【摘要】 目的观察miR-125a-5p在人多发性骨髓瘤细胞系U266中的表达变化及其对细胞增殖的影响,并探讨其机制是否与维生素D受体(VDR)有关。方法取生长状态良好的U266细胞、人正常骨髓原代细胞,采用实时荧光定量PCR法检测miR-125a-5p表达,采用Western blotting法检测VDR蛋白表达。Target Scan软件分析及双荧光素酶报告基因实验证实VDR是miR-125a-5p的作用靶点。将U266细胞分为inhibitor组、inhibitor+si-VDR组和空白对照组,分别转染miR-125a-5p-inhibitor+si-Con质粒、miR-125a-5p inhibitor+si-VDR质粒、miR-NC+si-Con质粒。采用CCK-8法检测各组转染后0、24、48、72 h的光密度(OD)值,集落形成实验对各组转染24 h的集落形成个数进行计数。结果 U266细胞、人正常骨髓原代细胞miR-125a-5p相对表达量分别为2.79±0.65、1,VDR蛋白相对表达量分别为0.27±0.06、1,二者比较P均<0.01。inhibitor组、inhibitor+si-VDR组和空白对照组培养48、72 h的OD值以及集落形成个数均依次升高,组间两两比较P均<0.05。结论 U266细胞中miR-125a-5p表达升高,miR-125a-5p可通过负性调控VDR表达而促进细胞增殖。更多还原

【Abstract】 Objective To observe the expression of miR-125a-5p in the human multiple myeloma cell line U266 and its effect on cell proliferation,and to explore whether its mechanism is related to vitamin D receptor (VDR). Methods The expression of miR-125a-5p in human multiple myeloma cell line U266 and human normal primary marrow cells was detected by real-time fluorescence quantitative PCR. Western blotting was used to detect the expression of VDR protein in U266 cells and human normal bone marrow primary cells. Target scan software analysis and double luciferase reporter gene experiment confirmed that VDR was the target of miR-125a-5p. U266 cells were divided into the inhibitor group,inhibitor+ si VDR group,and blank control group,which were transfected with miR-125a-5p-inhibitor and si-Con plasmid,miR-125a-5p inhibitor and si-VDR plasmid,and miR-NC and si-Con plasmid,respectively. CCK-8 was used to detect the OD values of three groups at 0,24,48 and 72 h after transfection. The number of colonies formed in two groups at 24 h after transfection was counted. Results The relative expression levels of miR-125a-5p and VDR protein in U266 cells were 2. 79 ± 0. 65 and 0. 27 ± 0. 06,respectively (both P < 0. 01). The OD value and the number of colony formation in the inhibitor group,inhibitor + si-VDR and blank control group increased in turn at 48 and 72 h,with statistically significant difference between these two groups (all P < 0. 05). Conclusion The expression of miR-125a-5p increases in U266 cells; miR-125a-5p can promote cell proliferation through negative regulation of VDR expression.更多还原