多发性骨髓瘤患者外周血单个核细胞微小RNA-202的表达及意义

摘要

Objective To establish a method of SYBR Green Ⅰ FQ-PCR for detecting the expression of miR-202 in peripheral blood mononuclear cells ( PBMC ) and analyze the expression of miR-202 and its clinical significance in MM.Methods Reverse transcription was performed with specific stemloop primer for miR-202,and then FQ-PCR was used to detected the expression of miR-202 in 21 MM patients and 20 healthy people.Data was presented as mean ± standard deviation ( (x) ± s ).Non-parametric Mann-Whitney test was used to analyze the difference between MM group and control group.In addition,1∶ 125 dilution of one test sample was detected by repeated 5 times,and the same sample was tested one time a day for 3 days,repeated 5 each time.The assessment of repeatability of measurements was done by calculating standard deviation and variation coefficient from threshold cycle (Ct).Results FQ-PCR detection of miR-202 in PBMC was amplified by the standard S-curve,with a single melting curve peak and no complex peak,which showed good specificity.The assay showed good reproducibility (intra-assay coefficient 1.2％ and inter-assay coefficient 3.2％ ) and high sensitivity ( 12.8 pmol/μ1).The expression of miR-202 in MM was 0.014 ( 0.007 - 0.221 ) and 1.844 ( 0.162 - 3.966 ) in normal controls.The expression level of miR-202 was significantly higher in MM than in normal controls (U =48.000,P ＜0.01 ).Conclusions FQ-PCR provide us a rapid,sensitive and specific method for detection of miR-202.The expression level of miR-202 is higher in MM than in normal controls.It is possible to play a role in MM progress and may be a useful marker to evaluate the development and treatment of MM.%目的 建立SYBR Green Ⅰ FQ-PCR检测人外周血单个核细胞(PBMC)中miR-202表达的方法,并初步探讨其检测MM的意义.方法 采用特异miR-202茎环引物逆转录后,用FQ-PCR对21例MM患者及20名健康人PBMC中miR-202表达水平做相对定量分析；结果用Mann-Whitney法进行两组间miR-202表达差异比较.此外,用1份1∶125稀释标本重复检测5次；及同一标本连续检测3d,每天检测1次,每次重复5管,根据循环阈值(Ct)计算标准差和变异系数(CV),以评价所建方法的重复性.结果 FQ-PCR检测PBMC中miR-202的扩增曲线呈标准的S型,熔解曲线峰值单一,未见杂峰,显示其特异性较好.批内CV为1.2％,批间CV为3.2％,当标本miR-202浓度被稀释为12.8 pmol/μl时,仍可稳定地得到扩增曲线.FQ-PCR检测MM组中miR-202表达量为1.844(0.162 ～3.966),健康对照组表达量为0.014(0.007～0.221),MM组中miR-202表达明显高于健康对照组(U=48.000,P＜0.01).结论 FQ-PCR是一种快速简便、特异性和重复性较好的检测miR-202的方法,MM患者PBMC中miR-202表达升高,其可能参与MM的发生过程,并有可能成为MM诊断和治疗的新指标.