调控猪ETV5基因miRNA的筛选鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2021-1057

摘要/Abstract

摘要：

ETV5是在精原干细胞自我更新和维持中起到关键作用的转录因子。为筛选出调控猪ETV5基因的miRNA,利用在线分析软件TargetScan、miRanda预测调控ETV5基因的miRNA,采用双荧光素酶活性检测对筛选出来的保守miRNA进行验证,筛选出miR-19后采用 miR-19 模拟物 mimics 和抑制物 inhibitor 分别转染猪胎儿成纤维细胞和睾丸支持细胞,荧光定量检测ETV5的表达,同时在猪胎儿成纤维细胞中对ETV5基因进行敲除,检测miR-19的表达变化。结果表明,miR-19可通过与ETV5 3' UTR区结合显著降低细胞中荧光素酶的活性（P<0.01）;细胞转染结果显示miR-19 mimcs 可显著降低猪胎儿成纤维细胞和睾丸支持细胞中ETV5的表达,miR-19 inhibitor 则结果相反（P<0.01）;在胎儿成纤维细胞中对ETV5基因进行敲除后,miR-19的表达极显著提高（P<0.01）。miR-19对猪ETV5基因的表达具有调控作用,为进一步开展miRNA调控猪ETV5基因影响猪生殖方面的研究提供参考。

关键词: miR-19, 猪, ETV5, 睾丸

Abstract:

E-twenty-six Variant Gene 5（ETV5）is a transcription factor that plays key role in the self-renewal and maintenance of spermatogonial stem cells. To screen the miRNAs regulating porcine ETV5 gene,the online analysis software TargetScan and MiRanda were used to predict the miRNAs that regulate ETV5,and dual luciferase activity detection was adapted to verify the screened conserved miRNAs. Then the verified miRNA mimics or inhibitor were transfected into porcine fetal fibroblasts and testicular Sertoli cells respectively,the expression of ETV5 was detected by real time fluorescence quantitative PCR. Concurrently,ETV5 gene was knocked out in the porcine fetal fibroblasts to detect the expression of miR-19. The results showed that miR-19 significantly reduced the activity of luciferase in the cells by binding to the 3`UTR region of ETV5（P<0.01）. Results of cell transfection demonstrated that miR-19 mimics significantly reduced the expression of ETV5 in the porcine fetal fibroblasts and testicular Sertoli cells（P<0.01）,while miR-19 inhibitor led to the opposite result（P<0.01）. After knockout of ETV5 gene in the fetal fibroblasts,the expression of the miR-19 significantly increased（P<0.01）. In sum,miR-19 may regulate the expression of porcine ETV5 gene,which provides a reference for further research on the effect of miRNA regulating porcine ETV5 gene on porcine reproduction.

Key words: miR-19, porcine, ETV5, testis

李虹仪, 彭国良, 肖正中, 张茂. 调控猪ETV5基因miRNA的筛选鉴定[J]. 生物技术通报, 2022, 38(5): 169-174.

LI Hong-yi, PENG Guo-liang, XIAO Zheng-zhong, ZHANG Mao. Screening and Identification of miRNA Regulating Porcine E-twenty-six Variant Gene 5[J]. Biotechnology Bulletin, 2022, 38(5): 169-174.

图/表 5

图1 调控猪ETV5基因的miRNA预测

Fig. 1 Prediction of miRNA regulating porcine ETV5 gene

图2 荧光素酶活性检测 **P<0.01,表示与对照组相比差异极显著,下同

Fig.2 Detection of luciferase activity ** indicate that there is a significant difference compared with the control group,P<0.01,the same below

图3 MiR-19调控胎儿成纤维细胞中ETV5 mRNA的表达

Fig.3 MIR-19 regulating the mRNA expression of ETV5 in the fetal fibroblasts

图4 MiR-19调控睾丸支持细胞中ETV5 mRNA的表达

Fig.4 MIR-19 regulating the mRNA expression of ETV5 in the Sertoli cells

图5 miR-19在ETV5敲除细胞中的表达量

Fig.5 Expression of miR-19 in the ETV5 knockout cells

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