朊病毒病相关miRNA在不同发病脏器和细胞中表达的变化

Expressions of prion disease-related miRNA in various pathogenic organs and cells

目的利用Real-time PCR法检测朊病毒病相关miRNA在不同发病脏器和细胞中的表达变化。方法提取羊痒病因子22L毒株感染的BALB/c小鼠大脑、海马、延髓、脾脏以及羊痒病因子22L毒株感染的鼠神经瘤ScN2a细胞总RNA,通过特殊设计的茎环结构的反转录引物进行反转录,应用ABI step one plus system定量PCR仪进行定量检测;对Real-time PCR进行特异性和重复性验证;采用SYBR Green荧光染料法,以U6 RNA作为内参照,2-△△Ct法进行miRNA表达相对定量分析,检测小鼠发病脏器中和细胞中miR-7a、miR-128、miR-135a、miR-146a、miR-152、miR-24-2*、miR-380-3p、miR-448-5p的表达。结果 Real-time PCR法检测朊病毒病相关miRNA特异性和重复性良好。各组中miRNA改变5倍以上的有发病终末期小鼠大脑中的miR-146a,海马中的miR-128、miR-135a、miR-152,延髓中的miR-448-5p,ScN2a细胞中的miR-146a,脾脏中的miR-152,其中脾脏中的miR-152显著上调,为18.98倍。结论朊病毒病中这些上调或下调的miRNA能调控细胞内多种代谢和信号传导途径发生异常,引起细胞及组织器官发生病理学变化。

Objective To analyze the expressions of prion disease-related miRNA in various pathogenic organs and cells by real-time quantitative PCR （RQ-PCR）. Methods Total RNA was isolated from the brain, hippocampus, medulla ob- longata, spleen of BALB/c mice as well as mouse neuroma ScNa cells, infected with seripe virus 22L strain, and reversely transcribed to template eDNA by using loop-stem structure reverse primer, then analyzed for miRNA expression by RQ- PCR using ABI step one plus system, based on which the real-time PCR was verified for specificity and reproducibility. The relative expression levels of miR-7a, miR-128, miR-135a, miR-146a, miR-152, miR-24-2＊, miR-380-3p and miR-448- 5p in pathogenic organs and cells were analyzed by 2C-AAO） method with SYBR Green, using U6 RNA as an internal refer- ence. Results Real-time PCR showed high specificity and reproducibility for determination of prion disease-related miRNA. The miRNA expression levels of miR-146a in brain, miR-128, miR-135a and miR-152 in hippocampus, miR-448- 5p in medulla oblongata, miR-152 in spleen of mice at terminal stage of onset, as well as miR-146a in SeN2a cells altered by more than 5 folds as compared with those in normal control group, of which the expression level of miR-152 in spleen increased by 18. 98 folds. Conclusion The up- and down-regulated miRNA in prion disease involved in a variety ofmetabolic and signal transduction pathways, resulting in pathological changes in cells and tissues.