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Protein & Peptide Letters


ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

General Research Article

miR-146a-5p Regulated Cell Proliferation and Apoptosis by Targeting SMAD3 and SMAD4

Author(s): Meiyu Qiu, Tao Li, Binhu Wang, Hongbin Gong and Tao Huang*

Volume 27, Issue 5, 2020

Page: [411 - 418] Pages: 8

DOI: 10.2174/0929866526666190911142926

open access plus


Background: microRNAs (miRNAs) are a small, endogenous non-coding RNAs that are involved in post-transcriptional gene regulation of many biological processes, including embryo implantation and placental development. In our previous study, miR-146a-5p was found expressed higher in the serum exosomes of pregnant sows than non-pregnant. The research on miR-146a-5p has been mainly related to human diseases, but there are few studies on its effects on the reproduction of sows in early pregnancy.

Objective: In this article, our motivation is to study the role of miR-146a-5p in the early pregnancy of sows on the cell proliferetion and apoptosis by targeting SMAD3 and SMAD4.

Methods: Bioinformatics software was used to identify the target genes of miR-146a-5p. The wildtype and mutant-type recombinant plasmids of dual-luciferase reporter with 3'-UTR of Smad3 or 3'- UTR of Smad4 were constructed, and co-transfected in porcine kidney cell (PK-15 cell) with miR- 146a-5p mimic, mimic-NC(M-NC), inhibitor and inhibitor-NC(IN-NC), then dual-luciferase activity analysis, qRT-PCR and Western blot were performed to verify the target genes. After the transfection of BeWo choriocarcinoma cell (BeWo cell) with miR-146a-5p mimic, M-NC, inhibitor and IN-NC, the mRNA expression of Caspase-3, BAX and Bcl-2 was measured using qRT-PCR, and the cell proliferation was measured using CCK-8 kit.

Results: The luciferase, mRNA and protein expression of Smad3 in PK-15 cells treated by Smad3- 3'-UTR-W co-transfected with miR-146a-5p mimic were significantly lower than that with miR- 146a-5p M-NC, and the results of Smad4 were similar to Smad3, but the protein expression had a trend to lower in mimic group. The expression level of Bcl-2 in the miR-146a-5p mimic group was significantly lower than that in the miR-146a-5p M-NC group, but the expression pattern of Caspase-3 was just opposite. The mimic of miR-146a-5p reduced the proliferation of BeWo cells, however the inhibitor increased.

Conclusion: Smad3 and Smad4 are the direct target genes of miR-146a-5p. The expression of Smad3 and Smad4 were affected by the mimic and inhibitor of miR-146a-5p. miR-146a-5p affects cell apoptosis and proliferation by regulating their target genes. This study provided new data to understand the regulation mechanism of early pregnancy in sows.

Keywords: Smad3, Smad4, miR-146a-5p, cell proliferation, apoptosis, luciferase.

Graphical Abstract
Tay, Y.; Zhang, J.; Thomson, A.M.; Lim, B.; Rigoutsos, I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature, 2008, 455(7216), 1124-1128.
[] [PMID: 18806776]
Loux, S.C.; Scoggin, K.E.; Bruemmer, J.E.; Canisso, I.F.; Troedsson, M.H.; Squires, E.L.; Ball, B.A. Evaluation of circulating miRNAs during late pregnancy in the mare. PLoS One, 2017, 12(4)e0175045
[] [PMID: 28388652]
Li, J.; Smyth, P.; Flavin, R.; Cahill, S.; Denning, K.; Aherne, S.; Guenther, S.M.; O’Leary, J.J.; Sheils, O. Comparison of miRNA expression patterns using total RNA extracted from matched samples of Formalin-Fixed Paraffin-Embedded (FFPE) cells and snap frozen cells. BMC Biotechnol., 2007, 7(1), 36.
[] [PMID: 17603869]
Fang, C.; Zhu, D.X.; Dong, H.J.; Zhou, Z.J.; Wang, Y.H.; Liu, L.; Fan, L.; Miao, K.R.; Liu, P.; Xu, W.; Li, J.Y. Serum microRNAs are promising novel biomarkers for diffuse large B cell lymphoma. Ann. Hematol., 2012, 91(4), 553-559.
[] [PMID: 21987025]
Lawrie, C.H.; Gal, S.; Dunlop, H.M.; Pushkaran, B.; Liggins, A.P.; Pulford, K.; Banham, A.H.; Pezzella, F.; Boultwood, J.; Wainscoat, J.S.; Hatton, C.S.; Harris, A.L. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br. J. Haematol., 2008, 141(5), 672-675.
[] [PMID: 18318758]
Bogunia-Kubik, K.; Wysoczańska, B.; Piątek, D.; Iwaszko, M.; Ciechomska, M.; Świerkot, J. Significance of polymorphism and expression of miR-146a and NFkB1 genetic variants in patients with rheumatoid arthritis. Arch. Immunol. Ther. Exp. (Warsz.), 2016, 64(1)(Suppl. 1), 131-136.
[] [PMID: 28083614]
Momen-Heravi, F.; Bala, S.; Bukong, T.; Szabo, G. Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages. Nanomedicine (Lond.), 2014, 10(7), 1517-1527.
[] [PMID: 24685946]
Vaksman, O.; Tropé, C.; Davidson, B.; Reich, R. Exosome-derived miRNAs and ovarian carcinoma progression. Carcinogenesis, 2014, 35(9), 2113-2120.
[] [PMID: 24925027]
Li, W.; Xi, Y.; Xue, S.; Wang, Y.; Wu, L.; Liu, H.; Lei, M. Sequence analysis of microRNAs during pre-implantation between Meishan and Yorkshire pigs. Gene, 2018, 646, 20-27.
[] [PMID: 29287711]
Zhang, X.; Yang, J.; Zhao, J.; Zhang, P.; Huang, X. MicroRNA-23b inhibits the proliferation and migration of heat-denatured fibroblasts by targeting Smad3. PLoS One, 2015, 10(7), e0131867
[] [PMID: 26153982]
Yang, P.; Wu, Z.; Ma, C.; Pan, N.; Wang, Y.; Yan, L. Endometrial miR-543 is downregulated during the implantation window in women with endometriosis-related infertility. Reprod. Sci., 2018, 26(7), 900-908.
[] [PMID: 30231774]
Wang, Y.; Ma, C.H.; Qiao, J. Differential expression of microRNA in eutopic endometrium tissue during implantation window for patients with endometriosis related infertility. Zhonghua Fu Chan Ke Za Zhi, 2016, 51(6), 436-441.
[PMID: 27356479]
Qu, J.; Zhu, Y.; Wu, X.; Zheng, J.; Hou, Z.; Cui, Y.; Mao, Y.; Liu, J. Smad3/4 binding to promoter II of P450arom so as to regulate aromatase expression in endometriosis. Reprod. Sci., 2017, 24(8), 1187-1194.
[] [PMID: 27920344]
Osterlund, C.; Fried, G. TGFbeta receptor types I and II and the substrate proteins Smad 2 and 3 are present in human oocytes. Mol. Hum. Reprod., 2000, 6(6), 498-503.
[] [PMID: 10825365]
Sirard, C.; de la Pompa, J.L.; Elia, A.; Itie, A.; Mirtsos, C.; Cheung, A.; Hahn, S.; Wakeham, A.; Schwartz, L.; Kern, S.E.; Rossant, J.; Mak, T.W. The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo. Genes Dev., 1998, 12(1), 107-119.
[] [PMID: 9420335]
Kuo, F.T.; Fan, K.; Ambartsumyan, G.; Menon, P.; Ketefian, A.; Bentsi-Barnes, I.K.; Pisarska, M.D. Relative expression of genes encoding SMAD signal transduction factors in human granulosa cells is correlated with oocyte quality. J. Assist. Reprod. Genet., 2011, 28(10), 931-938.
[] [PMID: 21766220]
Wang, H.; Liu, L.; Liu, X.; Zhang, M.; Li, X. Correlation between miRNAs and target genes in response to Campylobacter jejuni inoculation in chicken. Poult. Sci., 2018, 97(2), 485-493.
[] [PMID: 29253230]
Zou, L.; Xiong, X.; Yang, H.; Wang, K.; Zhou, J.; Lv, D.; Yin, Y. Identification of microRNA transcriptome reveals that miR-100 is involved in the renewal of porcine intestinal epithelial cells. Sci. China Life Sci., 2019, 62(6), 816-828.
[] [PMID: 31016537]
Gilchrist, G.C.; Tscherner, A.; Nalpathamkalam, T.; Merico, D.; LaMarre, J. MicroRNA expression during bovine oocyte maturation and fertilization. Int. J. Mol. Sci., 2016, 17(3), 396.
[] [PMID: 26999121]
Amanai, M.; Brahmajosyula, M.; Perry, A.C. A restricted role for sperm-borne microRNAs in mammalian fertilization. Biol. Reprod., 2006, 75(6), 877-884.
[] [PMID: 16943360]
Byrne, M.J.; Warner, C.M. MicroRNA expression in preimplantation mouse embryos from Ped gene positive compared to Ped gene negative mice. J. Assist. Reprod. Genet., 2008, 25(5), 205-214.
[] [PMID: 18347971]
Cui, X.S.; Shen, X.H.; Kim, N.H. Dicer1 expression in preimplantation mouse embryos: Involvement of Oct3/4 transcription at the blastocyst stage. Biochem. Biophys. Res. Commun., 2007, 352(1), 231-236.
[] [PMID: 17113034]
Tzur, G.; Levy, A.; Meiri, E.; Barad, O.; Spector, Y.; Bentwich, Z.; Mizrahi, L.; Katzenellenbogen, M.; Ben-Shushan, E.; Reubinoff, B.E.; Galun, E. MicroRNA expression patterns and function in endodermal differentiation of human embryonic stem cells. PLoS One, 2008, 3(11), e3726
[] [PMID: 19015728]
Zhang, P.; Wang, L.; Li, Y.; Jiang, P.; Wang, Y.; Wang, P.; Kang, L.; Wang, Y.; Sun, Y.; Jiang, Y. Identification and characterization of microRNA in the lung tissue of pigs with different susceptibilities to PCV2 infection. Vet. Res. (Faisalabad), 2018, 49(1), 18.
[] [PMID: 29448950]
Jiao, Y.; Huang, B.; Chen, Y.; Hong, G.; Xu, J.; Hu, C.; Wang, C. Integrated analyses reveal overexpressed Notch1 promoting porcine satellite cells’ proliferation through regulating the cell cycle. Int. J. Mol. Sci., 2018, 19(1), 271.
[] [PMID: 29337929]
Zhang, H.L.; Li, L.; Cheng, C.J.; Sun, X.C. Expression of miR-146a-5p in patients with intracranial aneurysms and its association with prognosis. Eur. Rev. Med. Pharmacol. Sci., 2018, 22(3), 726-730.
[PMID: 29461602]
Yuwen, D.L.; Sheng, B.B.; Liu, J.; Wenyu, W.; Shu, Y.Q. MiR-146a-5p level in serum exosomes predicts therapeutic effect of cisplatin in non-small cell lung cancer. Eur. Rev. Med. Pharmacol. Sci., 2017, 21(11), 2650-2658.
[PMID: 28678319]
Sun, Y.; Li, Y.; Wang, H.; Li, H.; Liu, S.; Chen, J.; Ying, H. miR-146a-5p acts as a negative regulator of TGF-β signaling in skeletal muscle after acute contusion. Acta Biochim. Biophys. Sin. (Shanghai), 2017, 49(7), 628-634.
[] [PMID: 28510617]
Zhong, H.; Wang, H.R.; Yang, S.; Zhong, J.H.; Wang, T.; Wang, C.; Chen, F.Y. Targeting Smad4 links microRNA-146a to the TGF-β pathway during retinoid acid induction in acute promyelocytic leukemia cell line. Int. J. Hematol., 2010, 92(1), 129-135.
[] [PMID: 20577838]
Drummond, A.E. TGFbeta signalling in the development of ovarian function. Cell Tissue Res., 2005, 322(1), 107-115.
[] [PMID: 15983782]
Stoikos, C.J.; Salamonsen, L.A.; Hannan, N.J.; O’Connor, A.E.; Rombauts, L.; Dimitriadis, E. Activin A regulates trophoblast cell adhesive properties: Implications for implantation failure in women with endometriosis-associated infertility. Hum. Reprod., 2010, 25(7), 1767-1774.
[] [PMID: 20457668]
Reddy, A.; Suri, S.; Sargent, I.L.; Redman, C.W.; Muttukrishna, S. Maternal circulating levels of activin A, inhibin A, sFlt-1 and endoglin at parturition in normal pregnancy and pre-eclampsia. PLoS One, 2009, 4(2), e4453
[] [PMID: 19412349]
Billiar, R.B.; St Clair, J.B.; Zachos, N.C.; Burch, M.G.; Albrecht, E.D.; Pepe, G.J. Localization and developmental expression of the activin signal transduction proteins Smads 2, 3, and 4 in the baboon fetal ovary. Biol. Reprod., 2004, 70(3), 586-592.
[] [PMID: 14585818]
Zhang, F.; Wang, J.; Chu, J.; Yang, C.; Xiao, H.; Zhao, C.; Sun, Z.; Gao, X.; Chen, G.; Han, Z.; Zou, W.; Liu, T. MicroRNA-146a induced by hypoxia promotes chondrocyte autophagy through Bcl-2. Cell. Physiol. Biochem., 2015, 37(4), 1442-1453.
[] [PMID: 26492575]
Zhou, X.; Su, S.; Li, S.; Pang, X.; Chen, C.; Li, J.; Liu, J. MicroRNA-146a down-regulation correlates with neuroprotection and targets pro-apoptotic genes in cerebral ischemic injury in vitro. Brain Res., , 2016, 1648(Pt A), 136-143.
[] [PMID: 27449900]
Zhou, C.; Jiang, C.Q.; Zong, Z.; Lin, J.C.; Lao, L.F. miR-146a promotes growth of osteosarcoma cells by targeting ZNRF3/GSK-3β/β-catenin signaling pathway. Oncotarget, 2017, 8(43), 74276-74286.
[] [PMID: 29088784]
Zou, Y.; Cai, Y.; Lu, D.; Zhou, Y.; Yao, Q.; Zhang, S. MicroRNA-146a-5p attenuates liver fibrosis by suppressing profibrogenic effects of TGFβ1 and lipopolysaccharide. Cell. Signal., 2017, 39, 1-8.
[] [PMID: 28739486]
Morikawa, M.; Derynck, R.; Miyazono, K. TGF-β and the TGF-β family: Context-dependent roles in cell and tissue physiology. Cold Spring Harb. Perspect. Biol., 2016, 8(5)a021873
[] [PMID: 27141051]
Li, X.; McFarland, D.C.; Velleman, S.G. Effect of Smad3-mediated transforming growth factor-β1 signaling on satellite cell proliferation and differentiation in chickens. Poult. Sci., 2008, 87(9), 1823-1833.
[] [PMID: 18753451]
Yao, G.; Yin, M.; Lian, J.; Tian, H.; Liu, L.; Li, X.; Sun, F. MicroRNA-224 is involved in transforming growth factor-β-mediated mouse granulosa cell proliferation and granulosa cell function by targeting Smad4. Mol. Endocrinol., 2010, 24(3), 540-551.
[] [PMID: 20118412]
Tomic, D.; Miller, K.P.; Kenny, H.A.; Woodruff, T.K.; Hoyer, P.; Flaws, J.A. Ovarian follicle development requires Smad3. Mol. Endocrinol., 2004, 18(9), 2224-2240.
[] [PMID: 15192076]

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