miR-1、miR-133b与肝泡型棘球蚴病患者肝纤维化的关系

Association between miR-1,miR-133b and liver fibrosis in patients with hepatic alveolar echinococcosis

目的探讨肝泡型棘球蚴病患者肝纤维化与微小核糖核酸(microRNA,miR)-1、miR-133b的相关性。方法选择2020年10月至2021年4月在新疆医科大学第一附属医院明确诊断为肝泡型棘球蚴病(9例)、肝硬化(9例)、肝细胞癌(5例)患者作为研究对象,并以同期健康志愿者为对照(10例),采集所有对象外周血样本制备血浆,经实时荧光定量PCR法检测外周血miR-1、miR-133b表达量;同时,自5例肝泡型棘球蚴病患者采集肝脏病灶周围组织(近端)、距病灶约5 cm处相应组织(远端),并采用免疫组织化学染色法检测肝泡型棘球蚴病患者肝脏病灶近、远端的细胞活化相关指标[细胞周期蛋白D1(CyclinD1)、细胞周期蛋白依赖性激酶1(CDK1)、α平滑肌肌动蛋白(α-SMA)],纤维化指标[Ⅰ、Ⅲ型胶原蛋白(CollagenⅠ、CollagenⅢ)],转化生长因子β1(TGF-β1)信号通路相关基因[TGF-β1,Ⅰ、Ⅱ型转化生长因子β1受体(TGF-β1RⅠ、TGF-β1RⅡ)]及其下游相关蛋白(SMAD2、SMAD3)表达情况。结果实时荧光定量PCR结果显示,肝泡型棘球蚴病、肝硬化、肝细胞癌和对照组人群外周血miR-1、miR-133b表达水平比较,差异均有统计学意义(H=16.54、28.40,均P<0.001);其中,肝泡型棘球蚴病组miR-1、miR-133b表达水平均高于对照组、肝硬化组(均P<0.05)。肝泡型棘球蚴病患者病灶近端和远端CDK1(0.46±0.02、0.42±0.01),α-SMA(0.54±0.09、0.51±0.07),TGF-β1(0.55±0.15、0.51±0.13),TGF-β1RⅠ(0.58±0.09、0.57±0.08),TGF-β1RⅡ表达水平(0.40±0.05、0.39±0.05)比较,差异均有统计学意义(t=5.56、3.17、3.18、4.27、5.65,P=0.005、0.034、0.034、0.024、0.011);CyclinD1、CollagenⅠ、CollagenⅢ、SMAD2、SMAD3表达水平比较,差异均无统计学意义(t=3.06、3.06、2.86、1.43、1.50,P=0.055、0.055、0.064、0.247、0.230)。Pearson相关分析结果显示,肝泡型棘球蚴病患者外周血miR-1与肝脏病灶近端TGF-β1RⅠ表达水平呈正相关(P=0.001);miR-1、miR-133b与CDK1、α-SMA、TGF-β1、TGF-β1RⅡ表达水平均无相关性(均P>0.05)。结论肝泡型棘球蚴病患者肝脏病灶近端TGF-β1信号通路相关因子表达上调;外周血miR-1和miR-133b表达上调,且miR-1与肝脏病灶近端TGF-β1RⅠ表达水平呈正相关,提示miR-1可能促进肝泡型棘球蚴病肝纤维化发生。

Objective To investigate the relationship between microRNA(miR)-1,miR-133b and hepatic fibrosis in patients with hepatic alveolar echinococcosis.Methods From October 2020 to April 2021,patients who were definitely diagnosed as hepatic alveolar echinococcosis(9 cases),cirrhosis(9 cases)and hepatocellular carcinoma(5 cases)in the First Affiliated Hospital of Xinjiang Medical University were selected as the research subjects,and healthy volunteers in the same period were taken as the control(10 cases).Peripheral blood samples of all subjects were collected to prepare plasma,and the expression levels of miR-1 and miR-133b in peripheral blood were detected by quantitative real-time PCR.At the same time,tissue samples around the liver lesion(proximal),and the corresponding tissues about 5 cm from the lesion(distal)were collected from 5 patients with hepatic alveolar echinococcosis,and immunohistochemical staining was used to detect the cell activation related indicators[cyclinD1,cyclin dependent kinase 1(CDK1),α-smooth muscle actin(α-SMA)],fibrosis indicators(CollagenⅠ,CollagenⅢ),transforming growth factor-β1(TGF-β1)signal pathway related genes[TGF-β1,TGF-β1 receptor typeⅠ/Ⅱ(TGF-β1RⅠ,TGF-β1RⅡ)]and its downstream related proteins(SMAD2,SMAD3).Results The quantitative real-time PCR results showed that there were significant differences in the expression levels of miR-1 and miR-133b in the peripheral blood of patients with hepatic alveolar echinococcosis,cirrhosis,hepatocellular carcinoma and the control group(H=16.54,28.40,P<0.001);the expression levels of miR-1 and miR-133b in hepatic alveolar echinococcosis group were higher than those in control group,cirrhosis group(P<0.05).The expression levels of CDK1(0.46±0.02,0.42±0.01),α-SMA(0.54±0.09,0.51±0.07),TGF-β1(0.55±0.15,0.51±0.13),TGF-β1RⅠ(0.58±0.09,0.57±0.08),and TGF-β1RⅡ(0.40±0.05,0.39±0.05)between the proximal and distal tissue of liver lesion in hepatic alveolar echinococcosis patients were statistically significantly different(t=5.56,3.17,3.18,4.27,5.65,P=0.005,0.034,0.034,0.024,0.011).There was no statistically significant difference in the expression levels of CyclinD1,CollagenⅠ,CollagenⅢ,SMAD2 and SMAD3 between the proximal and distal tissue of liver lesion in hepatic alveolar echinococcosis patients(t=3.06,3.06,2.86,1.43,1.50,P=0.055,0.055,0.064,0.247,0.230).Pearson correlation analysis showed that miR-1 in the patients'peripheral blood was positively correlated with TGF-β1RⅠin the proximal tissue of the liver lesion(P=0.001);there was no correlation between miR-1,miR-133b and CDK1,α-SMA,TGF-β1,TGF-β1RⅡ(P>0.05).Conclusions The expression of TGF-β1 signaling pathway related factors in the proximal tissue of liver lesion in patients with hepatic alveolar echinococcosis is up-regulated.The expression of miR-1 and miR-133b in peripheral blood is upregulated,and miR-1 is positively correlated with TGF-β1RⅠlevel in proximal tissue of liver lesion,suggesting that miR-1 may promote the occurrence of liver fibrosis in hepatic alveolar echinococcosis.