miR-196a降调RCC2对睾丸支持细胞增殖的促进作用
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摘要
为探究miR-196a对猪未成熟支持细胞增殖的影响及其机制,将miR-196a的激活剂(mimics)、抑制剂(inhibitor)及miR-ShNC转染至支持细胞,MTS方法检测细胞活力。发现miR-196a mimics能促进支持细胞的增殖,miR-196a inhibitor抑制细胞增殖;使用Targetscan软件预测miR-196a靶基因为RCC2 (regulator of chromosome condensation 2),将miR-196a的mimics及inhibitor转染支持细胞后使用RT-qPCR和Western blot法检测RCC2的表达,结果显示miR-196a下调RCC2的表达,RCC2是miR-196a的靶基因;使用RCC2的siRNA与miR-196a inhibitor共转染支持细胞,MTS方法观察支持细胞活力,发现干扰RCC2基因后,可促进支持细胞的增殖。本研究表明miR-196a通过下调RCC2促进支持细胞的增殖,为后续探索miR-196a在猪睾丸支持细胞发挥的作用提供一定的试验基础和理论依据。
To explore the effect of miR-196 a on immature sertoli cells proliferation and its mechanism,miR-196 a mimics,inhibitor and miRNA-ShNC were transfected into immature sertoli cells,the viability of sertoli cells was measured by MTS assay.It was found that miR-196 a mimics can promote the proliferation of sertoli cells,and miR-196 a inhibitor can inhibit cell proliferation;RCC2 was the candidate target gene of miR-196 a which was predicted by targetscan software,then the expression of RCC2 in miR-196 a mimics,inhibitor and miR-ShNC transfected cells were detected by RT-qPCR and Western blot,the results showed that miR-196 a downregulated the expression of RCC2,RCC2 was a target gene of miR-196 a;furthermore,the siRNA of RCC2 and miR-196 a inhibitor were co-transfected to sertoli cells,and then MTS assay was used to detect the viability of sertoli cells,MTS analysis showed that RCC2 promoted proliferation of sertoli cells.The present study showed that miR-196 a promotes the sertoli cells proliferation by targeting RCC2.It provided a certain experimental and theoretical basis for further exploration of the role of miR-196 a in sertoli cells of pig testis.
To explore the effect of miR-196 a on immature sertoli cells proliferation and its mechanism,miR-196 a mimics,inhibitor and miRNA-ShNC were transfected into immature sertoli cells,the viability of sertoli cells was measured by MTS assay.It was found that miR-196 a mimics can promote the proliferation of sertoli cells,and miR-196 a inhibitor can inhibit cell proliferation;RCC2 was the candidate target gene of miR-196 a which was predicted by targetscan software,then the expression of RCC2 in miR-196 a mimics,inhibitor and miR-ShNC transfected cells were detected by RT-qPCR and Western blot,the results showed that miR-196 a downregulated the expression of RCC2,RCC2 was a target gene of miR-196 a;furthermore,the siRNA of RCC2 and miR-196 a inhibitor were co-transfected to sertoli cells,and then MTS assay was used to detect the viability of sertoli cells,MTS analysis showed that RCC2 promoted proliferation of sertoli cells.The present study showed that miR-196 a promotes the sertoli cells proliferation by targeting RCC2.It provided a certain experimental and theoretical basis for further exploration of the role of miR-196 a in sertoli cells of pig testis.
引文
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[16] JIN C,ZHANG Y,LI J.Upregulation of MiR-196a promotes cell proliferation by downregulating p27(kip1) in laryngeal cancer[J].Biol Res,2016,49:40.
[17] SKOUFIAS D A,MOLLINARI C,LACROIX F B,et al.Human survivin is a kinetochore-associated passenger protein[J].J Cell Biol,2001,151(7):1575-1582.
[18] MATSUO M,NAKADA C,TSUKAMOTO Y,et al.MiR-29c is downregulated in gastric carcinomas and regulates cell proliferation by targeting RCC2[J].Mol Cancer,2013,12:15.
[19] MOLLINARI C,REYNAUD C,MARTINEAU-THUILLIER S,et al.The mammalian passenger protein TD-60 is an RCC1 family member with an essential role in prometaphase to metaphase progression[J].Dev Cell,2003,5(2):295-307.
[20] LUO L,YE L,LIU G,et al.Microarray-based approach identifies differentially expressed microRNAs in porcine sexually immature and mature testes[J].PloS One,2010,5(8):e11744.
[21] HU P,GUAN K,FENG Y,et al.MiR-638 inhibits immature sertoli cell growth by indirectly inactivating PI3K/AKT pathway via SPAG1 gene[J].Cell Cycle,2017,16(23):2290-2300.
[22] GUO J,LIU X,YANG Y,et al.MiR-375 down-regulation of the rearranged L-myc fusion and hypoxia-induced gene domain protein 1A genes and effects on Sertoli cell proliferation[J].Asian-Australas J Anim Sci,2018,31:1103-1109.
[2] AYDOGDU E,KATCHY A,TSOUKO E,et al.MicroRNA-regulated gene networks during mammary cell differentiation are associated with breast cancer[J].Carcinogenesis,2012,33(8):1502-1511.
[3] FARH K.K,GRIMSON A,JAN C,et al.The widespread impact of mammalian MicroRNAs on mRNA repression and evolution[J].Science,2005,310(5755):1817-1821.
[4] AMBROS V.The functions of animal microRNAs[J].Nature,2004,431(7006):350-355.
[5] OLIVETO S,MANCINO M,MANFRINI N,et al.Role of microRNAs in translation regulation and cancer[J].World J Biol Chem,2017,8(1):45-56.
[6] LAGOS-QUINTANA M,RAUHUT R,MEYER J,et al.New microRNAs from mouse and human[J].RNA,2003,9(2):175-179.
[7] WERNERSSON R,SCHIERUP M H,JORGENSEN F G,et al.Pigs in sequence space:a 0.66X coverage pig genome survey based on shotgun sequencing[J].BMC Genomics,2005(6):70.
[8] KIM Y J,BAE S W,YU S S,et al.MiR-196a regulates proliferation and osteogenic differentiation in mesenchymal stem cells derived from human adipose tissue[J].J Bone Miner Res,2008,24(5):816-825.
[9] LIAN C,SUN B,NIU S,et al.A comparative profile of the microRNA transcriptome in immature and mature porcine testes using Solexa deep sequencing[J].FEBS J,2012,279(6):964-975.
[10] NIELSEN M,HANSEN J H,HEDEGAARD J,et al.MicroRNA identity and abundance in porcine skeletal muscles determined by deep sequencing[J].Anim Genet,2010,41(2):159-168.
[11] LI G,LI Y,LI X,et al.MicroRNA identity and abundance in developing swine adipose tissue as determined by Solexa sequencing[J].J Cell Biochem,2011,112(5):1318-1328.
[12] CHEN S R,LIU Y X.Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling[J].Reproduction,2015,149:R159-167.
[13] GARCIA T X,FARMAHA J K,KOW S,et al.RBPJ in mouse sertoli cells is required for proper regulation of the testis stem cell niche[J].Development,2014,141:4468-4478.
[14] OATLEY J.M.,Brinster R.L.The germline stem cell niche unit in mammalian testes[J].Physiol Rev,2012,92(2):577-595.
[15] HUANG F,TANG J,ZHUANG X,et al.MiR-196a promotes pancreatic cancer progression by targeting nuclear factor kappa-B-inhibitor alpha[J].PLoS One,2014,9(2):e87897.
[16] JIN C,ZHANG Y,LI J.Upregulation of MiR-196a promotes cell proliferation by downregulating p27(kip1) in laryngeal cancer[J].Biol Res,2016,49:40.
[17] SKOUFIAS D A,MOLLINARI C,LACROIX F B,et al.Human survivin is a kinetochore-associated passenger protein[J].J Cell Biol,2001,151(7):1575-1582.
[18] MATSUO M,NAKADA C,TSUKAMOTO Y,et al.MiR-29c is downregulated in gastric carcinomas and regulates cell proliferation by targeting RCC2[J].Mol Cancer,2013,12:15.
[19] MOLLINARI C,REYNAUD C,MARTINEAU-THUILLIER S,et al.The mammalian passenger protein TD-60 is an RCC1 family member with an essential role in prometaphase to metaphase progression[J].Dev Cell,2003,5(2):295-307.
[20] LUO L,YE L,LIU G,et al.Microarray-based approach identifies differentially expressed microRNAs in porcine sexually immature and mature testes[J].PloS One,2010,5(8):e11744.
[21] HU P,GUAN K,FENG Y,et al.MiR-638 inhibits immature sertoli cell growth by indirectly inactivating PI3K/AKT pathway via SPAG1 gene[J].Cell Cycle,2017,16(23):2290-2300.
[22] GUO J,LIU X,YANG Y,et al.MiR-375 down-regulation of the rearranged L-myc fusion and hypoxia-induced gene domain protein 1A genes and effects on Sertoli cell proliferation[J].Asian-Australas J Anim Sci,2018,31:1103-1109.