摘要
目的研究环状RNA半胱氨酸丰富跨膜BMP调节因子1(CircCRIM1)调节miR-129-5p/鼠双微体基因2(MDM2)轴对卵巢癌细胞紫杉醇耐药性的影响。方法以实时荧光定量PCR检测人卵巢癌细胞株SKOV3及其紫杉醇耐药细胞株SKOV3-TR30中miR-129-5p与CircCRIM1、MDM2表达。体外培养的SKOV3-TR30细胞随机分为对照组、紫杉醇组、紫杉醇+阴性对照组、紫杉醇+CircCRIM1敲低组、紫杉醇+CircCRIM1敲低+miR-129-5p inhibitor组,分组转染处理后,以实时荧光定量PCR检测各组细胞miR-129-5p及CircCRIM1、MDM2、多药耐药相关蛋白3(MRP3)、P-糖蛋白(P-gp)表达;以MTT法和流式细胞实验检测各组细胞增殖率、凋亡率;以免疫印记法检测各组细胞MDM2、P-gp、MRP3及凋亡蛋白(Bcl-2、Bax、caspase-3)表达;以双荧光素酶报告基因实验检测SKOV3-TR30中CircCRIM1对miR-129-5p的靶向调节及miR-129-5p对MDM2的靶向调节。结果与SKOV3细胞相比,SKOV3-TR30细胞中CircCRIM1与MDM2 mRNA表达升高(P<0.05),miR-129-5p表达降低(P<0.05)。与对照组相比,紫杉醇组、紫杉醇+miR-129-5pmimics阴性对照组细胞各指标无明显变化(P>0.05)。与紫杉醇组相比,紫杉醇+阴性对照组细胞各指标无明显变化(P>0.05);紫杉醇+CircCRIM1敲低组细胞增殖率、CircCRIM1表达、MDM2、P-gp、MRP3 mRNA及蛋白表达、Bcl-2蛋白表达降低(P<0.05),细胞凋亡率、miR-129-5p表达、Bax及caspase-3蛋白表达升高(P<0.05),miR-129-5p inhibitor可逆转紫杉醇+CircCRIM1敲低组对细胞指标的作用。结论敲低CircCRIM1可通过上调miR-129-5p而降低MDM2表达,从而抑制卵巢癌细胞紫杉醇耐药性,促进其凋亡。
Objective To study the impact of circular RNA cysteine rich transmembrane BMP regulator 1(CircCRIM1)on paclitaxel resistance of ovarian cancer cells by regulating miR-129-5p/mouse double microgene 2(MDM2)axis.Methods The expression of miR-129-5p,CircCRIM1 and MDM2 in human ovarian cancer cell line SKOV3 and its paclitaxel-resistant cell line SKOV3-TR30 was detected by real-time quantitative PCR.The SKOV3-TR30 cells cultured in vitro were randomly grouped into control group,paclitaxel group,paclitaxel+negative control group,paclitaxel+CircCRIM1 knockdown group,and paclitaxel+CircCRIM1 knockdown+miR-129-5p inhibitor group.After grouping and transfection,real-time quantitative PCR was used to detect the expression of miR-129-5p,CircCRIM1,MDM2,multidrug resistance-related protein 3(MRP3)and P-glycoprotein(P-gp)in each group.The proliferation rate and apoptosis rate of each group were detected by MTT method and flow cytometry.The expression of MDM2,P-gp,MRP3 and apoptotic proteins(Bcl-2,Bax,caspase-3)in each group of cells was detected by western blotting,and the targeted regulation of CircCRIM1 on miR-129-5p and the targeted regulation of miR-129-5p on MDM2 in SKOV3-TR30 were detected by dual-luciferase reporter gene assay.Results Compared with SKOV3 cells,the expression of CircCRIM1 and MDM2 mRNA in SKOV3-TR30 cells increased(P<0.05),and the expression of miR-129-5p decreased(P<0.05).There was no significant difference in the above indexes between the paclitaxel group and the paclitaxel+miR-129-5p mimics negative control group with the control group(P>0.05).There was no significant difference in the above indexes between the paclitaxel+negative control group and the paclitaxel group(P>0.05).The cell proliferation rate,the expression of CircCRIM1,the mRNA and protein expression of MDM2,P-gp,and MRP3,and the protein expression of Bcl-2 in paclitaxel+CircCRIM1 knockdown group decreased(P<0.05).The apoptosis rate,the expression of miR-129-5p,the protein expression of Bax and caspase-3 increased(P<0.05),miR-129-5p inhibitor could reverse the effects of paclitaxel+CircCRIM1 knockdown group on cell indexes.Conclusion Knockdown of CircCRIM1 can reduce the expression of MDM2 by up-regulating miR-129-5p,thereby inhibiting the resistance of ovarian cancer cells to paclitaxel and promoting the apoptosis.
作者
罗祥力
蔡芃夷
唐文
LUO Xiangli;CAI Pengyi;TANG Wen(Department of Gynecology,Nanchong Central Hospital,Nanchong,Sichuan 637000,China;Department of Anatomy,Nanchong Health School,Nanchong,Sichuan 637000,China)
出处
《中国优生与遗传杂志》
2023年第2期262-268,共7页
Chinese Journal of Birth Health & Heredity
基金
南充市市校科技战略合作项目(18SXHZ0197)。