miR-34c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene
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摘要
MicroRNAs(miRNAs) are implicated in swine spermatogenesis via their regulations of cell proliferation, apoptosis, and differentiation. Recent studies indicated that miR-34 c is indispensable in the late steps of spermatogenesis. However, whether miR-34 c plays similar important roles in immature porcine Sertoli cells remain unknown. In the present study, we conducted two experiments using a completely randomised design to study the function roles of miR-34 c. The results from experiment I demonstrated that the relative expression level of miR-34 c in swine testicular tissues increased(P=0.0017) quadratically with increasing age, while the relative expression level of SMAD family member 7(SMAD7) decreased(P=0.0009) with curve. Furthermore, miR-34 c expression levels showed a significant negative correlation(P=0.013) with SMAD7 gene expression levels. The results from experiment II indicated that miR-34 c directly targets the SMAD7 gene using a luciferase reporter assay, and suppresses(P<0.05) SMAD7 mRNA and protein expressions in immature porcine Sertoli cells. Overexpression of miR-34 c inhibited(P<0.05) proliferation and enhanced(P<0.05) apoptosis in the immature porcine Sertoli cells, which was supported by the results from the Cell Counting Kit-8(CCK-8) assay, the 5-Ethynyl-2′-deoxyuridine(EdU) assay, and the Annexin V-FITC/PI staining assay. Furthermore, knockdown of SMAD7 via small interfering RNA(siR NA) gave a similar result. It is concluded that miR-34 c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene.
MicroRNAs(miRNAs) are implicated in swine spermatogenesis via their regulations of cell proliferation, apoptosis, and differentiation. Recent studies indicated that miR-34 c is indispensable in the late steps of spermatogenesis. However, whether miR-34 c plays similar important roles in immature porcine Sertoli cells remain unknown. In the present study, we conducted two experiments using a completely randomised design to study the function roles of miR-34 c. The results from experiment I demonstrated that the relative expression level of miR-34 c in swine testicular tissues increased(P=0.0017) quadratically with increasing age, while the relative expression level of SMAD family member 7(SMAD7) decreased(P=0.0009) with curve. Furthermore, miR-34 c expression levels showed a significant negative correlation(P=0.013) with SMAD7 gene expression levels. The results from experiment II indicated that miR-34 c directly targets the SMAD7 gene using a luciferase reporter assay, and suppresses(P<0.05) SMAD7 mRNA and protein expressions in immature porcine Sertoli cells. Overexpression of miR-34 c inhibited(P<0.05) proliferation and enhanced(P<0.05) apoptosis in the immature porcine Sertoli cells, which was supported by the results from the Cell Counting Kit-8(CCK-8) assay, the 5-Ethynyl-2′-deoxyuridine(EdU) assay, and the Annexin V-FITC/PI staining assay. Furthermore, knockdown of SMAD7 via small interfering RNA(siR NA) gave a similar result. It is concluded that miR-34 c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene.
MicroRNAs(miRNAs) are implicated in swine spermatogenesis via their regulations of cell proliferation, apoptosis, and differentiation. Recent studies indicated that miR-34 c is indispensable in the late steps of spermatogenesis. However, whether miR-34 c plays similar important roles in immature porcine Sertoli cells remain unknown. In the present study, we conducted two experiments using a completely randomised design to study the function roles of miR-34 c. The results from experiment I demonstrated that the relative expression level of miR-34 c in swine testicular tissues increased(P=0.0017) quadratically with increasing age, while the relative expression level of SMAD family member 7(SMAD7) decreased(P=0.0009) with curve. Furthermore, miR-34 c expression levels showed a significant negative correlation(P=0.013) with SMAD7 gene expression levels. The results from experiment II indicated that miR-34 c directly targets the SMAD7 gene using a luciferase reporter assay, and suppresses(P<0.05) SMAD7 mRNA and protein expressions in immature porcine Sertoli cells. Overexpression of miR-34 c inhibited(P<0.05) proliferation and enhanced(P<0.05) apoptosis in the immature porcine Sertoli cells, which was supported by the results from the Cell Counting Kit-8(CCK-8) assay, the 5-Ethynyl-2′-deoxyuridine(EdU) assay, and the Annexin V-FITC/PI staining assay. Furthermore, knockdown of SMAD7 via small interfering RNA(siR NA) gave a similar result. It is concluded that miR-34 c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene.
引文
Bouhallier F,Allioli N,Lavial F,Chalmel F,Perrard M H,Durand P,Samarut J,Pain B,Rouault J P.2010.Role of miR-34c microRNA in the late steps of spermatogenesis.RNA,16,720-731.
Choe N,Kwon J S,Kim Y S,Eom G H,Ahn Y K,Baik Y H,Park H Y,Kook H.2015.The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor.Cellular Signalling,27,1056-1065.
Cohen T V,Kollias H D,Liu N,Ward C W,Wagner K R.2015.Genetic disruption of Smad7 impairs skeletal muscle growth and regeneration.The Journal of Physiology,593,2479-2497.
Di Fusco D,Laudisi F,Dinallo V,Monteleone I,Di Grazia A,Marafini I,Troncone E,Colantoni A,Ortenzi A,Stolfi C,Picardo M,Monteleone G.2017.Smad7 positively regulates keratinocyte proliferation in psoriasis.The British Journal of Dermatology,177,1633-1643.
Hu P,Guan K,Feng Y,Ma C,Song H,Li Y,Xia X,Li J,Li F.2017.miR-638 Inhibits immature Sertoli cell growth by indirectly inactivating PI3K/AKT pathway via SPAG1 gene.Cell Cycle,16,2290-2300.
Itman C,Loveland K L.2008.SMAD expression in the testis:An insight into BMP regulation of spermatogenesis.Developmental Dynamics:An Official Publication of the American Association of Anatomists,237,97-111.
Jia J,Feng X,Xu W,Yang S,Zhang Q,Liu X,Feng Y,Dai Z.2014.MiR-17-5p modulates osteoblastic differentiation and cell proliferation by targeting SMAD7 in non-traumatic osteonecrosis.Experimental&Molecular Medicine,46,e107.
Jiao Z J,Yi W,Rong Y W,Kee J D,Zhong W X.2015.MicroR NA-1285 regulates 17β-estradiol-inhibited immature boar sertoli cell proliferation via adenosine monophosphateactivated protein kinase activation.Endocrinology,156,4059-4070.
Johnson L,Thompson D L,Varner Jr D D.2008.Role of Sertoli cell number and function on regulation of spermatogenesis.Animal Reproduction Science,105,23-51.
Kvansakul M,Hinds M G.2013.Structural biology of the Bcl-2 family and its mimicry by viral proteins.Cell Death&Disease,4,e909.
Li M,Yu M,Liu C,Zhu H,He X,Peng S,Hua J.2013.miR-34c works downstream of p53 leading to dairy goat male germline stem-cell(mGSCs)apoptosis.Cell Proliferation,46,223-231.
Liang X,Zhou D,Wei C,Luo H,Liu J,Fu R,Cui S.2012.MicroRNA-34c enhances murine male germ cell apoptosis through targeting ATF1.PLoS ONE,7,e33861.
Liu J,Zhou Y,Shi Z,Hu Y,Meng T,Zhang X,Zhang S,Zhang J.2016.microRNA-497 modulates breast cancer cell proliferation,invasion,and survival by targeting SMAD7.DNA and Cell Biology,35,521-529.
Liu T,Wu Y,Huang T,Zhang X,Cai Y.2017.miR-590 promotes the proliferation of HUMSCs and induces ECM synthesis by targeting Smad7.Oncology Letters,14,3941-3946.
Liu W M,Pang R T,Chiu P C,Wong B P,Lao K,Lee K F,Yeung W S.2012.Sperm-borne microRNA-34c is required for the first cleavage division in mouse.Proceedings of the National Academy of Sciences of the United States of America,109,490-494.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,25,402-408.
Lui W Y,Lee W M,Cheng C Y.2003a.TGF-βs:Their role in testicular function and Sertoli cell tight junction dynamics.International Journal of Andrology,26,147-160.
Lui W Y,Wong C H,Mruk D D,Cheng C Y.2003b.TGF-β3regulates the blood-testis barrier dynamics via the p38mitogen activated protein(MAP)kinase pathway:An in vivo study.Endocrinology,144,1139-1142.
Ma C,Song H,Guan K,Zhou J,Xia X,Li F.2016a.Characterization of swine testicular cell line as immature porcine Sertoli cell line.In Vitro Cellular&Developmental Biology.Animal,52,427-433.
Ma C,Song H,Yu L,Guan K,Hu P,Li Y,Xia X,Li J,Jiang S,Li F.2016b.miR-762 promotes porcine immature Sertoli cell growth via the ring finger protein 4(RNF4)gene.Scientific Reports,6,32783.
Mignard V,Lalier L,Paris F,Vallette F M.2014.Bioactive lipids and the control of Bax pro-apoptotic activity.Cell Death&Disease,5,e1266.
Ran M L,Chen B,Wu M S,Liu X C,He C Q,Yang A Q,Li Z,Xiang Y J,Li Z H,Zhang S W.2015.Integrated analysis of miRNA and mRNA expression profiles in development of porcine testes.RSC Advances,5,63439-63449.
Sanger F,Nicklen S,Coulson A R.1977.DNA sequencing with chain-terminating inhibitors.Proceedings of the National Academy of Sciences of the United States of America,74,5463-5467.
Song J,Wang Q,Luo Y,Yuan P,Tang C,Hui Y,Wang Z.2015.miR-34c-3p inhibits cell proliferation,migration and invasion of hepatocellular carcinoma by targeting MARCKS.International Journal of Clinical and Experimental Pathology,8,12728-12737.
Volkmann N,Marassi F M,Newmeyer D D,Hanein D.2014.The rheostat in the membrane:BCL-2 family proteins and apoptosis.Cell Death and Differentiation,21,206-215.
Walker S E,Lorsch J.2013.Sanger dideoxy sequencing of DNA.Methods in Enzymology,529,171-184.
Wang M,Liu C,Su Y,Zhang K,Zhang Y,Chen M,Ge M,Gu L,Lu T,Li N,Yu Z,Meng Q.2017.miRNA-34c inhibits myoblasts proliferation by targeting YY1.Cell Cycle,16,1661-1672.
Wu J,Bao J,Kim M,Yuan S,Tang C,Zheng H,Mastick GS,Xu C,Yan W.2014.Two miRNA clusters,miR-34b/c and miR-449,are essential for normal brain development,motile ciliogenesis,and spermatogenesis.Proceedings of the National Academy of Sciences of the United States of America,111,E2851-E2857.
Wu Z,Wu Y,Tian Y,Sun X,Liu J,Ren H,Liang C,Song L,Hu H,Wang L,Jiao B.2013.Differential effects of miR-34c-3p and miR-34c-5p on the proliferation,apoptosis and invasion of glioma cells.Oncology Letters,6,1447-1452.
Xiao X,Gaffar I,Guo P,Wiersch J,Fischbach S,Peirish L,Song Z,El-Gohary Y,Prasadan K,Shiota C,Gittes G K.2014.M2 macrophages promote beta-cell proliferation by up-regulation of SMAD7.Proceedings of the National Academy of Sciences of the United States of America,111,E1211-1220.
Yan X,Chen Y G.2011.Smad7:Not only a regulator,but also a cross-talk mediator of TGF-beta signalling.The Biochemical Journal,434,1-10.
Yan X,Liu Z,Chen Y.2009.Regulation of TGF-beta signaling by Smad7.Acta Biochimica et Biophysica Sinica,41,263-272.
Yang S,Wu B,Sun H,Ji F,Sun T,Zhao Y,Zhou D.2015.Interrupted E2F1-miR-34c-SCF negative feedback loop by hyper-methylation promotes colorectal cancer cell proliferation.Bioscience Reports,36,e00293.
Yao C,Sun M,Yuan Q,Niu M,Chen Z,Hou J,Wang H,Wen L,Liu Y,Li Z,He Z.2016.MiRNA-133b promotes the proliferation of human Sertoli cells through targeting GLI3.Oncotarget,7,2201-2219.
Yin X,Ma T,Han R,Ding J,Zhang H,Han X,Li D.2017.MiR-301b-3p/3584-5p enhances low-dose mono-n-butyl phthalate(MBP)-induced proliferation by targeting Rasd1in Sertoli cells.Toxicology in Vitro:An International Journal Published in Association with BIBRA,47,79-88.
Yu M,Mu H,Niu Z,Chu Z,Zhu H,Hua J.2014.miR-34c enhances mouse spermatogonial stem cells differentiation by targeting Nanos2.Journal of Cellular Biochemistry,115,232-242.
Yuan S,Tang C,Zhang Y,Wu J,Bao J,Zheng H,Xu C,Yan W.2015.mir-34b/c and mir-449a/b/c are required for spermatogenesis,but not for the first cleavage division in mice.Biology Open,4,212-223.
Zhang S,Yu M,Liu C,Wang L,Hu Y,Bai Y,Hua J.2012.MIR-34c regulates mouse embryonic stem cells differentiation into male germ-like cells through RARg.Cell Biochemistry and Function,30,623-632.
Choe N,Kwon J S,Kim Y S,Eom G H,Ahn Y K,Baik Y H,Park H Y,Kook H.2015.The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor.Cellular Signalling,27,1056-1065.
Cohen T V,Kollias H D,Liu N,Ward C W,Wagner K R.2015.Genetic disruption of Smad7 impairs skeletal muscle growth and regeneration.The Journal of Physiology,593,2479-2497.
Di Fusco D,Laudisi F,Dinallo V,Monteleone I,Di Grazia A,Marafini I,Troncone E,Colantoni A,Ortenzi A,Stolfi C,Picardo M,Monteleone G.2017.Smad7 positively regulates keratinocyte proliferation in psoriasis.The British Journal of Dermatology,177,1633-1643.
Hu P,Guan K,Feng Y,Ma C,Song H,Li Y,Xia X,Li J,Li F.2017.miR-638 Inhibits immature Sertoli cell growth by indirectly inactivating PI3K/AKT pathway via SPAG1 gene.Cell Cycle,16,2290-2300.
Itman C,Loveland K L.2008.SMAD expression in the testis:An insight into BMP regulation of spermatogenesis.Developmental Dynamics:An Official Publication of the American Association of Anatomists,237,97-111.
Jia J,Feng X,Xu W,Yang S,Zhang Q,Liu X,Feng Y,Dai Z.2014.MiR-17-5p modulates osteoblastic differentiation and cell proliferation by targeting SMAD7 in non-traumatic osteonecrosis.Experimental&Molecular Medicine,46,e107.
Jiao Z J,Yi W,Rong Y W,Kee J D,Zhong W X.2015.MicroR NA-1285 regulates 17β-estradiol-inhibited immature boar sertoli cell proliferation via adenosine monophosphateactivated protein kinase activation.Endocrinology,156,4059-4070.
Johnson L,Thompson D L,Varner Jr D D.2008.Role of Sertoli cell number and function on regulation of spermatogenesis.Animal Reproduction Science,105,23-51.
Kvansakul M,Hinds M G.2013.Structural biology of the Bcl-2 family and its mimicry by viral proteins.Cell Death&Disease,4,e909.
Li M,Yu M,Liu C,Zhu H,He X,Peng S,Hua J.2013.miR-34c works downstream of p53 leading to dairy goat male germline stem-cell(mGSCs)apoptosis.Cell Proliferation,46,223-231.
Liang X,Zhou D,Wei C,Luo H,Liu J,Fu R,Cui S.2012.MicroRNA-34c enhances murine male germ cell apoptosis through targeting ATF1.PLoS ONE,7,e33861.
Liu J,Zhou Y,Shi Z,Hu Y,Meng T,Zhang X,Zhang S,Zhang J.2016.microRNA-497 modulates breast cancer cell proliferation,invasion,and survival by targeting SMAD7.DNA and Cell Biology,35,521-529.
Liu T,Wu Y,Huang T,Zhang X,Cai Y.2017.miR-590 promotes the proliferation of HUMSCs and induces ECM synthesis by targeting Smad7.Oncology Letters,14,3941-3946.
Liu W M,Pang R T,Chiu P C,Wong B P,Lao K,Lee K F,Yeung W S.2012.Sperm-borne microRNA-34c is required for the first cleavage division in mouse.Proceedings of the National Academy of Sciences of the United States of America,109,490-494.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,25,402-408.
Lui W Y,Lee W M,Cheng C Y.2003a.TGF-βs:Their role in testicular function and Sertoli cell tight junction dynamics.International Journal of Andrology,26,147-160.
Lui W Y,Wong C H,Mruk D D,Cheng C Y.2003b.TGF-β3regulates the blood-testis barrier dynamics via the p38mitogen activated protein(MAP)kinase pathway:An in vivo study.Endocrinology,144,1139-1142.
Ma C,Song H,Guan K,Zhou J,Xia X,Li F.2016a.Characterization of swine testicular cell line as immature porcine Sertoli cell line.In Vitro Cellular&Developmental Biology.Animal,52,427-433.
Ma C,Song H,Yu L,Guan K,Hu P,Li Y,Xia X,Li J,Jiang S,Li F.2016b.miR-762 promotes porcine immature Sertoli cell growth via the ring finger protein 4(RNF4)gene.Scientific Reports,6,32783.
Mignard V,Lalier L,Paris F,Vallette F M.2014.Bioactive lipids and the control of Bax pro-apoptotic activity.Cell Death&Disease,5,e1266.
Ran M L,Chen B,Wu M S,Liu X C,He C Q,Yang A Q,Li Z,Xiang Y J,Li Z H,Zhang S W.2015.Integrated analysis of miRNA and mRNA expression profiles in development of porcine testes.RSC Advances,5,63439-63449.
Sanger F,Nicklen S,Coulson A R.1977.DNA sequencing with chain-terminating inhibitors.Proceedings of the National Academy of Sciences of the United States of America,74,5463-5467.
Song J,Wang Q,Luo Y,Yuan P,Tang C,Hui Y,Wang Z.2015.miR-34c-3p inhibits cell proliferation,migration and invasion of hepatocellular carcinoma by targeting MARCKS.International Journal of Clinical and Experimental Pathology,8,12728-12737.
Volkmann N,Marassi F M,Newmeyer D D,Hanein D.2014.The rheostat in the membrane:BCL-2 family proteins and apoptosis.Cell Death and Differentiation,21,206-215.
Walker S E,Lorsch J.2013.Sanger dideoxy sequencing of DNA.Methods in Enzymology,529,171-184.
Wang M,Liu C,Su Y,Zhang K,Zhang Y,Chen M,Ge M,Gu L,Lu T,Li N,Yu Z,Meng Q.2017.miRNA-34c inhibits myoblasts proliferation by targeting YY1.Cell Cycle,16,1661-1672.
Wu J,Bao J,Kim M,Yuan S,Tang C,Zheng H,Mastick GS,Xu C,Yan W.2014.Two miRNA clusters,miR-34b/c and miR-449,are essential for normal brain development,motile ciliogenesis,and spermatogenesis.Proceedings of the National Academy of Sciences of the United States of America,111,E2851-E2857.
Wu Z,Wu Y,Tian Y,Sun X,Liu J,Ren H,Liang C,Song L,Hu H,Wang L,Jiao B.2013.Differential effects of miR-34c-3p and miR-34c-5p on the proliferation,apoptosis and invasion of glioma cells.Oncology Letters,6,1447-1452.
Xiao X,Gaffar I,Guo P,Wiersch J,Fischbach S,Peirish L,Song Z,El-Gohary Y,Prasadan K,Shiota C,Gittes G K.2014.M2 macrophages promote beta-cell proliferation by up-regulation of SMAD7.Proceedings of the National Academy of Sciences of the United States of America,111,E1211-1220.
Yan X,Chen Y G.2011.Smad7:Not only a regulator,but also a cross-talk mediator of TGF-beta signalling.The Biochemical Journal,434,1-10.
Yan X,Liu Z,Chen Y.2009.Regulation of TGF-beta signaling by Smad7.Acta Biochimica et Biophysica Sinica,41,263-272.
Yang S,Wu B,Sun H,Ji F,Sun T,Zhao Y,Zhou D.2015.Interrupted E2F1-miR-34c-SCF negative feedback loop by hyper-methylation promotes colorectal cancer cell proliferation.Bioscience Reports,36,e00293.
Yao C,Sun M,Yuan Q,Niu M,Chen Z,Hou J,Wang H,Wen L,Liu Y,Li Z,He Z.2016.MiRNA-133b promotes the proliferation of human Sertoli cells through targeting GLI3.Oncotarget,7,2201-2219.
Yin X,Ma T,Han R,Ding J,Zhang H,Han X,Li D.2017.MiR-301b-3p/3584-5p enhances low-dose mono-n-butyl phthalate(MBP)-induced proliferation by targeting Rasd1in Sertoli cells.Toxicology in Vitro:An International Journal Published in Association with BIBRA,47,79-88.
Yu M,Mu H,Niu Z,Chu Z,Zhu H,Hua J.2014.miR-34c enhances mouse spermatogonial stem cells differentiation by targeting Nanos2.Journal of Cellular Biochemistry,115,232-242.
Yuan S,Tang C,Zhang Y,Wu J,Bao J,Zheng H,Xu C,Yan W.2015.mir-34b/c and mir-449a/b/c are required for spermatogenesis,but not for the first cleavage division in mice.Biology Open,4,212-223.
Zhang S,Yu M,Liu C,Wang L,Hu Y,Bai Y,Hua J.2012.MIR-34c regulates mouse embryonic stem cells differentiation into male germ-like cells through RARg.Cell Biochemistry and Function,30,623-632.