RNA干扰水牛MTPN基因对颗粒细胞增殖、凋亡及激素分泌的影响
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摘要
本研究旨在探讨敲低MTPN基因对体外培养水牛颗粒细胞增殖、凋亡及雌激素、孕酮分泌的影响。应用RNAi技术敲低颗粒细胞中MTPN基因的表达水平,通过实时荧光定量PCR方法检测体外培养水牛颗粒细胞中MTPN基因及增殖和周期相关基因的表达情况,CCK-8法检测细胞增殖,借助流式细胞仪检测细胞周期的分布,采用ELISA试剂盒检测细胞培养液中雌激素与孕酮含量。结果显示,经siRNA(si-MTPN)转染颗粒细胞后,MTPN基因相对表达量下降60%(P<0.01);细胞增殖受到显著抑制(P<0.05),G1期细胞数量下降,S期细胞数量上升,G2期细胞数量极显著上升(P<0.01),细胞被阻滞在G2期;增殖与周期相关基因Cyclin D2、Cytochrom C表达量显著上升(P<0.05),Caspase9、Fas基因表达量极显著上升(P<0.01);ELISA检测雌激素和孕酮分泌水平均显著下降(P<0.05)。综上表明,敲低MTPN基因能通过调控相关基因的表达抑制体外培养水牛颗粒细胞的增殖及雌激素、孕酮的分泌水平,为阐明MTPN基因参与家畜卵泡发生的分子机制提供参考。
This study was aimed to investigate the effect of MTPN gene knockdown on proliferation and apoptosis of buffalo granulosa cells in vitro and estrogen and progesterone secretion.The expression of MTPN gene in granulosa cells was knocked down by RNAi technology.The expression of MTPN gene and proliferation and cell cycle-related genes were detected by Real-time PCR,the cell proliferation and cell cycle distribution were detected by CCK-8 assay and flow cytometry,respectively.The estrogen and progesterone levels in the cell culture medium were determined by ELISA kit.The results showed that the relative expression of MTPN gene was decreased by 60% after siRNA(si-MTPN) transfection(P<0.01).The cell proliferation was significantly inhibited(P<0.05),the number of cells in G1 phase decreased,and the number of cells in S phase increased,the number of cells in G2 phase extremely significantly increased(P<0.01),and the cells were blocked in G2 phase.The expressions of Cyclin D2 and Cytochrom C genes were significantly up regulated(P<0.05),and the expressions of Caspase9 and Fas genes were extremely significantly up regulated(P<0.01).ELISA results showed that the levels of estrogen and progesterone secretion were significantly decreased(P<0.05).In conclusion,knockdown of MTPN gene could inhibit the proliferation of buffalo granulosa cells in vitro and the secretion of estrogen and progesterone by regulating the expression of related genes,which laid a reference for elucidating the molecular mechanism of MTPN gene involved in follicular development of livestock.
This study was aimed to investigate the effect of MTPN gene knockdown on proliferation and apoptosis of buffalo granulosa cells in vitro and estrogen and progesterone secretion.The expression of MTPN gene in granulosa cells was knocked down by RNAi technology.The expression of MTPN gene and proliferation and cell cycle-related genes were detected by Real-time PCR,the cell proliferation and cell cycle distribution were detected by CCK-8 assay and flow cytometry,respectively.The estrogen and progesterone levels in the cell culture medium were determined by ELISA kit.The results showed that the relative expression of MTPN gene was decreased by 60% after siRNA(si-MTPN) transfection(P<0.01).The cell proliferation was significantly inhibited(P<0.05),the number of cells in G1 phase decreased,and the number of cells in S phase increased,the number of cells in G2 phase extremely significantly increased(P<0.01),and the cells were blocked in G2 phase.The expressions of Cyclin D2 and Cytochrom C genes were significantly up regulated(P<0.05),and the expressions of Caspase9 and Fas genes were extremely significantly up regulated(P<0.01).ELISA results showed that the levels of estrogen and progesterone secretion were significantly decreased(P<0.05).In conclusion,knockdown of MTPN gene could inhibit the proliferation of buffalo granulosa cells in vitro and the secretion of estrogen and progesterone by regulating the expression of related genes,which laid a reference for elucidating the molecular mechanism of MTPN gene involved in follicular development of livestock.
引文
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[17] SHIMIZU T,KOSAKA N,MURAYAMA C,et al.Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary:Involvement of apoptosis and hormonal regulation[J].Animal Reproduction Science,2009,116(1-2):28-37.
[18] TIWARI M,PRASAD S,TRIPATHI A,et al.Apoptosis in mammalian oocytes:A review[J].Apoptosis,2015,20(8):1019-1025.
[19] BLONDIN P,SIRARD M A.Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes[J].Molecular Reproduction & Development,1995,41(1):54-62.
[20] SHERR C J,ROBERTS J M.CDK inhibitors:Positive and negative regulators of G1-phase progression[J].Genes & Development,1999,13(12):1501-1512.
[21] KOZAR K,CIEMERYCH M A,REBEL V I,et al.Mouse development and cell proliferation in the absence of D-cyclins[J].Cell,2004,118(4):477-491.
[22] 郭晓,徐铮,侯兰芬,等.慢性粒细胞性白血病中Cyclin D2与Cyclin E及p27 mRNA的表达和细胞周期分布[J].中华肿瘤防治杂志,2007,14(16):1235-1237. GUO X,XU Z,HOU L F,et al.Expression of Cyclin D2,Cyclin E and p27 mRNA and cell cycle distribution in chronic myeloid leukemia[J].Chinese Journal of Cancer Prevention and Treatment,2007,14(16):1235-1237.(in Chinese)
[23] 李奎,康相涛,刘英,等.固始鸡免疫器官内细胞凋亡基因Fas和FasL的动态表达[J].中国农业科学,2008,41(5):1489-1496. LI K,KANG X T,LIU Y,et al.Dynamic expression of apoptosis genes Fas and FasL in immune organs of Gushi chickens[J].Scientia Agricultura Sinica,2008,41(5):1489-1496.(in Chinese)
[24] CHEN M,GUERRERO A D,HUANG L,et al.Caspase-9-induced mitochondrial disruption through cleavage of anti-apoptotic BCL-2 family members[J].Journal of Biological Chemistry,2007,282(46):33888-33895.
[25] LIN L,YE Y,ZAKERI Z.P53,Apaf-1,caspase-3,and -9 are dispensable for Cdk5 activation during cell death[J]. Cell Death & Differentiation,2006,13(1):141-150.
[26] KNIGHT P G,SATCHELL L,GLISTER C.Intra-ovarian roles of activins and inhibins[J].Molecular & Cellular Endocrinology,2012,359(1-2):53-65.
[27] RIAZ H,DONG P,SHAHZAD M,et al.Constitutive and follicle-stimulating hormone-induced action of somatostatin receptor-2 on regulation of apoptosis and steroidogenesis in bovine granulosa cells[J].Journal of Steroid Biochemistry & Molecular Biology,2014,141(1):150-159.
[28] STOCCO D M,CLARK B J.Regulation of the acute production of steroids in steroidogenic cells[J].Endocrine Reviews,1996,17(3):221-244.
[29] 张晓娜,杨镒峰,常彤,等.雌激素、雄激素在卵泡发育中的作用及应用[J].特产研究,2017,39(3):50-54. ZHANG X N,YANG Y F,CHANG T,et al.The role of estrogen and/or androgen in the follicular development and its application[J].Special Wild Economic Animal and Plant Research,2017,39(3):50-54.(in Chinese)
[30] 张丽.常用生殖激素的功能及其在奶牛生产中的应用[J].山东畜牧兽医,2011,32(6):24-25. ZHANG L.The function of common reproductive hormones and its application in dairy cow production[J].Shandong Journal of Animal Science and Veterinary Medicine,2011,32(6):24-25.(in Chinese)
[2] HUANG Y L,WANG H J,CHEN F M,et al.Role of BCL2L10 in regulating buffalo (Bubalus bubalis) oocyte maturation[J].Theriogenology,2018,110:1-7.
[3] 崔保威,王复龙,崔昱清,等.我国水牛产业现状简析[J].肉类研究,2013,27(11):37-40. CUI B W,WANG F L,CUI Y Q,et al.A brief analysis of current status of the Chinese buffalo industry[J].Meat Research,2013,27(11):37-40.(in Chinese)
[4] DUNLOP C E,ANDERSON R A.The regulation and assessment of follicular growth[J].Scandinavian Journal of Clinical & Laboratory Investigation,2014,74(244):13-17.
[5] WEISSMAN A,BIRAN G,RAVHPN A,et al.Follicular growth and development under continuous GnRH antagonist administration[J]. Fertility & Sterility,2005,84(3):S320-S321.
[6] 金萱,刘以训.卵泡生长、分化和闭锁的调控[J].科学通报,2003,48(14):1506-1510. JIN X,LIU Y X.Regulation of follicular growth,differentiation and atresia[J].Chinese Science Bulletin,2003,48(14):1506-1510.(in Chinese)
[7] 吴玉萍,赵茴茴,周玉霞,等.颗粒细胞在卵母细胞发育成熟中的作用[J].国际生殖健康/计划生育杂志,2017,36(6):503-506. WU Y P,ZHAO H H,ZHOU Y X,et al.The role of granulosa cells in oocyte maturation[J].Journal of International Reproductive Health/Family Planning,2017,36(6):503-506.(in Chinese)
[8] 雷小灿,张海航,崔奎青,等.水牛BMP1基因对颗粒细胞增殖与凋亡的功能研究[J].畜牧兽医学报,2015,46(10):1766-1774. LEI X C,ZHANG H H,CUI K Q,et al.Functional analysis of buffalo BMP1 gene on proliferation and apoptosis of granulosa cells[J].ActaVeterinaria et Zootechnica Sinica,2015,46(10):1766-1774.(in Chinese)
[9] SEN S,KUNDU G,MEKHAIL N,et al.Myotrophin:Purification of a novel peptide from spontaneously hypertensive rat heart that influences myocardial growth[J].Journal of Biological Chemistry,1990,265(27):16635-16643.
[10] MARTINOVIC I,ABEGUNEWARDENE N,SEUL M,et al.Elevated monocyte chemoattractant protein-1 serum levels in patients at risk for coronary artery disease[J].Circulation Journal,2005,69(12):1484-1489.
[11] WANG L,WANG Y.Molecular characterization,expression patterns and subcellular localization of myotrophin (MTPN) gene in porcine skeletal muscle[J].Molecular Biology Reports,2012,39(3):2733-2738.
[12] 王洋,金一.siRNA及其运载系统研究进展[J].中国畜牧兽医,2017,44(5):1281-1288. WANG Y,JIN Y.Research progress on small interfering RNA and its delivery systems[J].China Animal Husbandry & Veterinary Medicine,2017,44(5):1281-1288.(in Chinese)
[13] GILCHRIST R B,RITTER L J,ARMSTRONG D T.Oocyte-somatic cell interactions during follicle development in mammals[J].Animal Reproduction Science,2004,82-83:431-436.
[14] CECCONI S,CICCARELLI C,BARBERI M,et al.Granulosa cell-oocyte interactions[J].European Journal of Obstetrics Gynecology & Reproductive Biology,2004,115(Suppl 1):S19.
[15] 刘慧萍,曾柳庭,张国民,等.颗粒细胞与卵母细胞自噬对卵泡发育和闭锁的调控[J].生殖与避孕,2017,37(1):67-74. LIU H P,ZENG L T,ZAHNG G M,et al.Progress in mechanism of granulosa and oocyte autophagy in ovarian follicular growth and atresia[J].Chinese Journal of Reproduction and Contraception,2017,37(1):67-74.(in Chinese)
[16] MANABE N,GOTO Y,MATSUDAMINEHATA F,et al.Regulation mechanism of selective atresia in porcine follicles:Regulation of granulosa cell apoptosis during atresia[J].Journal of Reproduction & Development,2004,50(5):493-514.
[17] SHIMIZU T,KOSAKA N,MURAYAMA C,et al.Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary:Involvement of apoptosis and hormonal regulation[J].Animal Reproduction Science,2009,116(1-2):28-37.
[18] TIWARI M,PRASAD S,TRIPATHI A,et al.Apoptosis in mammalian oocytes:A review[J].Apoptosis,2015,20(8):1019-1025.
[19] BLONDIN P,SIRARD M A.Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes[J].Molecular Reproduction & Development,1995,41(1):54-62.
[20] SHERR C J,ROBERTS J M.CDK inhibitors:Positive and negative regulators of G1-phase progression[J].Genes & Development,1999,13(12):1501-1512.
[21] KOZAR K,CIEMERYCH M A,REBEL V I,et al.Mouse development and cell proliferation in the absence of D-cyclins[J].Cell,2004,118(4):477-491.
[22] 郭晓,徐铮,侯兰芬,等.慢性粒细胞性白血病中Cyclin D2与Cyclin E及p27 mRNA的表达和细胞周期分布[J].中华肿瘤防治杂志,2007,14(16):1235-1237. GUO X,XU Z,HOU L F,et al.Expression of Cyclin D2,Cyclin E and p27 mRNA and cell cycle distribution in chronic myeloid leukemia[J].Chinese Journal of Cancer Prevention and Treatment,2007,14(16):1235-1237.(in Chinese)
[23] 李奎,康相涛,刘英,等.固始鸡免疫器官内细胞凋亡基因Fas和FasL的动态表达[J].中国农业科学,2008,41(5):1489-1496. LI K,KANG X T,LIU Y,et al.Dynamic expression of apoptosis genes Fas and FasL in immune organs of Gushi chickens[J].Scientia Agricultura Sinica,2008,41(5):1489-1496.(in Chinese)
[24] CHEN M,GUERRERO A D,HUANG L,et al.Caspase-9-induced mitochondrial disruption through cleavage of anti-apoptotic BCL-2 family members[J].Journal of Biological Chemistry,2007,282(46):33888-33895.
[25] LIN L,YE Y,ZAKERI Z.P53,Apaf-1,caspase-3,and -9 are dispensable for Cdk5 activation during cell death[J]. Cell Death & Differentiation,2006,13(1):141-150.
[26] KNIGHT P G,SATCHELL L,GLISTER C.Intra-ovarian roles of activins and inhibins[J].Molecular & Cellular Endocrinology,2012,359(1-2):53-65.
[27] RIAZ H,DONG P,SHAHZAD M,et al.Constitutive and follicle-stimulating hormone-induced action of somatostatin receptor-2 on regulation of apoptosis and steroidogenesis in bovine granulosa cells[J].Journal of Steroid Biochemistry & Molecular Biology,2014,141(1):150-159.
[28] STOCCO D M,CLARK B J.Regulation of the acute production of steroids in steroidogenic cells[J].Endocrine Reviews,1996,17(3):221-244.
[29] 张晓娜,杨镒峰,常彤,等.雌激素、雄激素在卵泡发育中的作用及应用[J].特产研究,2017,39(3):50-54. ZHANG X N,YANG Y F,CHANG T,et al.The role of estrogen and/or androgen in the follicular development and its application[J].Special Wild Economic Animal and Plant Research,2017,39(3):50-54.(in Chinese)
[30] 张丽.常用生殖激素的功能及其在奶牛生产中的应用[J].山东畜牧兽医,2011,32(6):24-25. ZHANG L.The function of common reproductive hormones and its application in dairy cow production[J].Shandong Journal of Animal Science and Veterinary Medicine,2011,32(6):24-25.(in Chinese)