我的梦想是做“百年老店”——记首都医科大学三博脑科医院神经外科于春江教授

doi:10.3969/j.issn.1006-7795.2021.01.029

摘要/Abstract

摘要： 目的探讨干预miRNA-181a(miR-181a)促进多发性骨髓瘤凋亡的机制。方法首先检测健康志愿者外周血、骨髓瘤患者骨髓、骨髓瘤细胞株MM1S中miR-181a的表达水平。再将miR-181a抑制剂(miR-181a inhibitor)及对照siRNA分别转染进MM1S细胞,回复实验进一步验证miR-181a功能。流式细胞术检测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、LC3 Ⅱ/LC3-Ⅰ、P62、Parkin。结果骨髓瘤及MM1S细胞株中miR-181a的相对表达水平显著升高(P<0.05),分别为1.57±0.09及1.64±0. 06。miRNA-181a 抑制剂作用下,MM1S细胞的凋亡率及Cleaved-caspase-3显著高于健康对照组及阴性转染对照组,电镜下自噬泡数目增加而且线粒体结构毁坏严重,线粒体膜电位水平低下伴有Bcl-2/Bax、LC3 Ⅱ/LC3-Ⅰ水平的显著升高,而P62水平的显著降低。过表达Parkin的回复实验显示,miRNA-181a 抑制剂对骨髓瘤细胞的作用明显降低。结论抑制miRNA-181a可以升高Parkin表达水平,进而促进该基因通路介导的线粒体自噬,抑制骨髓瘤细胞的增殖。

关键词: 多发性骨髓瘤, miRNA-181a, 线粒体自噬, 线粒体, 自噬, 凋亡

Abstract: Objective To investigate the role and possible molecular mechanism of anti miRNA 181a in the regulation of mitophagy and apoptosis on Multiple Myeloma(MM) cells. Methods The expression of miRNA 181a in human normal peripheral blood CD138+ cells,MM patients' bone marrow CD138+ cells and human myeloma cell lines MM1S was detected by fluorescence quantitative PCR (QPCR) . MiR 181a inhibitor(miR-181a inhibitor group) and negative control group (NC group) were transfected into MM1S cells, mitochondrial membrane potential(MMP) was examined by flow cytometry, autophagy and mitochondrial structure were observed by transmission electron microscope, and Western blotting was used to detect the expression of mitophagy related protein (Cleaved-caspase-3, Bcl-2/Bax, LC3 Ⅱ/LC3-Ⅰ, 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 overexpress 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, which significantly higher than normal peripheral blood cell (P<0.05). After transfection of miR-181a inhibitor,the apoptosis rate of miR-181a inhibitor group is significantly higher than the other two groups, the number of autophagic vesicles decreased with mitochondrial structure badly damaged were also observed in miR-181a inhibitor group. In miR-181a inhibitor group, relative expression of autophagy related protein Cleaved-caspase-3, the ratio of Bcl-2/Bax and LC3 Ⅱ/LC3-Ⅰincreased significantly, P62 decreased remarkable (P<0.01) . After the Parkin expression was rescued, MM1S cells' viability recovered. Conclusion Anti miRNA-181a could induce apoptosis on multiple myeloma cells via enhancing the level of mitophagy.

Key words: multiple myeloma, MiRNA-181a, mitophagy, mitochondria, autophagy, apoptosis

崔兴, 钟萍. 我的梦想是做“百年老店”——记首都医科大学三博脑科医院神经外科于春江教授[J]. 首都医科大学学报, 2021, 42(1): 167-169.

Cui Xing, Zhong Ping. Experimental study of anti miRNA-181a induces apoptosis via Mitophagy enhancing on Multiple Myeloma cells[J]. Journal of Capital Medical University, 2021, 42(1): 167-169.

参考文献

[1] Bartel D P.MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 2004,116(2):281-297.
[2] Chen Y,Gao D Y, Huang L. In vivo delivery of miRNAs for cancer therapy: challenges and strategies[J].Adv Drug Deliv Rev,2015,81(2):128-141.
[3] Kubiczkova L, Kryukov F, Slaby O,et al.Circulating serum microRNAs as novel diagnostic and prognostic biomarkers for multiplemyeloma and monoclonal gammopathy of undetermined significance[J].Haematologica,2014,99(3):511-518.
[4] Pichiorri F, Suh S S, Rocci A, et al.Downregulation of p53-inducible microRNAs 192, 194, and 215 impairs the p53/MDM2autoregulatory loop in multiple myeloma development[J].Cancer Cell, 2010,18(4):367-381.
[5] Abdi J, Rastgoo N, Li L,et al.Role of tumor suppressor p53 and micro-RNA interplay in multiple myeloma pathogenesis[J].J Hematol Oncol,2017,10(1):169.
[6] 王瑾,杨惠玲.微小RNA与肿瘤[J].国际内科学杂志,2007,34(3): 131-134.
[7] Duggal J, Harrison J S, Studzinski G P, et al. Involvement of microRNA181a in differentiation and cell cycle arrest induced by a plant-derived antioxidant carnosic acid and vitamin D analog doxercalciferol in human leukemia cells[J]. Microrna,2012,1(1):26-33.
[8] Tekirdag K A, Korkmaz G, Ozturk D G, et al.MIR181A regulates starvation- and rapamycin-induced autophagy through targeting of ATG5[J]. Autophagy,2013,9(3):374-385.
[9] 朱耀魁,程妮,张磊,等.microRNA-181家族与疾病的相关性研究进展[J].暨南大学学报(医学版),2012, 33(6): 547-552.
[10]Cheng M, Liu L, Lao Y,et al.MicroRNA-181a suppresses parkin-mediated mitophagy and sensitizes neuroblastoma cells to mitochondrial uncoupler-induced apoptosis[J]. Oncotarget,2016,7(27):42274-42287.
[11]Menghini R, Casagrande V, Marino A,et al. MiR-216a: a link between endothelial dysfunction and autophagy[J]. Cell Death Dis,2014,5:e1029.
[12]Ma K, Zhang H, Wei G, et al. Identification of key genes, pathways, and miRNA/mRNA regulatory networks of CUMS-induced depression in nucleus accumbens by integrated bioinformatics analysis[J]. Neuropsychiatr Dis Treat, 2019,15:685-700.
[13]Liu Z, Hua Y, Liu C, et al. MiRNA-494 inhibits apoptosis and promotes autophagy of acute myeloid leukemia cells by downregulating FGFR2[J]. Minerva Endocrinol,2019,44(4):410-413.
[14]尚晋,陈志忠,王志红,等.miRNA-132, miRNA-125b, miRNA-143和miRNA-145表达对多发性骨髓瘤细胞自噬及凋亡的影响[J].中国实验血液学杂志,2018,26 (6):1688-1694.
[15]Hoang B, Benavides A, Shi Y, et al. Effect of autophagy on multiple myeloma cell viability[J]. Mol Cancer Ther,2009,8(7): 1974-1984.
[16]Gocke C B, McMillan R, Wang Q, et al. IQGAP1 Scaffold-MAP Kinase Interactions Enhance Multiple Myeloma Clonogenic Growth and Self-Renewal[J].Mol Cancer Ther,2016,15(11):2733-2739.
[17]Zheng Z, Fan S, Zheng J, et al. Inhibition of thioredoxin activates mitophagy and overcomes adaptive bortezomib resistance in multiple myeloma[J]. J Hematol Oncol,2018,11(1):29.
[20]Springer M Z,Macleod K F. In brief: Mitophagy: mechanisms and role in human disease [J].J Pathol,2016,240(3):253-255.
[21]Zhang W, Ma Q, Siraj S, et al. Nix-mediated mitophagy regulates platelet activation and life span[J].Blood Adv,2019,3(15):2342-2354.
[22]Bowling J L, Skolfield M C, Riley W A, et al. Temporal integration of mitochondrial stress signals by the PINK1:Parkin pathway[J].BMC Mol Cell Biol,2019,20(1):33.
[23]Galluzzi L, Morselli E, Vitale I, et al. miR-181a and miR-630 regulate cisplatin-induced cancer cell death[J]. Cancer Res, 2010, 70(5): 1793-1803.
[24]Pekarsky Y, Santanam U, Cimmino A,et al. Tcl1 expression in chronic lymphocytic leukemia is regulated by miR-29 and miR-181[J]. Cancer Res, 2006,66(24):11590-11593.
[25]庄贤栩.Expression characteristics and prognosissignificance of miRNA-181a in acute myeloid leukemia with normal karyotype[J].中国医学文摘(内科学分册)(英文版),2017,4:231.
[26]Marcucci G, Radmacher M D, Maharry K, et al. MicroRNA expression in cytogenetically normal acute myeloid leukemia[J]. N Engl J Med, 2008, 358(18): 1919-1928.
[27]Cheng M, Liu L, Lao Y, et al. MicroRNA-181a suppresses parkin-mediated mitophagy and sensitizes neuroblastoma cells to mitochondrial uncoupler-induced apoptosis[J]. Oncotarget,2016,7(27):42274-42287.
[26]Roccaro A M, Sacco A, Thompson B, et al. MicroRNAs 15a and 16 regulate tumor proliferation in multiple myeloma[J]. Blood, 2009,113(26): 6669-6680.
[27]Yyusnita, Norsiah, Zakiah I, et al. MicroRNA (miRNA) expression profiling of peripheral blood samples in multiple myeloma patients using microarray[J].Malays J Pathol, 2012,34(2):133-143.
[28]Yuan R, Liu N, Yang J, et al. The expression and role of miR-181a in multiple myeloma[J]. Medicine (Baltimore), 2018,97(35):e12081.
[29]Chen L, Hu N, Wang C, et al. Long non-coding RNA CCAT1 promotes multiple myeloma progression by acting as a molecular sponge of miR-181a-5p to modulate HOXA1 expression[J]. Cell Cycle,2018,17(3):319-329.
[30]Shen X, Bai H, Zhu H, et al. Long non-coding RNA MEG3 functions as a competing endogenous RNA to regulate HOXA11 expression by sponging miR-181a in multiple myeloma[J]. Cell Physiol Biochem, 2018,49(1):87-100.
[31]Liu N, Yang J, Yuan R, et al. Effects of miR-181a on the biological function of multiple myeloma[J]. Oncol Rep, 2019,42(1):291-300.