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

Effect of Expression Levels of MiRNA-132,-125,-143 and-145 on Autophagy and Apoptosis of Multiple Myeloma Cells

目的:探讨多发性骨髓瘤细胞miRNA-132、miRNA-125b、miRNA-143和miRNA-145表达与自噬和凋亡的关系。方法:选取人骨髓瘤细胞株U266及正常CD138+浆细胞为研究对象,临床研究对象分为骨髓瘤组45例,健康对照组40例。实时定量PCR测定细胞中miRNA-132、miRNA-125b、miRNA-143和miRNA-145表达水平,Western blot测定自噬相关蛋白(LC3-Ⅱ,LC3-Ⅰ、P62、Beclin-1)表达量,凋亡相关分子(cleaved-Caspase3、cleaved-Caspase7、BCL-2、BAX)表达量;流式细胞术测定细胞凋亡率。分析miRNA表达水平与临床相关指标包括M蛋白、血红蛋白、β2-MG、乳酸脱氢酶、白蛋白、肌酐、血清钙的相关性。结果:与正常浆细胞对比,骨髓瘤细胞的miRNA-132和miRNA-125b表达显著增加(P <0.05),miRNA-143,miRNA-145表达显著降低(P <0.05);LC3-Ⅱ/LC3-Ⅰ显著增加(P <0.05),Beclin-1表达显著增加(P <0.05),P62表达显著降低(P <0.05);BAX、cleaved-Caspase3、cleaved-Caspase7表达显著降低(P <0.05),BCL-2表达显著增加(P<0.05);细胞凋亡率降低(P <0.05)。阳离子脂质体转染miRNA-125b mimic和miRNA-143 inhibitor后,正常浆细胞LC3-Ⅱ/LC3-Ⅰ显著增高(P <0.05),Beclin-1表达显著增高(P <0.05),P62表达也显著增加(P <0.05),细胞凋亡率降低(P <0.05);上述反应体系加入自噬抑制剂3-MA后,细胞凋亡率则无明显改变(P> 0.05)。miRNA-132、miRNA-125b、miRNA-143和miRNA-145表达在不同DS与ISS分期组,染色体核型异常组与正常核型组间均有显著性差异(P <0.05)。miRNA-125b和miRNA-143与β2-MG、白蛋白、血红蛋白水平均有显著相关性(P <0.05)。结论:miRNA-132、 miRNA-125b、 miRNA-143和miRNA-145表达与多发性骨髓瘤患者临床特征密切相关,miRNA-125b过表达和miRNA-143表达下调通过上调自噬水平抑制骨髓瘤细胞凋亡。

Objective: To investigate relationship of miRNA-132, miRNA-256, miRNA-143 and miRNA-145 level with antophagy and apoptosis of multiple mgeloma cells.Methods: Human myeloma cell line U266 and normal CD138+plasma cells were selected and used for study and detection, the 45 cases of MM were enrolled in MM group, and 40 normal persons were sellectod in control group.The expression of miRNA-132, miRNA-125b, miRNA-143 and miRNA-145 were measured by using qPCR, the expressions of autophagy-related protein (LC3- Ⅱ , LC3- Ⅰ , P62, beclin-1) and apoptosisrelated molecules (cleaved-Caspase3, cleaved-Caspase7, BCL-2, BAX) were measured by using Western blot, respectively.The rate of apoptosis was measured by using flow cytometry.The correlation of miRNA expression level with clinicalrelated indexes including M protein, hemoglobin, β2-MG, lactate dehydrogenase, albumin, creatinine and serum calcium was analyzed.Results: Compared with normal plasma cells, the expression of miRNA-132 and miRNA-125b in myeloma cells increased significantly (P<0.05), and the expression of miRNA-143 and miRNA-145 decreased significantly (P<0.05),but the expression of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 increased significantly (P<0.05).The expression of P62.BAX, cleaved-Caspase 3 and cleaved-Caspase 7 decreased significantly (P<0.05), the BCL-2 expression increased significantly (P<0.05),but the rate of apoptosis decreased (P<0.05).After transfection with miRNA-125b mimic or miRNA-143 inhibitor by using the cationic liposomes, the LC3- Ⅱ /LC3- Ⅰ of normal plasma cells increased significantly (P<0.05), the expression of Beclin-1 significantly increased (P<0.05), the expression of P62 decreased significantly (P<0.05), and the apoptosis rate decreased (P<0.05).However, the apoptosis rate was not significantly changed after addition of the autophagic inhibitor 3-MA in the reaction system(P>0.05).The expressions of miRNA-132, miRNA-125b, miRNA-143 and miRNA-145 were significantly different between DS and ISS staging group, also between the patients with abnormal and normal chromosome karyotype (P<0.05).The miRNA-125b and miRNA-143 significantly correlated with the levels of β2-MG, albumin and hemoglobin (P<0.05).Conclusion: The expressions of miRNA-132,miRNA-125b, miRNA-143 and miRNA-145 in patients with multiple myeloma closely relate with the clinical characteristics.Both over-expression of miRNA-125b and down-expression of miRNA-143 inhibit the apoptosis of myeloma cells by up-regulation of autophagy.