腺病毒介导shRNA干扰绵羊MSTN基因效果及对生肌调节因子和干扰素反应基因表达的影响

doi:10.11843/j.issn.0366-6964.2017.10.006

摘要/Abstract

摘要：

旨在进一步揭示MSTN在绵羊成肌细胞中的调控机制，制备可有效失活MSTN基因的工具，为通过RNA干扰技术提高绵羊产肉量提供方法和理论依据。本研究以绵羊成肌细胞为试验材料，构建特异靶向绵羊MSTN基因的shRNA干扰质粒载体，将干扰效果好的质粒进一步包装为重组腺病毒，转染细胞后采用qRT-PCR和Western blot检测MSTN基因以及生肌调节因子和干扰素反应基因的表达。结果表明，质粒ShR218和ShR511干扰MSTN基因效率分别达到35%和48%，双元干扰质粒ShR3+4干扰效率最高达到85%。成功包装shRNA重组腺病毒载体Sh511和Sh3+4，病毒滴度达到1×10⁸ pfu·mL^-1，对成肌细胞的感染效率达到90%以上。Sh511和Sh3+4对MSTN基因mRNA的表达抑制分别达到53%和76%，对蛋白表达抑制分别达到55%和64%。MSTN基因沉默后，伴随着Myf5、MyoD、MyoG、Myf6基因mRNA水平的极显著性下调（P<0.01），但只引起MyoG蛋白水平极显著升高（P<0.01），未引起Myf5、MyoD、Myf6蛋白水平的显著变化；腺病毒感染成肌细胞未引起OAS1基因mRNA水平的显著变化，但引起IFNGR1基因mRNA水平的极显著升高（P<0.01），对二者蛋白水平均无显著影响。本研究成功构建靶向MSTN基因的shRNA腺病毒载体，能有效抑制成肌细胞MSTN的mRNA和蛋白表达，并影响生肌调节因子Myf5、MyoD、MyoG、Myf6基因和干扰素受体基因IFNGR1表达。

关键词: 绵羊, MSTN, 成肌细胞, RNA干扰

Abstract:

The aim of this study was to further reveal the regulation mechanism of MSTN in sheep myoblasts, prepare the tool silencing MSTN, and provide the methods and theoretical basis for increasing the yield of muscle mass by using RNA interference technology. In this study, sheep myoblasts were used as experimental materials. The short hairpin RNA(shRNA) expression plasmid vector specific targeting to MSTN gene were constructed, then the plasmids with better interference effect were packaged for recombinant adenovirus, the expression of MSTN, myogenic regulatory factor and interferon response genes in myoblasts after infected by recombinant adenovirus were detected by qRT-PCR and Western blot. The result showed that the interference efficiency of plasmid ShR218, ShR511 were 35% and 48%,respectively,the interference efficiency of ShR3+4 was 85%.Recombinant adenovirus vector Sh511 and Sh3+4 were successfully packaged, the virus titer reached 1×10⁸ pfu·mL^-1, the infection rate to myoblasts reached more than 90%. The mRNA expression of MSTN gene had been decreased by 53% and 76%, and the protein level had been decreased by 55% and 64% through Sh511 and Sh3+4, respectively. The knockdown of MSTN gene was accompanied with expression downregulation of myogenic regulatory factor Myf5, MyoD, MyoG and Myf6 at mRNA level(P<0.01), but at protein level only MyoG was significantly increased(P<0.01), no significant changes in Myf5, MyoD and Myf6. Adenovirus infecting myoblasts did not cause significant changes at OAS1 mRNA level, but caused significant increase at IFNGR1 mRNA level (P<0.01), while had no significant effect on their protein levels. In this study, shRNA adenovirus vector targeting to MSTN gene was successfully constructed, which can effectively inhibit the mRNA and protein expression of MSTN in myoblasts, and affect the expression of Myf5, MyoD, MyoG, Myf6 and IFNGR1 genes.

Key words: sheep, MSTN, myoblasts, RNAi

中图分类号:

S826.2

王红娜, 孙洪新, 张英杰, 刘月琴, 谷振慧, 王苏瑶. 腺病毒介导shRNA干扰绵羊MSTN基因效果及对生肌调节因子和干扰素反应基因表达的影响[J]. 畜牧兽医学报, 2017, 48(10): 1833-1842.

WANG Hong-na, SUN Hong-xin, ZHANG Ying-jie, LIU Yue-qin, GU Zhen-hui, WANG Su-yao. Adenovirus-mediated shRNA Interference of MSTN Gene and Its Effect on Expression of Myogenic Regulatory Factor and Interferon Response Gene in Sheep[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(10): 1833-1842.

参考文献

[1] MCPHERRON A C, LAWLER A M, LEE S J. Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member[J]. Nature, 1997, 387(6628): 83-90.
[2] ARTHUR P F. Double muscling in cattle: a review[J]. Aust J Agr Res, 1995, 46(8): 1493-1515.
[3] WILLIAMS M S. Myostatin mutation associated with gross muscle hypertrophy in a child[J]. N Engl J Med, 2004, 351(10): 1030-1031.
[4] CLOP A, MARCQ F, TAKEDA H, et al. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep[J]. Nat Genet, 2006, 38(7): 813-818.
[5] MOSHER D S, QUIGNON P, BUSTAMANTE C D, et al. A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs[J]. PLoS Genet, 2007, 3(5): e79.
[6] WELLE S, BHATT K, PINKERT C A, et al. Muscle growth after postdevelopmental myostatin gene knockout[J]. Am J Physiol Endocrinol Metab, 2007, 292(4): E985-E991.
[7] UEDA R. RNAi: a new technology in the post-genomic sequencing era[J]. J Neurogenet, 2001, 15(3-4): 193-204.
[8] MAGEE T R, ARTAZA J N, FERRINI M G, et al. Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass[J]. J Gene Med, 2006, 8(9): 1171-1181.
[9] SATO F, KUROKAWA M, YAMAUCHI N, et al. Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA[J]. Am J Physiol Cell Physiol, 2006, 291(3): C538-C545.
[10] JAIN H, SINGH S, KADAM M, et al. Knockdown of the myostatin gene by RNA interference in caprine fibroblast cells[J]. J Biotechnol, 2010, 145(2): 99-102.
[11] TESSANNE K, GOLDING M C, LONG C R, et al. Production of transgenic calves expressing an shRNA targeting myostatin[J]. Mol Reprod Dev, 2012, 79(3): 176-185.
[12] 盛鹏程, 朱瑞良, 苗向阳. 绵羊Myostatin基因shRNA慢病毒载体的构建与鉴定[J]. 中国兽医学报, 2012, 32(4): 628-632.
SHENG P C, ZHU R L, MIAO X Y. Construction and identification of lentivial RNA interference vector of sheep myostatin receptor gene[J]. Chinese Journal of Veterinary Science, 2012, 32(4): 628-632. (in Chinese)
[13] LIU C X, LI W R, ZHANG X M, et al. Knockdown of endogenous myostatin promotes sheep myoblast proliferation[J]. In vitro Cell Dev Biol Anim, 2014, 50(2): 94-102.
[14] WALTHER W, STEIN U. Viral vectors for gene transfer: a review of their use in the treatment of human diseases[J]. Drugs, 2000, 60(2): 249-271.
[15] THOMAS C E, EHRHARDT A, KAY M A. Progress and problems with the use of viral vectors for gene therapy[J]. Nat Rev Genet, 2003, 4(5): 346-358.
[16] 王红娜, 张英杰, 刘月琴. 绵羊成肌细胞的纯化、培养、鉴定及其成肌诱导分化研究[J]. 河北农业大学学报, 2016, 39(1): 94-98, 102.
WANG H N, ZHANG Y J, LIU Y Q. A study of purification, culture, identification and differentiation of sheep myoblast cells[J]. Journal of Agricultural University of Hebei, 2016, 39(1): 94-98, 102. (in Chinese)
[17] LIU C X, LI W R, ZHANG X M, et al. The critical role of myostatin in differentiation of sheep myoblasts[J]. Biochem Biophys Res Commun, 2012, 422(3): 381-386.
[18] 孔庆辉, 晁燕, 夏明哲, 等. 黄河裸裂尻鱼MSTN基因RNA干扰研究[J]. 中国实验动物学报, 2016, 24(4): 344-350.
KONG Q H, CHAO Y, XIA M Z, et al. Inhibitory effect of RNA interference of MSTN gene expression on the downstream genes in Schizopygopsis pylzovi[J]. Acta Laboratorium Animalis Scientia Sinica, 2016, 24(4): 344-350. (in Chinese)
[19] 刘建军, 张茂雷, 朱芳芳. 实时荧光定量PCR筛选有效RNA干扰方法的建立[J]. 检验医学与临床, 2010, 7(10): 903-905.
LIU J J, ZHANG M L, ZHU F F. Establishment of a method in detecting the effect of RAN interference on time flurogenic quantitative polymerase chain reaction[J]. Laboratory Medicine and Clinic, 2010, 7(10): 903-905. (in Chinese)
[20] LOUBOUTIN J P, WANG L L, WILSON J M. Gene transfer into skeletal muscle using novel AAV serotypes[J]. J Gene Med, 2005, 7(4): 442-451.
[21] MERENTIE M, LOTTONEN-RAIKASLEHTO L, PARVIAINEN V, et al. Efficacy and safety of myocardial gene transfer of adenovirus, adeno-associated virus and lentivirus vectors in the mouse heart[J]. Gene Ther, 2016, 23(3): 296-305.
[22] LU J, REN H X, SHENG X H, et al. Transcript characteristic of myostatin in sheep fibroblasts[J]. J Cell Biochem, 2012, 113(8): 2652-2660.
[23] LU J, WEI C H, ZHANG X N, et al. The effect of myostatin silencing by lentiviral-mediated RNA interference on goat fetal fibroblasts[J]. Mol Biol Rep, 2013, 40(6): 4101-4108.
[24] PATEL A K, TRIPATHI A K, PATEL U A, et al. Myostatin knockdown and its effect on myogenic gene expression program in stably transfected goat myoblasts[J]. In vitro Cell Dev Biol Anim, 2014, 50(7): 587-596.
[25] PATEL U A, PATEL A K, JOSHI C G. Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector-mediated RNAi[J]. Biotechnol Prog, 2015, 31(2): 452-459.
[26] KUMAR R, SINGH S P, KUMARI P, et al. Small interfering RNA (siRNA)-mediated knockdown of myostatin Influences the expression of myogenic regulatory factors in caprine foetal myoblasts[J]. Appl Biochem Biotechnol, 2014, 172(3): 1714-1724.
[27] 孙洪新, 王红娜, 张英杰, 等. BMPR-IB基因的克隆及其在绒山羊成纤维细胞中的表达[J]. 畜牧兽医学报, 2016, 47(6): 1124-1132.
SUN H X, WANG H N, ZHANG Y J, et al. Cloning and expression of BMPR-IB gene in cashmere goat fibroblast cells[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(6): 1124-1132. (in Chinese)
[28] GANTIER M P, WILLIAMS B R G. The response of mammalian cells to double-stranded RNA[J]. Cytokine Growth Factor Rev, 2007, 18(5-6): 363-371.
[29] 张义兵, 桂建芳. 鱼类干扰素反应及分子调控[J]. 生物工程学报, 2011, 27(5): 675-683.
ZHANG Y B, GUI J F. Fish interferon response and its molecular regulation: a review[J]. Chinese Journal of Biotechnology, 2011, 27(5): 675-683. (in Chinese)
[30] TRIPATHI A K, APARNATHI M K, VYAVAHARE S S, et al. Myostatin gene silencing by RNA interference in chicken embryo fibroblast cells[J]. J Biotechnol, 2012, 160(3-4): 140-145.
[31] SINGH N K, SINGH S, JAIN S K, et al. Evaluation of interferon response induced by anti-myostatin shRNA constructs in goat (Capra hircus) fetal fibroblasts by quantitative real time-polymerase chain reaction[J]. Anim Biotechnol, 2012, 23(3): 174-183.
[32] TRIPATHI A K, RAMANI U V, PATEL A K, et al. Short hairpin RNA-induced myostatin gene silencing in caprine myoblast cells in vitro[J]. Appl Biochem Biotechnol, 2013, 169(2): 688-694.