多发性骨髓瘤患者血浆微小RNA-17-5p的表达及其对肿瘤发生发展的作用

Expression of miR-17-5p in the plasma of patients with multiple myeloma and its role in tumorigenesis and development

目的探讨多发性骨髓瘤(MM)患者血浆微小RNA(miR)-17-5p的表达及其对肿瘤发生发展的作用。方法纳入2013年4月至2018年4月在河南省肿瘤医院血液科就诊的意义未明单克隆丙种球蛋白血症(MGUS)患者、MM患者及20名健康志愿者,采用逆转录聚合酶链反应(qRT-PCR)法检测血浆循环miR-17-5p和骨髓单个核细胞中miR-17-5p的表达量。其中MM组患者分为初诊未治MM(NDMM)组、完全缓解MM(CRMM)组以及复发难治MM(RRMM)组。分析各组miR-17-5p的表达差异;用相关性分析评估NDMM患者的血浆miR-17-5p与血清M蛋白和骨髓浆细胞比例之间的关系;用受试者工作特征(ROC)曲线评估血浆miR-17-5p作为MM诊断相关的分子标志物的可能性;过表达或敲低miR-17-5p的表达后,细胞计数试剂盒(CCK-8)法检测miR-17-5P对MM细胞系增殖的影响,裸鼠皮下成瘤实验体内检测miR-17-5p对MM细胞增殖的影响。结果纳入MGUS患者10例,其中男6例、女4例。MM患者92例,其中男54例、女38例;MM患者中,NDMM组22例、CRMM组11例、RRMM组59例。NDMM组和RRMM组骨髓单个核细胞中miR-17-5p的表达量高于健康对照组[1.37(0.47,4.87)、2.68(1.02,5.02)比1.00(1.00,1.00),均P<0.05],且血浆miR-17-5p的表达水平也高于健康对照组[1.85(0.92,3.51)、2.79(1.22,5.04)比1.00(1.00,1.00),均P<0.05];miR-17-5p在KMS-11、RPMI-8226、H929、MM-1R、U266B1等MM细胞系中的表达量均高于健康对照组的骨髓单个核细胞(3.96±0.68、1.58±0.32、3.51±0.55、5.08±0.76、3.22±0.75比1.00±0,均P<0.05);血浆miR-17-5p表达与血清M蛋白和骨髓浆细胞比例均呈正相关(r=0.50,P<0.05;r=0.60,P<0.01);ROC曲线显示血浆miR-17-5p作为诊断MM的相关分子标志物的特异度为0.591,灵敏度为0.900(ROC曲线下面积=0.74,cut-off值为0.491);CCK-8结果提示miR-17-5p过表达使RPMI-8226和NCI-H929等MM细胞系72 h的增殖较对照组增高(1.37±0.11比1.07±0.09,2.14±0.09比1.82±0.11,均P<0.05),miR-17-5p低表达使NCI-H929和MM-1R等MM细胞系72 h的增殖较对照组降低(1.38±0.09比1.83±0.11,1.45±0.10比1.73±0.09,均P<0.05)。裸鼠皮下成瘤实验提示,miR-17-5p过表达组肿瘤体积大于对照组[(1865±181)比(1389±227)mm^(3),P<0.05],miR-17-5p低表达组肿瘤体积小于对照组[(1006±171)比(1389±227)mm^(3),P<0.05]。结论miR-17-5p可能作为MM疾病发展状态相关的血浆分子标志物,在MM细胞系中起癌基因作用。

Objective To investigate the expression of miR-17-5p in the plasma of patients with multiple myeloma(MM)and its role in tumorigenesis and development.Methods Patients diagnosed with unidentified monoclonal gammopathy of undetermined significance(MGUS)or MM in Cancer Hospital of Zhengzhou University from April 2013 to April 2018 were enrolled,as well as 20 healthy volunteers.Reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-17-5p in plasma circulation and bone marrow mononuclear cells.There were 22 cases with newly diagnosed MM(NDMM),11 cases with complete remission MM(CRMM)and 59 case with recurrent refractory MM(RRMM).The expression levels of miR-17-5p in each group were analyzed.The correlation analysis was used to evaluate the relationship between plasma miR-17-5p and the proportion of serum M protein and bone marrow plasma cells in patients with untreated multiple myeloma.Receiver operating characteristic(ROC)curve was used to evaluate the possibility of plasma miR-17-5p as a molecular marker related to MM diagnosis.After over expression or knockdown of miR-17-5p expression,CCK-8 method was used to detect the effect of miR-17-5p on the proliferation of MM cell line.The effect of miR-17-5p on the proliferation of MM cells was detected in nude mice subcutaneous tumorigenesis experiment.Results The expression of miR-17-5p in bone marrow mononuclear cells in NDMM and RRMM group were higher than those in healthy volunteers[1.37(0.47,4.87),2.68(1.02,5.02)vs 1.00(1.00,1.00),all P<0.05],and the expression levels of miR-17-5p in plasma were also higher than those in healthy control group[1.85(0.92,3.51),2.79(1.22,5.04)vs 1.00(1.00,1.00),all P<0.05].The expression of miR-17-5p in MM cell lines such as KMS-11,RPMI-8226,H929,MM-1R,U266B1 were higher than that in bone marrow mononuclear cells of healthy control group(3.96±0.68,1.58±0.32,3.51±0.55,5.08±0.76,3.22±0.75 vs 1.00±0,all P<0.05);Plasma miR-17-5p was positively correlated with the ratio of serum M protein and bone marrow plasma cells(r=0.50,P<0.05;r=0.60,P<0.01).ROC curve showed that the specificity was 0.591 and the sensitivity was 0.900 of plasma miR-17-5p as a molecular marker related to diagnosis(area under ROC curve=0.74,cut-off value:0.491).CCK-8 results showed that over expression of miR-17-5p increased the proliferation of RPMI-8226 and NCI-H929 cell lines at 72 hours compared with the control group(1.37±0.11 vs 1.07±0.09,2.14±0.09 vs 1.82±0.11,both P<0.05),and low expression of miR-17-5p reduced the proliferation of NCI-H929 and MM-1R cell lines at 72 hours compared with the control group(1.38±0.09 vs 1.83±0.11,1.45±0.10 vs 1.73±0.09,both P<0.05).The subcutaneous tumorigenesis experiment in nude mice showed that the tumor volume of miR-17-5p over expression group was larger than that of the control group[(1865±181)vs(1389±227)mm^(3),P<0.05],and the tumor volume of miR-17-5p low expression group was smaller than that of the control group[(1006±171)vs(1389±227)mm^(3),P<0.05].Conclusion miR-17-5p may play an oncogene role in MM cell lines as a plasma molecular marker related to the development of MM disease.