miR-138-5p过表达在骨肉瘤MG63细胞及多药耐药细胞MG63/Dox对阿霉素敏感性中作用

Role of miR-138-5p overexpression in improving the sensitivity of MG63 and MG63/Dox cells to doxorubicin

目的探讨骨肉瘤MG63细胞及骨肉瘤多药耐药细胞MG63/Dox对阿霉素的敏感性与miR-138-5p过表达的关系。方法采用脂质体将miR-138-5p mimics、miR-con分别转染至骨肉瘤MG63细胞及骨肉瘤多药耐药MG63/Dox细胞,分为miR-138-5p过表达MG63细胞组与MG63细胞对照组、miR-138-5p过表达MG63/Dox细胞组与MG63/Dox细胞对照组。采用实时荧光定量PCR法检测4组细胞miR-138-5p和多药耐药基因1(multidrug resistance 1, MDR1) mRNA相对表达量;采用MTT法检测4组细胞不同浓度阿霉素(0、1、10、20、40、80μg/L)下的增殖抑制率;采用Western blot法检测4组细胞P-糖蛋白(P-glycoprotein, P-gp)蛋白相对表达量。结果 miR-138-5p过表达MG63细胞组(10.15±2.12)、miR-138-5p过表达MG63/Dox细胞组(6.69±1.38)miR-138-5p相对表达量分别高于MG63细胞对照组(1.00±0.18)、MG63/Dox细胞对照组(1.00±0.13)(t=7.449,P<0.001;t=7.110,P=0.001),miR-138-5p过表达细胞转染成功。10、20、40、80μg/L阿霉素下,miR-138-5p过表达MG63细胞组(0.41±0.04、0.51±0.04、0.67±0.06、0.81±0.07)、miR-138-5p过表达MG63/Dox细胞组(0.45±0.04、0.63±0.08、0.78±0.06、0.92±0.06)细胞增殖抑制率分别高于MG63细胞对照组(0.25±0.04、0.34±0.05、0.48±0.04、0.61±0.06)、MG63/Dox细胞对照组(0.34±0.03、0.45±0.04、0.57±0.05、0.62±0.07)(P<0.05),0、1μg/L阿霉素下,miR-138-5p过表达MG63细胞组与MG63细胞对照组、miR-138-5p过表达MG63/Dox细胞组与MG63/Dox细胞对照组细胞增殖抑制率比较差异均无统计学意义(P>0.05)。miR-138-5p过表达MG63细胞组、miR-138-5p过表达MG63/Dox细胞组MDR1 mRNA(0.55±0.08、0.38±0.10)及P-gp蛋白相对表达量(0.58±0.08、0.30±0.07)分别低于MG63细胞对照组(1.00±0.15、1.01±0.14)、MG63/Dox细胞对照组(1.00±0.11、0.69±0.04)(P<0.05)。结论 miR-138-5p过表达可抑制耐药相关分子MDR1 mRNA和P-gp蛋白的表达,增强骨肉瘤细胞MG63和耐药细胞MG63/Dox对阿霉素的敏感性。

Objective To investigate the relationships of miR-138-5 p overexpression with the sensitivities of osteosarcoma MG63 cells and multidrug-resistant osteosarcoma MG63/Dox cells to doxorubicin. Methods MiR-138-5 p mimics and miR-con were transfected into osteosarcoma MG63 cells and multidrug-resistant osteosarcoma MG63/Dox cells by liposomes, respectively, and were divided into MG63 overexpression group, MG63 control group, MG63/Dox overexpression group and MG63/Dox control group. The relative expressions of miR-138-5 p and multidrug resistance 1(MDR1) mRNA were detected by real-time fluorescence quantitative PCR in four groups. MTT assay was used to detect the proliferation inhibition rates of doxorubicin in concentrations of 0, 1, 10, 20, 40 and 80 μg/L. Western blot was performed to detect the relative expression of P-glycoprotein(P-gp) in four groups. Results The relative expression of miR-138-5 p was higher in MG63 overexpression group(10.15±2.12) and MG63/Dox overexpression group(6.69±1.38) than that in MG63 control group(1.00±0.18) and MG63/Dox control group(1.00±0.13)(t=7.449, P<0.001;t=7.110, P=0.001), showing a successful transfection by miR-138-5 p overexpression. When the doxorubicin concentrations were 10, 20, 40 and 80 μg/L, the proliferation inhibition rates were higher in MG63 overexpression group(0.41±0.04, 0.51±0.04, 0.67±0.06, 0.81±0.07) and MG63/Dox overexpression group(0.45±0.04, 0.63±0.08, 0.78±0.06, 0.92±0.06) than those in MG63 control group(0.25±0.04, 0.34±0.05,0.48±0.04,0.61±0.06)and MG63/Dox control group(0.34±0.03,0.45±0.04,0.57±0.05,0.62±0.07)(P<0.05).When doxorubicin concentrations were 0 and 1μg/L,there were no significant differences in the proliferation inhibition rates between MG63 overexpression group and MG63 control group,and between MG63/Dox overexpression group and MG63/Dox control group(P>0.05).The relative expressions of MDR1 mRNA and P-gp protein were lower in MG63 overexpression group(0.55±0.08,0.58±0.08)and MG63/Dox overexpression group(0.38±0.10,0.30±0.07)than those in MG63 control group(1.00±0.15,1.01±0.14)and MG63/Dox control group(1.00±0.11,0.69±0.04)(P<0.05).Conclusion The overexpressed miR-138-5 Pcan inhibit the expressions of MDR1 mRNA and P-gp protein,and enhance the sensitivities of MG63 and MG63/Dox cells to doxorubicin.