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

参考文献 27

[1]	Alankarage D, Lavery R, Svingen T, et al. SOX9 regulates expression of the male fertility gene Ets variant factor 5(ETV5)during mammalian sex development[J]. Int J Biochem Cell Biol, 2016, 79:41-51. doi: S1357-2725(16)30214-X pmid: 27498191
[2]	Gutierrez-Aguilar R, Kim DH, Casimir M, et al. The role of the transcription factor ETV5 in insulin exocytosis[J]. Diabetologia, 2014, 57(2):383-391. doi: 10.1007/s00125-013-3096-5 pmid: 24190582
[3]	Chen C, Ouyang W, Grigura V, et al. ERM is required for transcriptional control of the spermatogonial stem cell niche[J]. Nature, 2005, 436(7053):1030-1034. doi: 10.1038/nature03894 URL
[4]	Zhang XY, Zhao X, Li GL, et al. Establishment of Etv5 gene knockout mice as a recipient model for spermatogonial stem cell transplantation[J]. Biol open. 2021, 10(1):bio056804.
[5]	Schlesser HN, Simon L, Hofmann MC, et al. Effects of ETV5(ets variant gene 5)on testis and body growth, time course of spermatogonial stem cell loss, and fertility in mice[J]. Biol Reprod, 2008, 78(3):483-489. doi: 10.1095/biolreprod.107.062935 pmid: 18032421
[6]	Tyagi G, Carnes K, Morrow C, et al. Loss of Etv5 decreases proliferation and RET levels in neonatal mouse testicular germ cells and causes an abnormal first wave of spermatogenesis[J]. Biol Reprod, 2009, 81(2):258-266.
[7]	Eo J, Shin H, Kwon S, et al. Complex ovarian defects lead to infertility in Etv5-/- female mice[J]. Mol Hum Reprod, 2011, 17(9):568-576. doi: 10.1093/molehr/gar021 URL
[8]	Jamsai D, Clark BJ, Smith SJ, et al. A missense mutation in the transcription factor ETV5 leads to sterility, increased embryonic and perinatal death, postnatal growth restriction, renal asymmetry and polydactyly in the mouse[J]. PLoS One, 2013, 8(10):e77311. doi: 10.1371/journal.pone.0077311 URL
[9]	张廷焕, 张利娟, 陈四清, 等. 猪miR-378种子序列的多态性对其功能以及胴体性状的影响[J]. 生物技术通报, 2021, 37(6):154-162. doi: 10.13560/j.cnki.biotech.bull.1985.2020-1165
	Zhang TH, Zhang LJ, Chen SQ, et al. Effects of the polymorphism of the seed sequence in porcine miR-378 on its function and carcass traits[J]. Biotechnol Bull, 2021, 37(6):154-162.
[10]	罗芳, 李朝晖, 赵小阳. 精原干细胞的自我更新及分化调控的研究现状与展望[J]. 发育医学电子杂志, 2020, 8(4):378-384.
	Luo F, Li ZH, Zhao XY. Research and prospect of self-renewal and differentiation regulation for spermatogonial stem cell[J]. J Dev Med:Electron Version, 2020, 8(4):378-384.
[11]	Wu X, Goodyear SM, Tobias JW, et al. Spermatogonial stem cell self-renewal requires ETV5-mediated downstream activation of Brachyury in mice[J]. Biol Reprod, 2011, 85(6):1114-1123. doi: 10.1095/biolreprod.111.091793 URL
[12]	Niu Z, Goodyear SM, Rao S, et al. MicroRNA-21 regulates the self-renewal of mouse spermatogonial stem cells[J]. PNAS, 2011, 108(31):12740-12745. doi: 10.1073/pnas.1109987108 URL
[13]	梅星星, 李小勇, 吴际. 一组miRNAs在睾丸发育中的表达及miR-125a对精原干细胞发育的调节作用[J]. 上海交通大学学报:医学版, 2015, 35(5):625-630.
	Mei XX, Li XY, Wu J. Expressions of a group of miRNAs during testis development and regulation effect of miR-125a on development of spermatogonial stem cells[J]. J Shanghai Jiao Tong Univ:Med Sci, 2015, 35(5):625-630.
[14]	He Z, Jiang J, Kokkinaki M, et al. MiRNA-20 and mirna-106a regulate spermatogonial stem cell renewal at the post-transcriptional level via targeting STAT3 and Ccnd1[J]. Stem Cells, 2013, 31(10):2205-2217. doi: 10.1002/stem.1474 URL
[15]	Cui N, Hao G, Zhao Z, et al. MicroRNA-224 regulates self-renewal of mouse spermatogonial stem cells via targeting DMRT1[J]. J Cell Mol Med, 2016, 20(8):1503-1512. doi: 10.1111/jcmm.12838 pmid: 27099200
[16]	Wang Y, Li X, Gong X, et al. MicroRNA-322 regulates self-renewal of mouse spermatogonial stem cells through Rassf8[J]. Int J Biol Sci, 2019, 15(4):857-869. doi: 10.7150/ijbs.30611 URL
[17]	Li Q, Li H, Liang J, et al. Sertoli cell-derived exosomal MicroRNA-486-5p regulates differentiation of spermatogonial stem cell through PTEN in mice[J]. J Cell Mol Med, 2021, 25(8):3950-3962. doi: 10.1111/jcmm.16347 URL
[18]	Fu H, Zhou F, Yuan Q, et al. miRNA-31-5p mediates the proliferation and apoptosis of human spermatogonial stem cells via targeting JAZF₁ and cyclin A2[J]. Mol Ther Nucleic Acids, 2019, 14:90-100. doi: 10.1016/j.omtn.2018.11.004 URL
[19]	Zhou F, Yuan Q, Zhang W, et al. MiR-663a stimulates proliferation and suppresses early apoptosis of human spermatogonial stem cells by targeting NFIX and regulating cell cycle[J]. Mol Ther Nucleic Acids, 2018, 12:319-336. doi: 10.1016/j.omtn.2018.05.015 URL
[20]	Zhang J, Cao H, Xie J, et al. The oncogene Etv5 promotes MET in somatic reprogramming and orchestrates epiblast/primitive endoderm specification during mESCs differentiation[J]. Cell Death Dis, 2018, 9(2):224. doi: 10.1038/s41419-018-0335-1 URL
[21]	Mus LM, Lambertz I, Claeys S, et al. The ETS transcription factor ETV5 is a target of activated ALK in neuroblastoma contributing to increased tumour aggressiveness[J]. Sci Rep, 2020, 10(1):218. doi: 10.1038/s41598-019-57076-5 URL
[22]	Bullock M, Lim G, Zhu Y, et al. ETS factor ETV5 activates the mutant telomerase reverse transcriptase promoter in thyroid cancer[J]. Thyroid, 2019, 29(11):1623-1633. doi: 10.1089/thy.2018.0314 pmid: 31452441
[23]	Meng D, Li Z, Ma X, et al. ETV5 overexpression contributes to tumor growth and progression of thyroid cancer through PIK3CA[J]. Life Sci, 2020, 253:117693. doi: 10.1016/j.lfs.2020.117693 URL
[24]	Mao Z, Feng M, Li Z, et al. ETV5 regulates hepatic fatty acid metabolism through PPAR signaling pathway[J]. Diabetes, 2021, 70(1):214-226. doi: 10.2337/db20-0619 URL
[25]	Liu Y, Zhang Y. ETV5 is essential for neuronal differentiation of human neural progenitor cells by repressing NEUROG2 expression[J]. Stem Cell Rev Rep, 2019, 15(5):703-716. doi: 10.1007/s12015-019-09904-4 URL
[26]	王烁程, 陈晓丽, 张林波, 等. 支持细胞对精原干细胞增殖、分化调控的研究进展[J]. 畜牧兽医学报, 2019, 50(2):253-260.
	Wang SC, Chen XL, Zhang LB, et al. The progress of the study on the regulation of the proliferation, differentiation of spermatogonial stem cells by the Sertoli cells[J]. Chin J Animal Vet Sci, 2019, 50(2):253-260.
[27]	梁洺源, 朱化彬, 陈晓丽, 等. 支持细胞调控精原干细胞增殖、分化和凋亡的研究进展[J]. 畜牧兽医学报, 2016, 47(2):225-231.
	Liang MY, Zhu HB, Chen XL, et al. The study progress of the proliferation, differentiation and apoptosis of spermatogonial stem cells under the regulation of Sertoli cells[J]. Chin J Animal Vet Sci, 2016, 47(2):225-231.