞(U251、CRT、HS683、H4),并将U251、H4细胞分为对照组、si-NC组、si-circGFRA1组、si-circGFRA1+inhibitor NC组、si-circGFRA1+miR-138-5p inhibitor组.用qRT-PCR法测定组织、细胞circGFRA1、miR-138-5p、ANXA2 mRNA水平;CCK-8法、流式细胞术、Transwell小室法分别测定U251、H4细胞吸光度(A450)值、凋亡率、侵袭细胞数目;双荧光素酶分析miR-138-5p和circGFRA1、ANXA2的靶向关系,Western blot法测定U251、H4细胞ANXA2蛋白表达.结果 与对照组比较,胶质瘤组织circGFRA1、ANXA2 mRNA表达升高,miR-138-5p表达降低(均P<0.05);与人神经星形胶质细胞株比较,U251、CRT、HS683、H4细胞的circGFRA1和ANXA2 mRNA表达水平升高,miR-138-5p表达降低(均P<0.05);与对照组比较,si-circGFRA1组U251、H4细胞的circGFRA1、ANXA2 mRNA与蛋白表达水平、A450值、侵袭细胞数降低,miR-138-5p、凋亡率均升高(均P<0.05);与si-circGFRA1组比较,si-circGFRA1+miR-138-5p inhibitor组U251、H4细胞circGFRA1、ANXA2 mRNA和蛋白表达、A450值、侵袭细胞数升高,miR-138-5p、凋亡率降低(均P<0.05);miR-138-5p与circGFRA1、ANXA2均存在靶向关系;与mimics NC组比较,miR-138-5p mimics组U251、H4细胞ANXA2 mRNA和蛋白水平下降(均P<0.05).结论 沉默circGFRA1可能通过调控miR-138-5p/ANXA2信号通路抑制胶质瘤恶性进展.
Abstract
Aim To investigate the influences of circGFRA1 on proliferation,apoptosis and invasion of glioma cells,and analyze the role of miR-138-5p/annexin A2(ANXA2)axis in it.Methods From January 2020 to January 2022,72 patients with brain glioma treated in our hospital and 72 patients undergoing internal decompression of brain injury were selected,and brain glioma tissues and normal brain tissues were taken respectively,in addition,human neuroastrocyte cell lines,human glioma cells U251,CRT,HS683,H4 were cultured in vitro,U251 and H4 cells were divided into a control group,a si-NC group,a si circGFRA1 group,a si-circGFRA1+inhibitor NC group,and a si-circGFRA1+miR-138-5p inhibitor group,the levels of circGFRA1,miR-138-5p and ANXA2 mRNA in tissues and cells were measured by qRT-PCR,the A450 value,apoptosis rate and number of invasive cells of U251 and H4 cells were measured by CCK-8 method,flow cytometry and Transwell chamber method respectively,the targeting of miR-138-5p with circGFRA1 and ANXA2 was analyzed by double luciferase,and the expression of ANXA2 protein in U251 and H4 cells was measured by Western blot method.Results Compared with the control group,the mRNA expression of circGFRA1 and ANXA2 in glioma tissues increased,and the expression of miR-138-5p decreased(P<0.05).Compared with the human neuroastrocytoma cell line,the expression of U251,CRT,HS683,H4 circGFRA1,ANXA2 mRNA in human glioma cells increased,and the expression of miR-138-5p decreased(P<0.05).Compared with the control group,the mRNA and protein expression of circGFRA1 and ANXA2,A450 value,and number of invasive cells in U251 and H4 cells in the si-circGFRA1 group decreased,miR-138-5p and apoptosis rate increased(P<0.05).Compared with the si circGFRA1 group,the mRNA and protein expression of circGFRA1 and ANXA2,A450 value,and number of invasive cells in U251 and H4 cells in the si-circGFRA1+miR-138-5p inhibitor group increased,miR-138-5p and apoptosis rate decreased(P<0.05).miR-138-5p had a targeting relationship with circGFRA1 and ANXA2.Compared with the mimics NC group,the mRNA and protein levels of ANXA2 in U251 and H4 cells in the miR-138-5p mimics group decreased(P<0.05).Conclusion Silencing circGFRA1 may inhibit the malignant progression of glioma by regulating the miR-138-5p/ANXA2 axis.
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