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2025年8月2日 星期六
农业生物技术学报  2023, Vol. 31 Issue (7): 1333-1344    DOI: 10.3969/j.issn.1674-7968.2023.07.001
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
miR-10b靶向调节PTEN基因及其蛋白对猪卵巢颗粒细胞凋亡的调控作用
章会斌, 刘阳光, 尚金男, 韩政, 周忍, 许黎明, 徐启隆, 郑先瑞, 殷宗俊, 张晓东*
安徽农业大学 动物科技学院/地方畜禽遗传资源保护与生物育种安徽省重点实验室,合肥 230036
The Effect of MiR-10b Targeting PTEN Gene and Its Protein on the Apoptosis of Porcine (Sus scrofa) Ovarian Granulosa Cells
ZHANG Hui-Bin, LIU Yang-Guang, SHANG Jin-Nan, HAN Zheng, ZHOU Ren, XU Li-Ming, XU Qi-Long, ZHENG Xian-Rui, YIN Zong-Jun, ZHANG Xiao-Dong*
College of Animal Science and Technology/Anhui Provincial Key Laboratory of Genetic Resources Protection and Biological Breeding of Local Livestock and Poultry, Anhui Agricultural University, Hefei 230036, China
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摘要 miRNA (microRNA, miR)是可参与基因转录后调控的内源性非编码小 RNA,miR-10b 是猪(Sus scrofa) 卵巢闭锁过程中的一个关键调控因子,而其靶向基因同源性磷酸酶-张力蛋白(phosphatase and tensin homolog, PTEN)基因在细胞凋亡中起着重要作用。为探讨 miR-10b和PTEN基因对猪卵巢颗粒细 胞 (pig ovarian granulosa cells, pGCs) 凋亡的调控作用,本研究分别用miR-10b 的模拟物及抑制物转染 pGCs,构建过表达和抑制 miR-10b 的 pGCs 模型,采用流式细胞术检测过表达和抑制 miR-10b对pGCs凋亡的影响。结果显示,过表达 miR-10b 极显著增加了 pGCs 凋亡率(P<0.01),抑制 miR-10b 表达后 pGCs 凋亡率极显著降低(P<0.01)。基于生物信息学分析可知,miR-10b 能直接结合PTEN 3'UTR 区。进一步构建通过荧光素酶报告基因载体,发现相对于pmirGLO-PTEN-WT 与 mimic NC共转染组,pmirGLO- PTEN-WT与miR-10b mimic 共转染后的荧光活性更低(P<0.01),pmirGLO-PTEN-MUT 与 miR-10b mimic共转染,或与 mimic NC 共转染后,荧光活性无显著差异(P>0.05),pmiGLO 与 miR-10b mimic 共转染,或与 mimic NC 共转染的荧光也无显著差异(P>0.05),通过双荧光素酶报告基因系统再次证实了 miR-10b 能靶向结合 PTEN 3'UTR 区,抑制 PTEN 基因表达。利用 qPCR 及 Westren blot 检测 PTEN 基因及其蛋白的 表达水平,发现 miR-10b 可以抑制 PTEN 基因及其蛋白的表达水平。此外,在抑制 miR-10b 表达后,引入 siRNA 下调 PTEN 基因的表达,可以缓解 miR-10b 对 pGCs 凋亡的促进作用。本研究证实了 miR-10b 可以通过抑制靶基因 PTEN 的表达来调控 pGCs 凋亡,为深入了解 pGCs 的凋亡机制和研究卵泡发育提供了数据支持。
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章会斌
刘阳光
尚金男
韩政
周忍
许黎明
徐启隆
郑先瑞
殷宗俊
张晓东
关键词 miR-10b同源性磷酸酶-张力蛋白(PTEN)颗粒细胞    
Abstract:MicroRNA (miRNA) are a class of endogenous non-coding small RNAs that can be involved in post-transcriptional regulation of genes. miR-10b has recently been shown to be a key regulator during ovarian atresia in pig (Sus scrofa), while phosphatase and tonic homolog genes (PTEN) play an important role in apoptosis. In order to investigate the regulatory effects of miR-10b and PTEN on apoptosis in pig ovarian granulosa cells (pGCs), the miR-10b mimic and inhibitor were transfected into pGCs to performed miR-10b overexpression and repression, respectively. The results showed that the apoptosis rate of pGCs was significantly increased after overexpression of miR-10b (P<0.01). In contrast, inhibition of miR-10b expression decreased the apoptosis of pGCs (P<0.01). To further determine the function of miR-10b, bioinformatics analysis was carried out to predict target gene of miR-10b, the result showed that 3'UTR of PTEN contained the target sites for miR-10b. By luciferase reporter assay, the firefly luciferase activity was reduced when co-transfection of pmirGLO-PTEN-WT and miR-10b mimic compared to co-transfection of pmirGLO-PTEN-WT and mimic NC (P<0.01), but no significant difference in the firefly luciferase activity between co-transfection of pmirGLO-PTEN-MUT and miR-10b mimic compared to co-transfection of pmirGLO-PTEN-MUT and mimic NC (P>0.05). In addition, co-transfection of pmirGLO and mimic NC was no significantly different compared to co-transfection of pmirGLO and miR-10b mimic (P>0.05). To better understand the mechanism of miR-10b during apoptosis of pGCs, effect of miR-10b on the expression of PTEN was verified and found that expression of PTEN gene and its protein decreased significantly after overexpression of miR-10b (P<0.01). In contrast, when miR-10b expression was inhibited by miR-10b inhibitor, the PTEN was increased compared to the inhibitor NC group. After inhibiting the expression of miR-10b, and siRNA was transfected to down-regulate the expression of PTEN gene, which partially alleviated the promoting effect of miR-10b on pGCs apoptosis. Together, it was demonstrated that miR-10b could directly bind PTEN 3'UTR and inhibit the expression level of PTEN, and miR-10b could regulate apoptosis of pGCs by inhibiting the expression of the target gene PTEN. This study demonstrates that miR-10b can regulate apoptosis by inhibiting the expression of the target gene PTEN, which provides data to support an in-depth understanding of the apoptotic mechanism of pGCs and the study of follicle development.
Key wordsmiR-10b    Phosphatase and tensin homolog (PTEN)    Granulosa cell    Pig
收稿日期: 2022-04-12     
基金资助:国家重点研发计划项目(2021YFD1301200); 国家自然科学基金(31972531); 安徽省高校协同创新项目(GXXT-2021-055)
通讯作者: *xdzhang1983@163.com   
引用本文:   
章会斌, 刘阳光, 尚金男, 韩政, 周忍, 许黎明, 徐启隆, 郑先瑞, 殷宗俊, 张晓东. miR-10b靶向调节PTEN基因及其蛋白对猪卵巢颗粒细胞凋亡的调控作用[J]. 农业生物技术学报, 2023, 31(7): 1333-1344.
ZHANG Hui-Bin,LIU Yang-Guang,SHANG Jin-Nan,HAN Zheng,ZHOU Ren,XU Li-Ming,XU Qi-Long,ZHENG Xian-Rui,YIN Zong-Jun,ZHANG Xiao-Dong . The Effect of MiR-10b Targeting PTEN Gene and Its Protein on the Apoptosis of Porcine (Sus scrofa) Ovarian Granulosa Cells. 农业生物技术学报, 2023, 31(7): 1333-1344.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.07.001     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I7/1333
 
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