牦牛miR-378前体克隆及组织表达分析
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摘要
旨在克隆牦牛miR-378的前体序列,阐明其组织表达规律,结合bta-miR-378靶基因的生物信息学预测和分析,探讨miR-378在牦牛生长发育过程中的调控功能。采用PCR方法成功克隆类乌齐牦牛miR-378前体序列,实时荧光定量PCR(RT-qPCR)检测miR-378-3p在各组织中的表达模式,结合生物信息学软件TargetScan、DAVID以及数据库NCBI、miRbase等对miR-378进行保守性分析、靶基因预测及其生物学功能分析。结果表明,miR-378在各物种间高度保守,且miR-378-3p在各组织中广泛表达,其中在臀大肌中表达水平最高,显著高于其他组织(P<0.01),在臀脂中的表达高于卵巢、大脑、乳腺和肝脏。获得的272个靶基因主要参与细胞分化、细胞发育、大分子代谢等多个生物学过程,涉及孕酮介导的卵母细胞成熟、促性腺激素释放激素(GnRH)信号通路等,由此推测,miR-378可能在卵泡发育、卵母细胞成熟过程中起关键作用,进而影响母牦牛的繁殖性能。
This work aims to clone the precursor sequence of yak miR-378,to elucidate its tissue expression pattern,and to explore theregulatory function of miR-378 in the growth and development of yak combined with bioinformatics prediction and analysis of bta-miR-378 targetgene. PCR was employed to successfully clone the miR-378 precursor sequence of leiwuqi yak,and real-time quantitative PCR(RT-qPCR)was used to detect the expression patterns of miR-378-3p in various tissues. Further,bioinformatics software and databases such as TargetScan,DAVID,NCBI,miRbase,etc were together applied to perform conservative analysis,target gene prediction and biological function analysisof miR-378. The results showed that miR-378 was highly conserved among different species and miR-378-3p was widely expressed in all tissues,the expression level was the highest in gluteus maximus,which was significantly higher than that in other tissues(P < 0.01). Expression inhip fat was higher than in ovary,brain,breast and liver. The obtained 272 target genes were mainly involved in many biological processessuch as cell differentiation,cell development,and macromolecular metabolism,involved in progesterone-mediated oocyte maturation andgonadotropin releasing hormone(GnRH)signaling pathways. It is speculated that miR-378 may play a key role in follicular development andoocyte maturation,which may affect the reproductive performance of female calves.
This work aims to clone the precursor sequence of yak miR-378,to elucidate its tissue expression pattern,and to explore theregulatory function of miR-378 in the growth and development of yak combined with bioinformatics prediction and analysis of bta-miR-378 targetgene. PCR was employed to successfully clone the miR-378 precursor sequence of leiwuqi yak,and real-time quantitative PCR(RT-qPCR)was used to detect the expression patterns of miR-378-3p in various tissues. Further,bioinformatics software and databases such as TargetScan,DAVID,NCBI,miRbase,etc were together applied to perform conservative analysis,target gene prediction and biological function analysisof miR-378. The results showed that miR-378 was highly conserved among different species and miR-378-3p was widely expressed in all tissues,the expression level was the highest in gluteus maximus,which was significantly higher than that in other tissues(P < 0.01). Expression inhip fat was higher than in ovary,brain,breast and liver. The obtained 272 target genes were mainly involved in many biological processessuch as cell differentiation,cell development,and macromolecular metabolism,involved in progesterone-mediated oocyte maturation andgonadotropin releasing hormone(GnRH)signaling pathways. It is speculated that miR-378 may play a key role in follicular development andoocyte maturation,which may affect the reproductive performance of female calves.
引文
[1]Xie Z, Kasschau KD, Carrington AJC. Negative feedback regulationof in by microRNA-Guided mRNA degradation[J]. CurrentBiology, 2003, 13(9):784-789.
[2]Thermann R, Hentze MW. Drosophila miR2 induces pseudopolysomes and inhibits translation initiation[J]. Nature, 2012,447(7146):875-878.
[3]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronicgene lin-4 encodes small RNAs with antisense complementarity tolin-14[J]. Cell, 1993, 75(5):843.
[4]Tong HL, Jiang RY, Liu TT, et al. bta-miR-378 promote thedifferentiation of bovine skeletal muscle-derived satellite cells[J].Gene, 2018, 668:246-251.
[5]Kasashima K, Nakamura Y, Kozu T. Altered expression profilesof microRNAs during TPA-induced differentiation of HL-60cells[J]. Biochem Biophys Res Commun, 2004, 322(2):403-410.
[6]Chen QG, Zhou W, Han T, et al. MiR-378 suppresses prostatecancer cell growth through downregulation of MAPK1 in vitro and in vivo[J]. Tumour Biol, 2016, 37(2):1-9.
[7]Zeng M, Zhu L, Li L, et al. miR-378 suppresses the proliferation,migrationandinvasionofcoloncancercellsbyinhibitingSDAD1[J]. Cellular&Molecular Biology Letters, 2017, 22(1):12.
[8]Fu H, Zhang J, Pan T, et al. miR-378a enhances the sensitivityof liver cancer to sorafenib by targeting VEGFR, PDGFRβandc-Raf[J]. Molecular Medicine Reports, 2018, 17(3):4581-4588.
[9]Ji KX, Cui F, Qu D, et al. MiR-378 promotes the cell proliferationof non-small cell lung cancer by inhibiting FOXG1[J]. EuropeanReview for Medical&Pharmacological Sciences, 2018, 22(4):1011.
[10]Li S, Yang F, Wang M, et al. miR-378 functions as an oncomiRNA by targeting the ST7L/Wnt/β-catenin pathway in cervicalcancer[J]. International Journal of Molecular Medicine, 2017,40(4):1047.
[11]Chai SH, Noor SM, Nagoor NH. MiR-378 and MiR-1827 regulatetumor invasion, migration and angiogenesis in human Lungadenocarcinoma by Targeting RBX1 and CRKL, respectively[J].Journal of Cancer, 2018, 9(2):331-345.
[12]Tan D, Zhou C, Han S, et al. MicroRNA-378 enhances migrationand invasion in cervical cancer by directly targeting autophagyrelated protein 12[J]. Molecular Medicine Reports, 2018, 17(5):6319-6326.
[13]Chan JK, Kiet TK, Blansit K, et al. MiR-378 as a biomarker forresponse to anti-angiogenic treatment in ovarian cancer[J].Gynecologic Oncology, 2014, 133(3):568-574.
[14]Ma TH, Cheng L, Wang B, et al. MIR-378 induces apoptsis ofgranulosa cells during follicle development in cattle[J]. Journalof Animal&Plant Sciences, 2017, 27(5):1738-1742.
[15]Lu Z, Du L, Liu R, et al. MiR-378 and BMP-Smad can influencethe proliferation of sheep myoblast[J]. Gene, 2018, 674:143-150.
[16]张阳阳. miR-378在牛前体脂肪细胞分化的作用与机制[D].长春:吉林大学, 2014.
[17]茹松伟,申卫红,杨鹏程,等. microRNA靶基因预测算法研究概况及发展趋势[J].生命科学, 2007, 19(5):562-567.
[18]Yong Z, Li C, Hu L, et al. miR-378 activates the pyruvatePEP futile cycle and enhances lipolysis to ameliorate obesity inmice[J]. Ebiomedicine, 2016, 5:93-104.
[19]Wei X, Hui L, Zhang B, et al. miR-378a-3p promotes differentiationand inhibits proliferation of myoblasts by targeting HDAC4 inskeletal muscle development[J]. RNA Biology, 2016, 13(12):1300-1309.
[20]Nunu H, Jiu W, Weidong X, et al. MiR-378a-3p enhancesadipogenesis by targeting mitogen-activated protein kinase 1[J].Biochemical&Biophysical Research Communications, 2015, 457(1):37-42.
[21]Liu SY, Zhang YY, Gao Y, et al. MiR-378 plays an important rolein the differentiation of bovine preadipocytes[J]. Cellular Physiology&Biochemistry, 2015, 36(4):1552-1562.
[22]Xu S, Linhermelville K, Yang BB, et al. Micro-RNA378(miR-378)regulates ovarian estradiol production by targeting aromatase[J].Endocrinology, 2011, 152(10):3941-3951.
[23]Toms D, Xu S, Pan B, et al. Progesterone receptor expressionin granulosa cells is suppressed by microRNA-378-3p[J].Molecular&Cellular Endocrinology, 2015, 399:95-102.
[24]Ma T, Jiang H, Gao Y, et al. Microarray analysis of differentiallyexpressed microRNAs in non-regressed and regressed bovinecorpus Luteum tissue;microRNA-378 may suppress Luteal cellapoptosis by targeting the interferon gamma receptor 1 gene[J].Journal of Applied Genetics, 2011, 52(4):481-486.
[25]徐盛玉.营养水平对后备母猪卵巢microRNA-378表达影响及microRNA-378作用机制研究[D].成都:四川农业大学,2012.
[26]熊显荣,兰道亮,李键,等.牦牛卵巢小RNA高通量测序及生物信息学分析[J].畜牧兽医学报, 2016, 47(1):55-63.
[27]潘阳阳,崔燕,樊江峰,等.胰岛素样生长因子-1(IGF-1)对牦牛卵丘细胞热休克蛋白70(HSP70)表达的影响及其与细胞凋亡的关联性分析[J].农业生物技术学报, 2015, 23(9):1207-1216.
[28]李莉华,高琪,王晓艳,等. miR-378靶向胰岛素样生长因子1类受体抑制肝癌细胞生长[J].中华肝脏病杂志, 2013, 21(8):609-613.
[2]Thermann R, Hentze MW. Drosophila miR2 induces pseudopolysomes and inhibits translation initiation[J]. Nature, 2012,447(7146):875-878.
[3]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronicgene lin-4 encodes small RNAs with antisense complementarity tolin-14[J]. Cell, 1993, 75(5):843.
[4]Tong HL, Jiang RY, Liu TT, et al. bta-miR-378 promote thedifferentiation of bovine skeletal muscle-derived satellite cells[J].Gene, 2018, 668:246-251.
[5]Kasashima K, Nakamura Y, Kozu T. Altered expression profilesof microRNAs during TPA-induced differentiation of HL-60cells[J]. Biochem Biophys Res Commun, 2004, 322(2):403-410.
[6]Chen QG, Zhou W, Han T, et al. MiR-378 suppresses prostatecancer cell growth through downregulation of MAPK1 in vitro and in vivo[J]. Tumour Biol, 2016, 37(2):1-9.
[7]Zeng M, Zhu L, Li L, et al. miR-378 suppresses the proliferation,migrationandinvasionofcoloncancercellsbyinhibitingSDAD1[J]. Cellular&Molecular Biology Letters, 2017, 22(1):12.
[8]Fu H, Zhang J, Pan T, et al. miR-378a enhances the sensitivityof liver cancer to sorafenib by targeting VEGFR, PDGFRβandc-Raf[J]. Molecular Medicine Reports, 2018, 17(3):4581-4588.
[9]Ji KX, Cui F, Qu D, et al. MiR-378 promotes the cell proliferationof non-small cell lung cancer by inhibiting FOXG1[J]. EuropeanReview for Medical&Pharmacological Sciences, 2018, 22(4):1011.
[10]Li S, Yang F, Wang M, et al. miR-378 functions as an oncomiRNA by targeting the ST7L/Wnt/β-catenin pathway in cervicalcancer[J]. International Journal of Molecular Medicine, 2017,40(4):1047.
[11]Chai SH, Noor SM, Nagoor NH. MiR-378 and MiR-1827 regulatetumor invasion, migration and angiogenesis in human Lungadenocarcinoma by Targeting RBX1 and CRKL, respectively[J].Journal of Cancer, 2018, 9(2):331-345.
[12]Tan D, Zhou C, Han S, et al. MicroRNA-378 enhances migrationand invasion in cervical cancer by directly targeting autophagyrelated protein 12[J]. Molecular Medicine Reports, 2018, 17(5):6319-6326.
[13]Chan JK, Kiet TK, Blansit K, et al. MiR-378 as a biomarker forresponse to anti-angiogenic treatment in ovarian cancer[J].Gynecologic Oncology, 2014, 133(3):568-574.
[14]Ma TH, Cheng L, Wang B, et al. MIR-378 induces apoptsis ofgranulosa cells during follicle development in cattle[J]. Journalof Animal&Plant Sciences, 2017, 27(5):1738-1742.
[15]Lu Z, Du L, Liu R, et al. MiR-378 and BMP-Smad can influencethe proliferation of sheep myoblast[J]. Gene, 2018, 674:143-150.
[16]张阳阳. miR-378在牛前体脂肪细胞分化的作用与机制[D].长春:吉林大学, 2014.
[17]茹松伟,申卫红,杨鹏程,等. microRNA靶基因预测算法研究概况及发展趋势[J].生命科学, 2007, 19(5):562-567.
[18]Yong Z, Li C, Hu L, et al. miR-378 activates the pyruvatePEP futile cycle and enhances lipolysis to ameliorate obesity inmice[J]. Ebiomedicine, 2016, 5:93-104.
[19]Wei X, Hui L, Zhang B, et al. miR-378a-3p promotes differentiationand inhibits proliferation of myoblasts by targeting HDAC4 inskeletal muscle development[J]. RNA Biology, 2016, 13(12):1300-1309.
[20]Nunu H, Jiu W, Weidong X, et al. MiR-378a-3p enhancesadipogenesis by targeting mitogen-activated protein kinase 1[J].Biochemical&Biophysical Research Communications, 2015, 457(1):37-42.
[21]Liu SY, Zhang YY, Gao Y, et al. MiR-378 plays an important rolein the differentiation of bovine preadipocytes[J]. Cellular Physiology&Biochemistry, 2015, 36(4):1552-1562.
[22]Xu S, Linhermelville K, Yang BB, et al. Micro-RNA378(miR-378)regulates ovarian estradiol production by targeting aromatase[J].Endocrinology, 2011, 152(10):3941-3951.
[23]Toms D, Xu S, Pan B, et al. Progesterone receptor expressionin granulosa cells is suppressed by microRNA-378-3p[J].Molecular&Cellular Endocrinology, 2015, 399:95-102.
[24]Ma T, Jiang H, Gao Y, et al. Microarray analysis of differentiallyexpressed microRNAs in non-regressed and regressed bovinecorpus Luteum tissue;microRNA-378 may suppress Luteal cellapoptosis by targeting the interferon gamma receptor 1 gene[J].Journal of Applied Genetics, 2011, 52(4):481-486.
[25]徐盛玉.营养水平对后备母猪卵巢microRNA-378表达影响及microRNA-378作用机制研究[D].成都:四川农业大学,2012.
[26]熊显荣,兰道亮,李键,等.牦牛卵巢小RNA高通量测序及生物信息学分析[J].畜牧兽医学报, 2016, 47(1):55-63.
[27]潘阳阳,崔燕,樊江峰,等.胰岛素样生长因子-1(IGF-1)对牦牛卵丘细胞热休克蛋白70(HSP70)表达的影响及其与细胞凋亡的关联性分析[J].农业生物技术学报, 2015, 23(9):1207-1216.
[28]李莉华,高琪,王晓艳,等. miR-378靶向胰岛素样生长因子1类受体抑制肝癌细胞生长[J].中华肝脏病杂志, 2013, 21(8):609-613.