miR-145-3p在多发性骨髓瘤中表达及其对多发性骨髓瘤细胞凋亡与自噬作用机制的研究
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摘要
目的探讨微小m RNA-145-3p(miR-145-3p)在多发性骨髓瘤(MM)中表达及其对MM细胞凋亡与自噬作用机制。方法 2014年6月至2017年5月我院收治的MM患者60例(MM组)、健康体检者30例(正常组),采用荧光定量PCR法检测两组血浆样本及细胞株(MM细胞株选择U266、RPMI-8266、LP-1、H929,以CD138+浆细胞为对照组)中miR-145-3p表达水平,后选择毒性最低的MM细胞株(LP-1)进行miR-145-3p mimic或inhibitor转染(分别为mimic组、inhibitor组),并设立对照,以流式细胞仪进行凋亡检测[吸光度值(OD值)],并采用westren blot检测转染细胞凋亡与自噬相关标志物[肿瘤蛋白21(P21)、unc-51样激酶1(ULK1)、溶酶体相关膜蛋白2(LAMP2)]。结果 MM组血浆及细胞株(U266、RPMI-8266、LP-1、H929)中miR-145-3p水平均低于正常组(P <0. 05);转染后随时间延长,OD值均增加,且转染后3 d及5 d OD值大小均为mimic组<对照组 Objective To explore the expression of miR-145-3p in multiple myeloma( MM) and underlying mechanisms of its effect on cell apoptosis and autophagy.Methods 60 MM patients and 30 healthy subjects admitted in our hospital from June 2014 to May 2017 were selected as study subjects. The fluorescence quantitative PCR method was used to determine the expression levels of miR-145-3p in the plasma samples and MM cell lines( U266,RPMI-8266,LP-1 and H929),and CD138+plasma cells were used as control.Afterwards,the LP-1,the lowest toxic MM cell line was selected for miR-145-3p mimic or inhibitor transfection designed as the mimic group and the inhibitor group,respectively.At the same time,a control was set up. Apoptosis [absorbance value/optical density( OD value) ]was detected by flow cytometry.Western blot was used to detect the related markers of apoptosis and autophagy such as tumor protein 21( P21),unc-51 like kinase 1( ULK1) and lysosomal associated membrane protein 2( LAMP2).Results The expression levels of miR-145-3p in the plasma of the MM group and the cell lines were lower than those of the normal group( P < 0. 05).After transfection,the OD value was increased with the time,and the OD value on the 3-days and 5-dys after transfection was the mimic group< the control group < the inhibitor group( P < 0. 05).The level of P21 in the mimic group was higher than that in the inhibitor group and the control group while the levels of ULK1 and LAMP2 were lower than those in the inhibitor group and the control group( P <0. 05).Conclusion Expression of miR-145-3p in MM is low,and the targeted regulation of P21/ULK1/LAMP2 signaling pathway may inhibit the cell apoptosis and promote the cell autophagy.
引文
[1]杨玛丽,肖翠,魏榕,等.葫芦素E抑制多发性骨髓瘤细胞增殖及与阿霉素的协同效应[J].成都医学院学报,2016,11(2):150-154,175.
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[11]Kato M,Kurozumi A,Goto Y,et al. Abstract 1459:Dual-strand tumor-suppressor micro RNA-145(mi R-145-5p and mi R-145-3p)are involved in castration-resistant prostate cancer pathogenesis[J].Cancer Research,2017,77(13):1459-1459.
[12]杨雅芝,张小燕,万倩,等.外泌小体内mi RNA在多发性骨髓瘤进展中的作用及可能机制[J].中国实验血液学杂志,2017,25(1):301-305.
[13]Qian HR,Yang Y.Functional role of autophagy in gastric cancer[J].Oncotarget,2016,7(14):17641.
[14]吴洪坤.miR-145-3p靶向HDAC4调控多发性骨髓瘤细胞凋亡与自噬的作用及机制研究[D].第二军医大学,2016.
[15]张旗.miR-145在多发性骨髓瘤细胞中的功能及其机制研究[D].吉林大学,2015.
[16]Zhang Q,Yan W,Bai Y,et al. Synthetic mi R-145 mimic inhibits multiple myeloma cell growth in vitro and in vivo[J].Oncology Reports,2015,33(1):448-456.
[2]沈继春,李阳,刘晓梅,等.多发性骨髓瘤患者mi R-21和mi R-32的表达及临床应用[J].中国实验诊断学,2015,19(7):1123-1126.
[3]丰茂晓,古春明.mi R-19a和mi R-92a在多发性骨髓瘤中的功能及其信号通路分析[J].中国病理生理杂志,2015,31(8):1505-1509.
[4]朱荷艳,李慧慧.MicroRNA-145在急性髓系白血病患者中的表达及意义[J].重庆医学,2016,45(3):377-379.
[5]沈树平,陆亚倩,郑旸,等.microRNA-145调控大鼠骨髓间充质干细胞成骨分化的实验研究[J].口腔医学,2017,37(8):693-697.
[6]中国医师协会血液科医师分会,中华医学会血液学分会,中国多发性骨髓瘤工作组,等.中国多发性骨髓瘤诊治指南(2013年修订)[J].中华内科杂志,2013,52(9):791-795.
[7]袁杰,申娴娟,景蓉蓉,等.多发性骨髓瘤相关信号通路与mi RNA相互调控的研究进展[J].临床检验杂志,2015,33(9):698-702.
[8]徐艳妮,肖翠容,黄英丹,等.循环血清中mi RNA作为多发性骨髓瘤诊断标志的研究[J].中国实验血液学杂志,2017,25(2):471-475.
[9]Xia H,Yamada S,Aoyama M,et al.Prognostic impact of micro RNA-145 down-regulation in adult T-cell leukemia/lymphoma[J].Human Pathology,2014,45(6):1192-1198.
[10]Mataki H,Seki N,Mizuno K,et al.Dual-strand tumor-suppressor micro RNA-145(mi R-145-5p and mi R-145-3p)coordinately targeted MTDH in lung squamous cell carcinoma[J]. Oncotarget,2016,7(44):72084-72098.
[11]Kato M,Kurozumi A,Goto Y,et al. Abstract 1459:Dual-strand tumor-suppressor micro RNA-145(mi R-145-5p and mi R-145-3p)are involved in castration-resistant prostate cancer pathogenesis[J].Cancer Research,2017,77(13):1459-1459.
[12]杨雅芝,张小燕,万倩,等.外泌小体内mi RNA在多发性骨髓瘤进展中的作用及可能机制[J].中国实验血液学杂志,2017,25(1):301-305.
[13]Qian HR,Yang Y.Functional role of autophagy in gastric cancer[J].Oncotarget,2016,7(14):17641.
[14]吴洪坤.miR-145-3p靶向HDAC4调控多发性骨髓瘤细胞凋亡与自噬的作用及机制研究[D].第二军医大学,2016.
[15]张旗.miR-145在多发性骨髓瘤细胞中的功能及其机制研究[D].吉林大学,2015.
[16]Zhang Q,Yan W,Bai Y,et al. Synthetic mi R-145 mimic inhibits multiple myeloma cell growth in vitro and in vivo[J].Oncology Reports,2015,33(1):448-456.