CircBACH1调节miR-140-5p/MDM2轴对多发性骨髓瘤细胞增殖、凋亡和化疗敏感性的影响

Impacts of CircBACH1 on proliferation,apoptosis and chemosensitivity of multiple myeloma cells by regulating miR-140-5p/MDM2 axis

目的探讨CircBACH1对多发性骨髓瘤(MM)细胞增殖、凋亡和化疗敏感性的影响以及在此过程中对miR-140-5p/MDM2轴的调节机制。方法收集MM组和正常对照组骨髓浆细胞,将人MM细胞系U266细胞分为未转染(Control)组、si-NC组、si1-CircBACH1组、si2-CircBACH1组、si-CircBACH1+anti-miR-NC组、si-CircBACH1+anti-miR-140-5p组。实时荧光定量PCR检测细胞中CircBACH1、miR-140-5p、鼠双微体2(MDM2)mRNA的表达;CCK-8法检测细胞增殖活性;流式细胞术检测细胞凋亡及对硼替佐米的敏感性;Western blot检测细胞中B细胞淋巴因子2(Bcl-2)、Bcl-2相关X蛋白(Bax)、裂解的胱天蛋白酶(cleaved caspase)-3蛋白的表达;双萤光素酶报告基因实验验证CircBACH1、miR-140-5p、MDM2三者的靶向关系。结果与正常对照组相比,MM组的骨髓浆细胞及U266细胞中CircBACH1、MDM2 m RNA表达升高,miR-140-5p表达降低(P<0.05);与Control组和si-NC组相比,siCircBACH1组细胞中CircBACH1、MDM2的mRNA表达、细胞增殖活性、Bcl-2蛋白表达均降低,miR-140-5p的表达、细胞凋亡率、化疗敏感性及cleaved caspase-3、Bax表达均升高(P<0.05);而回补实验中,si-CircBACH1对MM细胞的影响被anti-miR-140-5p逆转,且MDM2的表达也升高(P<0.05)。双萤光素酶基因报告实验验证了CircBACH1、MDM2均与miR-140-5p存在靶向关系。结论敲低CircBACH1能够上调miR-140-5p表达,下调MDM2的表达,抑制MM细胞的增殖,促进MM细胞凋亡,提高化疗敏感性。

Objective To investigate the effect of CircBACH1 on proliferation,apoptosis and chemotherapy sensitivity of multiple myeloma(MM)cells and the regulatory mechanism on miR-140-5p/MDM2 axis during this process.Methods Bone marrow plasma cells were collected from the MM patient group and normal control group.Human MM cell line U266 cells were divided into the untransfected group,the si-NC group,the si1-CircBACH1 group,the si2-CircBACH1 group,the si-CircBACH1+anti-miR-NC group and the si-CircBACH1+anti-miR-140-5p group.The mRNA expressions of CircBACH1,miR-140-5p and mouse double microsoma 2(MDM2)were detected by real-time quantitative PCR.Cell proliferation activity was detected by CCK-8 assay.Flow cytometry was used to detect apoptosis and sensitivity to bortezomib.The expression levels of B-cell lymphofactor 2(Bcl-2),Bcl-2 associated X protein(Bax)and cleaved caspase-3 were detected by Western blot assay.Dual luciferase reporter gene assay was used to verify the targeting relationship of CircBACH1,miR-140-5p and MDM2.Results Compared with the normal control group,the mRNA expression levels of CircBACH1 and MDM2 were increased in bone marrow plasma cells and U266 cells in the MM group,while the expression of miR-140-5p was decreased(P<0.05).Compared with the control group and the si-NC group,the mRNA expression of CircBACH1 and MDM2,cell proliferation activity and Bcl-2 protein expression were decreased in the transfected si CircBACH1 group.The expression of miR-140-5p,cell apoptosis rate,chemotherapy sensitivity,cleaved caspase-3 and Bax expressions were increased(P<0.05).In the anti-miR-140-5p supplementation experiment,the effect of si CircBACH1 on MM cells was reversed,and the expression of MDM2 was increased(P<0.05).Dual luciferase gene reporting experiment verified the targeting relationship between CircBACH1 and MDM2 and miR-140-5p.Conclusion Knock down of CircBACH1 can up-regulate the expression of miR-140-5p,down-regulate the expression of MDM2,inhibit the proliferation of MM cells,promote the apoptosis of MM cells and improve the chemosensitivity.