Taqman探针实时荧光定量PCR检测肝脏疾病患者血清中miR-122的表达水平及其临床意义

Detection of miR-122 expression in serum by Taqman probe real-time fluorescence quantitative PCR and its clinical significance

目的:运用Taqman探针实时荧光定量聚合酶链式反应(PCR)检测肝脏疾病患者血清中miR-122的表达水平并探讨其临床意义。方法:设计miR-122及U6 snRNA的茎环引物和Taqman探针,运用Taqman探针实时荧光定量PCR检测27例肝癌术前(HCC)患者、15例乙型肝炎(hepatitis B)患者、15例丙型肝炎(hepatitis C)患者、15例正常对照者(HC)、11例肝癌术后(PHCC)患者及10例肝癌术后复发(recurrence)患者血清中miR-122的表达水平,并分析miR-122与肝脏疾病相关标志物的关系。结果:Taqman探针实时荧光定量PCR方法能检测血清中miR-122的表达。HCC、hepatitis B、hepatitis C及recurrence患者血清中miR-122的表达水平均高于HC和PH-CC患者(P<0.05),hepatitis C患者血清miR-122表达水平高于HCC、hepatitis B和recurrence患者(P<0.05),但HCC、hepatitis B和recurrence患者血清miR-122的表达水平无明显差异(P>0.05),PHCC患者血清miR-122的表达水平比HCC和recurrence患者低(P<0.05)。血清乙型肝炎病毒表面抗原(HBsAg)阳性和(或)乙型肝炎病毒e抗原(HBeAg)阳性患者血清miR-122的表达水平高于阴性者(P<0.05)。血清丙型肝炎病毒抗体(HCV-Ab)阳性患者血清miR-122的表达水平高于阴性者(P<0.05)。血清丙氨酸氨基转移酶(ALT)与miR-122的表达水平有正相关性(r=0.34,P<0.05)。血清甲胎蛋白(AFP)≥400μg/L组血清miR-122的表达高于AFP<400μg/L组(P<0.05)。结论:Taqman探针实时荧光定量PCR适用于检测血清miR-122的表达水平。在HCC、hepatitis B、hepatitisC及recurrence患者血清中miR-22均有不同程度的增高,尤其是hepatitis C患者,且PHCC患者血清miR-122表达下降,复发后升高。血清miR-122的表达与肝脏疾病的某些指标有关,提示血清miR-122可作为肝脏疾病,特别是肝癌早期诊断、手术疗效及预后判断的新指标。

AIM： To detect the serum level of miR-122 expression by the technique of Taqman probe real-time fluorescence quantitative PCR for identifying its clinical significance. METHODS： The stem-loop RT primer, PCR primer and Taqman probe of miR-122 and U6 snRNA were designed. The expression of miR-122 in the serum samples of 27 cases of preoperative hepatocellular carcinoma （ HCC）, 15 cases of hepatitis B, 15 cases of hepatitis C, 15 cases of healthy con- trol （ HC）, 11 cases of postoperative hepatoceUular carcinoma （PHCC） and 10 cases of recurrence after postoperative hep- atocellular carcinoma was detected by Taqman probe real-time fluorescence quantitative PCR. U6 snRNA was used as the internal control. RESULTS ： The results showed that the method of Taqman probe real-time fluorescence quantitative PCR could detect the amplification signal of serum miR-122. The expression level of serum miR-122 in the patients with HCC, hepatitis B, hepatitis C and recurrence was higher than that in HC and the patients with PHCC. Meanwhile, the serum lev- el of miR-122 in the patients with hepatitis C was higher than that in the patients with HCC, hepatitis B and recurrence.However, no difference of miR-122 expression level among HCC, hepatitis B and recurrent patients was observed. The miR-122 level was lower in PHCC patients than that in HCC and recurrent patients. In hepatitis B virus surface antigen （HBsAg） and/or hepatitis B virus e antigen （HBeAg） positive patients, the miR-122 level was higher than that in the neg- ative ones. The miR-122 level in hepatitis C antibody （HCV-Ab） positive patients was raised compared with the negative ones. The serum level of alanine aminotransferase （ALT） was positively correlated with the serum level of miR-122 （ r = 0. 34, P 〈 0.05 ）. The miR-122 expression level in the patients with serum AFP I〉 400 I^g/L was higher than that in the patients with serum AFP 〈 400 ixg/L. CONCLUSION： The method of Taqman probe real-time fluorescence quantitative PCR can detect the serum level of miR-122 expression. Serum miR-122 might be used as a new biomarker of liver disea- ses, especially in the early diagnosis of primary hepatoeellular carcinoma, the curative effect of surgical operation and the prognosis.