摘要
目的探讨干预微RNA-181a(miR-181a)促进多发性骨髓瘤(MM)细胞凋亡的机制。方法检测健康志愿者外周血、MM患者骨髓、MM细胞株MM1S中miR-181a的表达水平。分别转染miR-181a抑制剂、对照siRNA入MM1S细胞,回复实验进一步验证miR-181a功能。流式细胞术检测细胞凋亡率和线粒体膜电位,透射电镜观察线粒体超微结构及自噬泡,Western blotting检测cleaved-caspase-3、Bcl-2/Bax、LC3Ⅱ/LC3Ⅰ、P62、Parkin蛋白的表达。结果MM患者骨髓和MM1S细胞株中,miR-181a相对表达水平显著升高(1.57±0.09和1.64±0.06,P<0.05)。miRNA-181a抑制剂作用下,MM1S细胞的凋亡率及cleaved-caspase-3表达水平显著高于对照组及阴性对照组,电镜下自噬泡数目增加,线粒体结构毁坏严重,线粒体膜电位水平低下,Bcl-2/Bax、LC3Ⅱ/LC3Ⅰ表达水平显著升高,而P62表达水平显著降低。敲减Parkin的回复实验显示,miRNA-181a抑制剂对MM细胞的作用明显降低。结论抑制miRNA-181a可增强Parkin表达,促进Parkin通路介导的线粒体自噬,从而促进MM细胞凋亡。
Objective To investigate the role and possible molecular mechanism of anti-microRNA(miRNA)-181a in the regulation of mitophagy and apoptosis on multiple myeloma(MM)cells.Methods In the expression of miRNA-181a in human normal peripheral blood CD138+cells,MM patients’bone marrow CD138+cells and human myeloma cell lines MM1S were detected by fluorescence quantitative PCR.The miR-181a inhibitor(miR-181a inhibitor group)and a negative control(NC group)were transfected into MM1S cells.Mitochondrial membrane potential(MMP)was examined by flow cytometry while autophagy and mitochondrial structure were observed by transmission electron microscope.Western blotting was used to detect the expression of mitophagy related protein(cleaved-caspase-3,Bcl-2/Bax,LC3Ⅱ/LC3Ⅰ,P62,and Parkin)in three groups.Overexpression Parkin of miR-181a inhibitor group as rescue test and Western blotting was used to detect mitophagy related protein expression after overexpressing Parkin.Results The relative expression levels of miR-181a in MM patients’bone marrow and MM1S cell lines were 1.57±0.09 and 1.64±0.06,significantly higher than normal peripheral blood cells(P<0.05).After transfection of miR-181a inhibitor,the apoptosis rate of miR-181a inhibitor group was significantly higher than the other two groups.The number of autophagic vesicles that decreased with badly damaged mitochondrial structure were also observed in miR-181a inhibitor group.In the miR-181a inhibitor group,relative expression of autophagy related protein cleavedcaspase-3,the ratio of Bcl-2/Bax and LC3Ⅱ/LC3Ⅰincreased significantly,P62 decreased remarkably(P<0.01).After the Parkin expression was rescued,MM1S cells’viability recovered.Conclusion Anti-miRNA-181a could induce apoptosis on MM cells via enhancing the level of mitophagy.
作者
崔兴
徐龙进
CUI Xing;XU Longjin(Department of Hematology,Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250011,China;Shandong Center for Disease Control and Prevention,Jinan 250014,China)
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2022年第1期6-11,共6页
Journal of China Medical University
基金
国家自然科学基金(81774080)
泰山学者青年专家人才项目(tsqn201812145)
山东省重点研发计划(2019GSF108162)。
