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
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.
[1] Al-Kindi A Y, Mahmoud Y, Woller M J.2001. Ultrastructural changes in granulosa cells and plasma steroid levels af‐ ter administration of luteinizing hormone-releasing hor‐ mone in the Western painted turtle, Chrysemys picta[J]. Tissue & Cell, 33(4): 361-367. [2] Almaimani R A, Aslam A, Ahmad J, et al.2022. In vivo and In vitro enhanced tumoricidal effects of metformin, ac‐ tive vitamin d(3), and 5-fluorouracil triple therapy against colon cancer by modulating the PI3K/Akt/PTEN/mTOR network[J]. Cancers, 14(6): 1538. [3] Bahena-Ocampo I, Espinosa M, Ceballos-Cancino G, et al.2016. miR-10b expression in breast cancer stem cells supports self-renewal through negative PTEN regula‐ tion and sustained AKT activation[J]. EMBO Reports, 17(5): 648-658. [4] Cao R, Wu W J, Zhou X L, et al.2015. Expression and pre‐ liminary functional profiling of the let-7 family during porcine ovary follicle atresia[J]. Molecules and Cells, 38(4): 304-311. [5] Cheng Y, Yu C, Li W, et al.2020. Matrine inhibits prolifera‐ tion, invasion, and migration and induces apoptosis of colorectal cancer cells via miR-10b/PTEN pathway[J]. Cancer Biotherapy and Radiopharmaceuticals, 37(10): 871-881. [6] Chun S Y, Eisenhauer K M, Minami S, et al.1996. Hormonal regulation of apoptosis in early antral follicles: Follicle- stimulating hormone as a major survival factor[J]. Endo‐ crinology, 137(4): 1447-1456. [7] Di Cristofano A, Pesce B, Cordon-Cardo C, et al.1998. Pten is essential for embryonic development and tumour sup‐ pression[J]. Nature Genetics, 19(4): 348-355. [8] Donadeu F X, Schauer S N, Sontakke S D.2012. Involve‐ ment of miRNAs in ovarian follicular and luteal devel‐ opment[J]. Journal of Endocrinology, 215(3): 323-334. [9] Fan H, He J, Bai Y, et al.2022. Baicalin improves the func‐ tions of granulosa cells and the ovary in aged mice through the mTOR signaling pathway[J]. Journal of Ovarian Research, 15(1): 34. [10] Fan Y, Chang Y, Wei L, et al.2019. Apoptosis of mural granu‐ losa cells is increased in women with diminished ovari‐ an reserve[J]. Journal of Assisted Reproduction and Ge‐ netics, 36(6): 1225-1235. [11] Gao S, De Geyter C, Kossowska K, et al.2007. FSH stimu‐ lates the expression of the ADAMTS-16 protease in ma‐ ture human ovarian follicles[J] Molecular Human Re‐production, 13(7): 465-471. [12] Gao Y, Chen J, Ji R, et al.2021. USP25 regulates the prolifer‐ ation and apoptosis of ovarian granulosa cells in poly‐ cystic ovary syndrome by modulating the PI3K/AKT pathway via deubiquitinating PTEN[J]. Frontiers in Cell and Developmental Biology, 9: 779718. [13] Guo L, Huang Q, Zhao J, et al.2021. microRNA-10b pro‐ motes the apoptosis of bovine ovarian granulosa cells by targeting plasminogen activator inhibitor-1[J]. Theri‐ ogenology, 176: 206-216. [14] Han L L, Yin X R, Zhang S Q.2018. miR-650 promotes the metastasis and epithelial-mesenchymal transition of he‐patocellular carcinoma by directly inhibiting LATS2 ex‐pression[J]. Cellular Physiology and Biochemistry, 51(3): 1179-1192. [15] Hossain M M, Sohel M M, Schellander K, et al.2012. Char‐ acterization and importance of microRNAs in mammali‐ an gonadal functions[J]. Cell & Tissue Research, 349(3): 679-690. [16] Hsueh A J, Billig H, Tsafriri A.1994. Ovarian follicle atresia: A hormonally controlled apoptotic process[J]. Endo‐ crine Reviews, 15(6): 707-724. [17] Imbar T, Eisenberg I.2014. Regulatory role of microRNAs in ovarian function[J]. Fertility and Sterility, 101(6): 1524-1530. [18] Ke X, Li L, Li J, et al.2022. Anti-oncogenic PTEN induces ovarian cancer cell senescence by targeting P21[J]. Cell Biology International, 46(1): 118-128. [19] Lee M S, Liu C H, Lee T H, et al.2010. Association of cre‐ atin kinase B and peroxiredoxin 2 expression with age and embryo quality in cumulus cells[J]. Journal of As‐ sisted Reproduction and Genetics, 27(11): 629-639. [20] Lee R C, Feinbaum R L, Ambros V.1993. The C. elegans het‐erochronic gene lin-4 encodes small RNAs with anti‐sense complementarity to lin-14[J]. Cell, 75(5): 843-854. [21] Lee Y, Kim M, Han J, et al.2004. MicroRNA genes are tran‐ scribed by RNA polymerase Ⅱ[J]. EMBO Journal, 23(20): 4051-4060. [22] Li Q, Du X, Pan Z, et al.2018. The transcription factor SMAD4 and miR-10b contribute to E2 release and cell apoptosis in ovarian granulosa cells by targeting CYP19A1[J]. Molecular and Cellular Endocrinology, 476: 84-95. [23] Li Y, Fang Y, Liu Y, et al.2015. MicroRNAs in ovarian func‐tion and disorders[J]. Journal of Ovarian Research, 8:51. [24] Liu J, Du X, Zhou J, et al.2014. MicroRNA-26b functions as a proapoptotic factor in porcine follicular granulosa cells by targeting Sma-and Mad-related protein 4[J]. Bi‐ ology of Reproduction, 91(6): 146. [25] Liu J, Zeng L, Zhuang S, et al.2020. Cadmium exposure dur‐ ing prenatal development causes progesterone disrup‐ tors in multiple generations via steroidogenic enzymes in rat ovarian granulosa cells[J]. Ecotoxicology and En‐ vironmental Safety, 201: 110765. [26] Ma L, Teruya-Feldstein J, Weinberg R A.2007. Tumour inva‐ sion and metastasis initiated by microRNA-10b in breast cancer[J]. Nature, 449(7163): 682-688. [27] Matsuda-Minehata F, Inoue N, Goto Y, et al.2006. The regula‐ tion of ovarian granulosa cell death by pro- and anti- apoptotic molecules[J]. Journal of Reproduction and De‐ velopment, 52(6): 695-705. [28] McGinnis L K, Luense L J, Christenson L K.2015. MicroR‐ NA in ovarian biology and disease[J]. Cold Spring Har‐ bor Perspectives in Medicine, 5(9): a022962. [29] Peng J Y, An X P, Fang F, et al.2016. MicroRNA-10b sup‐ presses goat granulosa cell proliferation by targeting brain-derived neurotropic factor[J]. Domestic Animal Endocrinology, 54: 60-67. [30] Salilew-Wondim D, Ahmad I, Gebremedhn S, et al.2014. The expression pattern of microRNAs in granulosa cells of subordinate and dominant follicles during the early lute‐ al phase of the bovine estrous cycle[J]. PLOS ONE, 9(9): e106795. [31] Scott R, Zhang M, Seli E.2018. Metabolism of the oocyte and the preimplantation embryo: Implications for assist‐ ed reproduction[J]. Current Opinion in Obstetrics & Gy‐ necology, 30(3): 163-170. [32] Siraj M A, Jacobs A T, Tan G T.2022. Altersolanol B, a fun‐ gal tetrahydroanthraquinone, inhibits the proliferation of estrogen receptor-expressing (ER+) human breast adeno‐ carcinoma by modulating PI3K/AKT, p38/ERK MAPK and associated signaling pathways[J]. Chemico-Biologi‐ cal Interactions, 359: 109916. [33] Song X, He J, Shi B, et al.2022. Hypoxic microenvironment- induced reduction in PTEN-L secretion promotes non- small cell lung cancer metastasis through PI3K/AKT pathway[J]. Evidence-Based Complementary and Alter‐ native Medicine, 2022: 6683104. [34] Tu J, Yang Y, Albert Cheung H, et al.2017. Conserved miR-10 family represses proliferation and induces apoptosis in ovarian granulosa cells[J]. Scientific Reports, 7:41304 [35] Wu L, Chen Y, Chen Y, et al.2019. Effect of HIF-1α/miR-10b-5p/PTEN on hypoxia-induced cardiomyocyte apoptosis[J]. Journal of American Heart Association, 8(18): e011948. [36] Wu N, Gu T, Lu L, et al.2019. Roles of miRNA-1 and miR‐ NA-133 in the proliferation and differentiation of myo‐ blasts in duck skeletal muscle[J]. Journal of Cellular Physiology, 234(4): 3490-3499. [37] Xu X, Shen H R, Yu M, et al.2022. MicroRNA let-7i inhibits granulosa-luteal cell proliferation and oestradiol biosyn‐ thesis by directly targeting IMP2[J]. Reproductive Bio‐medicine Online, 44(5): 803-816. [38] Yan T, Wang X, Wei G, et al.2021. Exosomal miR-10b-5p mediates cell communication of gastric cancer cells and fibroblasts and facilitates cell proliferation[J]. Journal of Cancer, 12(7): 2140-2150. [39] Yao J, Huang R, Li M, et al.2021. PTEN expression in hu‐ man granulosa cells is associated with ovarian responses and clinical outcomes in IVF[J]. Reproductive Sciences, 28(7): 1910-1921. [40] Yao W, Pan Z, Du X, et al.2018. miR-181b-induced SMAD7 downregulation controls granulosa cell apoptosis through TGF- β signaling by interacting with the TGF‐ BR1 promoter[J]. Journal of Cellular Physiology, 233(9): 6807-6821. [41] Zhu W, Yang M, Shang J, et al.2019. MiR-222 inhibits apop‐tosis in porcine follicular granulosa cells by targeting the THBS1 gene[J]. Animal Science Journal, 90(6): 719-727.
