温郁金提取物抑制miR-103a表达对MPP+诱导的SK-N-SH细胞增殖凋亡的影响

Effect of Extract of Curcuma Wenyujin Inhibiting miR-103a Expression on Proliferation and Apoptosis of SK-N-SH Cells Induced by MPP

目的:探讨温郁金提取物是否通过抑制微小RNA(miRNA)-103a的表达影响帕金森细胞的增殖与凋亡。方法:将SK-N-SH细胞分为对照组、模型组、温郁金提取物低、中、高剂量组(以下简称低、中、高剂量组)、miR-103a组、miR-NC组、高剂量+miR-NC组、高剂量+miR-103a组。对照组不作处理,模型组及低、中、高剂量组给予1-甲基4-苯基吡啶离子(MPP+)诱导,低、中、高剂量组分别给予8、16、24μg/mL的温郁金提取物。miR-103a组转染miR-103a mimic并给予MPP+,miR-NC组转染miR-103a mimic阴性对照(miR-NC)并给予MPP+,高剂量+miR-NC组转染miR-NC并给予24μg/mL温郁金提取物,高剂量+miR-103a组转染miR-103a mimic并给予24μg/mL温郁金提取物。流式细胞术检测各组细胞周期与凋亡,蛋白免疫印迹试验检测天冬氨酸特异性半胱氨酸蛋白酶3(Caspase3)、细胞核相关抗原Ki-67蛋白表达,比色法检测丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)水平,实时荧光定量PCR(qRT-PCR)检测miR-103a表达。结果:与对照组比较,模型组细胞G0-G1期细胞比例、细胞凋亡率、Caspase3蛋白表达、miR-103a表达及MDA含量增加(P<0.05),S期细胞比例、Ki-67蛋白表达、GSH-Px活性减少(P<0.05)。与模型组比较,中、高剂量组G0-G1期细胞比例、细胞凋亡率、Caspase3蛋白表达、miR-103a表达及MDA含量减少(P<0.05),S期细胞比例、Ki-67蛋白表达、GSH-Px活性增加(P<0.05),且呈浓度依赖性(P<0.05)。与miR-NC组比较,miR-103a组细胞G0-G1期细胞比例、细胞凋亡率、Caspase3蛋白表达、miR-103a表达及MDA含量增加(P<0.05),S期细胞比例、Ki-67蛋白表达、GSH-Px活性减少(P<0.05)。与高剂量+miR-NC组比较,高剂量+miR-103a组细胞G0-G1期细胞比例、细胞凋亡率、Caspase3蛋白表达、miR-103a表达及MDA含量增加(P<0.05),S期细胞比例、Ki-67蛋白表达、GSH-Px活性减少(P<0.05)。结论:温郁金提取物可通过下调miR-103a表达,促进MPP+诱导的SK-N-SH细胞增殖,抑制其凋亡并减轻氧化应激。

Objective:To discuss whether the extract of curcuma wenyujin has an effect on proliferation and apoptosis of Parkinson cells through inhibiting microRNA(miRNA)-103 a expression.Methods:SK-N-SH cells were divided into the control group,the model group,the extract of curcuma wenyujin groups of low,medium and high dose(low-dose group,medium-dose group,and high-dose group for short),the miR-103 a group,the miR-NC group,the high-dose+miR-NC group and the high-dose+miR-103 a group.The control group was not treated,low-dose,medium-dose,and high-dose groups as well as the model group were induced by 1-methyl-4-phenylpyridiniumion(MPP+);low-dose,medium-dose,and high-dose groups were given 8,16,and 24μg/mL extract of curcuma wenyujin respectively.The miR-103 a group was transfected with miR-103 a mimic and given MPP+.The miR-NC group was transfected with miR-103 a mimic negative control(miR-NC)and given MPP+.The high-dose+miR-NC group was transfected with miR-NC and given 24μg/mL extract of curcuma wenyujin.The high-dose+miR-103 a group was transfected with miR-103 a mimic and given 24μg/mL extract of curcuma wenyujin.Flow cytometry was used to detect cell cycle and apoptosis in each group.Western blot was used to detect the expression of protein of cysteinyl aspartate specific proteinase3(Caspase3)and nucleus-related antigen Ki-67.Colorimetric method was applied to measuring levels of malondialdehyde(MDA)and glutathione peroxidase(GSH-Px).Real time fluorescent quantitative PCR(qRT-PCR)was used to detect miR-103 a expression.Results:When compared with those in the control group,in the model group,the G0-G1 phase cell proportion,cell apoptosis rate,expression of protein of Caspase3,miR-103 a expression,and MDA content were increased(P<0.05);the S phase cell proportion,expression of protein of Ki-67,and GSH-Px activity were decreased(P<0.05).When compared with those in the model group,in the medium-dose and high-dose groups,the G0-G1 phase cell proportion,cell apoptosis rate,expression of protein of Caspase3,miR-103 a expression,and MDA content were decreased(P<0.05);the S phase cell proportion,expression of protein of Ki-67,and GSH-Px activity were increased(P<0.05),and showed a concentration-dependent manner(P<0.05).When compared with those in the miR-NC group,in the miR-103 a group,the G0-G1 phase cell proportion,cell apoptosis rate,expression of protein of Caspase3,miR-103 a expression,and MDA content were increased(P<0.05);the S phase cell proportion,expression of protein of K-i67,and GSH-Px activity were decreased(P<0.05).When compared with those in the high-dose+miR-NC group,in the high-dose+miR-103 a group,the G0-G1 phase cell proportion,cell apoptosis rate,expression of protein of Caspase3,miR-103 a expression,and MDA content were increased(P<0.05);the S phase cell proportion,expression of protein of K-i67,and GSH-Px activity were decreased(P<0.05).Conclusion:Extract of curcuma wenyujin can promote proliferation of SK-N-SH cells induced by MPP+,inhibit their apoptosis,and relieve oxidative stress through down-regulating miR-103 a expression.