细粒棘球蚴感染绵羊血清miRNA差异表达及诊断价值评价

Effect of all-trans tretinoic acid on the activity and growth of Echinococcus multilocularis protoscolices in vitro

目的 筛选细粒棘球蚴感染后绵羊血清中差异表达的miRNA,评价其在棘球蚴病诊断中的潜在价值。方法 10只绵羊分为感染组(5只)和对照组(5只),感染组每只绵羊灌胃2 000个细粒棘球绦虫虫卵,对照组绵羊给予生理盐水。感染后60 d,收集两组绵羊外周血,提取血清总RNA,利用小RNA高通量测序鉴定并筛选差异表达的miRNA。选取5个差异表达miRNA进行实时荧光定量逆转录PCR (qRT-PCR)验证,并采用Medcale软件绘制受试者工作特征(ROC)曲线,计算曲线下面积(AUC),筛选具有潜在诊断价值(AUC≥0.7)的miRNA。采用qRT-PCR检测15份细粒棘球蚴感染绵羊血清样品中具有潜在诊断价值的miRNA的转录水平,并计算曲线下面积(AUC)以及敏感性和特异性。采用miRanda和RNAhybrid软件对差异表达miRNA的靶基因进行预测,并进行基因本体(GO)富集分析和京都基因与基因组百科全书(KEGG)路径富集分析。结果 测序结果显示,共鉴定出26个差异性表达的miRNAs,其中21个上调表达,5个下调表达。qRT-PCR结果显示,感染组oar-miR-191、oar-let-7a、oar-miR-150、oar-miR-26a、oar-miR-21等5个相对丰度较高的差异miRNA的相对表达水平分别为2.22±0.31、2.12±0.24、2.42±0.35、2.09±0.15、3.23±0.83,均高于对照组(1.00±0.11),其中oar-miR-191、oarlet-7a、oar-miR-150、oar-miR-26a的相对表达水平与对照组差异有统计学意义(t=3.960、4.766、4.096、9.126,均P <0.05)。ROC曲线分析发现,oar-let-7a、oar-miR-26a、oar-miR-21的AUC均小于0.7。oar-miR-191的AUC为0.858(95%CI为0.719~0.997,P <0.05),具有较高的诊断价值,其灵敏度为71.43%,特异性为85.71%;oar-miR-150的AUC为0.738 (95%CI为0.550~0.926,P <0.05),具有一定的诊断意义,其灵敏度为53.33%,特异性为86.67%。GO显著性富集分析结果显示,转录水平上升两倍以上的miRNA靶基因主要与应激反应、细胞表面分子、蛋白质结合等功能相关;KEGG通路富集分析结果表明,转录水平上升至少两倍的miRNA靶基因主要富集在炎症反应、细胞自噬和细胞凋亡等关键信号通路上。结论 本研究筛选出oar-miR-191和oar-miR-150在细粒棘球蚴感染诊断中具有较好的灵敏度和特异性,有望成为细粒棘球病诊断的潜在生物标志物。

Objective To investigate the effect of all-trans retinoic acid(ATRA) on the activity and growth of Echinococcus multilocularis protoscoleces in vitro.Methods The cysts were dissected aseptically from the gerbils and the active E. multilocularis protoscolices were obtained by grinding, filtering, and cleaning. The protoscolices were divided into different concentrations of the ATRA groups(10, 25, 50, and 100 μmol/L groups with the corresponding final concentration of ATRA), the dimethyl sulfoxide(DMSO) group(the final concentration of DMSO was 0.1%), and the blank group(the same amount of complete medium was added). After 9 days of intervention, eosin staining was used to observe the activity and morphological changes of the groups under the microscope. The survival rate of the protoscolices was calculated, and the survival curve was drawn. E. multilocularis microcysts were obtained by co-culturing the protoscolices with rat hepatocellular carcinoma RH35 cells for 5-6 weeks, and the microcysts were treated with different final concentrations of ATRA(10, 100 μmol/L) for 9 days. The activity and morphological changes of the microcysts were observed under a microscope, and the DMSO group and blank group were set up. After 48 h of intervention, the EdU positive rate was detected by EdU cell imaging and the relative expression of Caspase-3 in protoscolices was detected using the Caspase-3 kit. After 24 hours of intervention, a ROS detection kit was used to quantify the levels of reactive oxygen species(ROS) in each group. An independent sample t-test was used to compare the two groups of samples, and a one-way analysis of variance(ANOVA) was used to compare the difference between the ATRA and DMSO groups of different concentrations.Results The volume of dead protoscoleces in the 10, 25,50, and 100 μmol/L groups significantly decreased and could be stained by eosin dye. With the increase in time and drug concentration, the outline blurred, light transmission weakened, and small hook detachment increased. On day 4,the survival rates of the protoscoleces in the 10, 25, 50, and 100 μmol/L groups were(87.33 ± 4.90) %,(74.00 ±2.08) %,(64.33 ± 2.03) % and(53.33 ± 1.86) %, respectively. They were all lower than those in the DMSO group[(95.67 ± 1.20) %](F = 98.41, P < 0.05). On day 9, the survival rates of protoscoleces in the 10, 25, and 50 μmol/L groups were(62.00 ± 2.64)%,(36.33 ± 2.52)% and(7.67 ± 1.53)%, which were lower than those in the DMSO group[(85.67 ± 2.08)%](F = 1154.34, P < 0.05). When different concentrations of ATRA were co-cultured with multilocular echinococcus microcysts for 9 days, with the increase in ATRA concentration, the vesicles grew slowly, and the intracapsular structure became cloudy. The germinal layer and stratum corneum of microcysts were intact in the blank group and the DMSO group. EdU cell imaging showed red and blue fluorescence in ATRA groups with different concentrations.The EdU positive rates in the 10, 25, 50, and 100 μmol/L groups were(51.63 ± 3.09) %,(42.09 ± 1.36) %,(38.46 ±0.65) %, and(25.23 ± 1.32) %, respectively. All of them were lower than those in the DMSO group [(58.32 ± 0.91)%](F = 168.59, P < 0.05). The caspase-3 activity of the 10, 25, 50, and 100 μmol/L groups was(19.23 ± 2.27),(26.27 ±3.45),(43.29 ± 2.10) and(72.80 ± 1.40) U/L, respectively, showing a dose-dependent increase. All of them were higher than the DMSO group [(14.22 ± 0.52) U/L](F = 404.08, P < 0.05). The green fluorescence of ROS in the ATRA group was stronger than that in the DMSO group. The fluorescence intensity of the 10, 25, 50, and 100 μmol/L groups was260.96 ± 2.52, 282.10 ± 7.40, 330.30 ± 12.46 and 346.10 ± 6.39, respectively. All of them were higher than the DMSO group(236.03 ± 6.89)(F = 80.53, P < 0.05).Conclusion ATRA can inhibit the activity and growth of E. multilocularis protoscoleces in vitro, induce significant ROS accumulation in protoscolices, inhibit protoscolices proliferation, and promote apoptosis.