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| 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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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.
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Received: 12 April 2022
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Corresponding Authors:
*xdzhang1983@163.com
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