Construction of IGF1 Gene Interference Vector in Guanling Cattle (Bos taurus) and Its Effect on Myogenic Cells
SONG Lin-Jin1,2, XU Hou-Qiang1,2,*, LI Yong1,3, SUN Jin-Kui1,2
1 Key Laboratory of Genetic Breeding and Reproduction of Plateau Mountain Animals, Ministry of Education, Guizhou University/Key Laboratory of Animal Genetic Breeding and Reproduction, Guizhou Province, Guiyang 550025, China; 2 School of Animal Science, Guizhou University, Guiyang 550025, China; 3 School of Life Sciences, Guizhou University, Guiyang 550025, China
Abstract:Insulin like growth factor 1 (IGF1) is a single-chain peptide hormone that affects muscle
development by regulating the synthesis of skeletal muscle proteins, and is important for cell proliferation and
other physiological functions. Three healthy adult (2~3 years old) Guanling cattle (Bos taurus) were selected
and 12 tissue samples were collected from heart, liver, spleen, lung, kidney, foreleg, hindleg, longest dorsal
muscle, rhomboid, gluteus, stomach and small intestine for RNA extraction, respectively, and the longest
dorsal muscle was successfully cultured in Guanling myogenic cells. The relative mRNA expression of IGF1
gene in different tissues was detected by qPCR; the physical and chemical properties, secondary and tertiary
structures, and subcellular localization of IGF1 protein in Guanling bovine were analyzed by online software;
meanwhile, three pairs of short hairpin RNA (shRNA) interference sequences and one pair of negative control
sequences of IGF1 gene were designed by online software, and sequenced with pGPU6-GFP-Neo. IGF1
interference vector was sequenced and transfected into adult myoblasts. qPCR was used to detect and screen
the most efficient interfering vector and analyze the effect of the interfering vector on IGF1 gene expression.
The relative mRNA expression of cyclin-dependent kinases 2 (CDK2) and cyclin-dependent kinases 1 (Cyclin
D1) was measured by qPCR, and the proliferation of myogenic cells was detected by CCK8. The results
showed that IGF1 gene was expressed in 12 tissues including heart, foreleg, hindleg, longest back, rhomboid
and gluteus muscles of Guanling cattle, and the relative mRNA expression of liver was the highest, which was
significantly higher than that of other tissues (P<0.01). The IGF1 protein had a molecular formula of
C744H1186N214O216S15, a relative molecular mass of 17.06581 kD and a theoretical isoelectric point of 9.36, and
was a basic unstable protein; After the IGF1 gene was inhibited, the expression of cyclin D1 and CDK2, was
significantly lower than that of shRNA-NC (P<0.01). The results of cell proliferation detection by CCK8
method showed that the proliferation of myogenic cells at 24, and 72 h was significantly lower than that of the
control group after IGF1 gene inhibited (P<0.05), and was inhibited for extremely significant difference at 48
h (P<0.01). Silencing IGF1 gene expression in vitro affected the proliferative capacity of myogenic cells and
inhibited the expression of genes related to myogenic cell proliferation. This study provides basic data for
further research on the effect of IGF1 gene on the muscle growth and development of Guanling cattle and its
regulatory mechanism
宋林锦, 许厚强, 李永, 孙金魁. 关岭牛IGF1基因干扰载体构建及其对成肌细胞的影响[J]. 农业生物技术学报, 2022, 30(5): 896-907.
SONG Lin-Jin, XU Hou-Qiang, LI Yong, SUN Jin-Kui. Construction of IGF1 Gene Interference Vector in Guanling Cattle (Bos taurus) and Its Effect on Myogenic Cells. 农业生物技术学报, 2022, 30(5): 896-907.
[1] 白波 .2000. 胰岛素样生长因子与胎儿生长发育[J]. 国外医学(儿科学分册), 27(1): 4-7. (Bai B.2000. Insulin-like growth factor and fetal growth and development[J]. Foreign Medicine (Pediatrics), 27(1): 4-7.) [2] 安静 .2013. IGF1 在绵羊成肌细胞增殖与分化中的作用[D]. 硕士学位论文, 新疆农业大学, 导师: 刘明军, pp. 44-45. (An J.2013. The role of IGF1 in the proliferation and differentiation of sheep myoblasts[D]. Thesis for M. S., Xinjiang Agricultural University, Supervisor: Liu M J, pp. 44-45.) [3] 丁军莉 .2019. IGF1 对梅花鹿鹿茸间充质细胞增殖分化的调控[D]. 硕士学位论文, 吉林大学, 导师: 岳占碰, pp. 30-31. (Ding J L.2019. Regulation of IGF1 on the prolifera tion and differentiation of sika deer antler mesenchymal cells[D]. Thesis for M. S., Jilin University, Supervisor: Yue Z P, pp. 30-31.) [4] 龚亚凡 .2020. 胰岛素样生长因子 1 (IGF1)在鸡心肌损伤中的作用机制研究[D]. 硕士学位论文, 东北农业大学, 导师 : 张子威 , pp. 48-49. (Gong Y F.2020. The mecha‐ nism of insulin-like growth factor 1 (IGF1) in myocardi‐ al injury in chickens[D]. Thesis for M.S., Northeast Agricultural University, Supervisor: Zhang Z W, pp. 48-49.) [5] 韩利军, 乔虎云, 梁炳生 . 2020. 肌卫星细胞研究新进展[J]. 山西医药杂志, 49(12): 1508-1513. (Han L J, Qiao H Y, Liang B S.2020. New progress in muscle satellite cell research[J]. Shanxi Medical Journal, 49(12): 1508-1513.) [6] 卢劲晔, 王锦峰, 刘静 . 2014. 胰岛素样生长因子-1 对细胞周期的影响[J]. 畜牧与兽医 , 46(02): 100-104. (Lu J Y, Wang J F, Liu J.2014. Effects of insulin-like growth factor-1 on cell cycle[J]. Animal Husbandry and Veterinary Medicine, 46(02): 100-104.) [7] 宋海燕, 苏少锋, 王希生, 等 . 2018. GH, IGF1 及其受体基因在蒙古马不同器官中的表达差异[J]. 农业生物技术学报, 26(4): 670-678. (Song H Y, Su S F, Wang X S, et al.2018. Expression differences of GH, IGF1 and its recep‐tor genes in different organs of Mongolian horses[J]. Chinese Journal of Agricultural Biotechnology, 26(4): 670-678.) [8] 唐文, 陈祥, 洪磊, 等 . 2021. 山羊 IGF1 基因过表达载体构建及其对子宫上皮细胞的影响[J]. 农业生物技术学报, 29(6): 1150-1160. (Tang W, Chen X, Hong L, et al.2021. Construction of goat IGF1 gene overexpression vector and its effect on uterine epithelial cells[J]. Chinese Journal of Agricultural Biotechnology, 29(6): 1150-1160.) [9] 韦云芳, 李飞翔, 马卫国, 等 . 2020. 昆明犬 IGF-1 基因 CDS 区克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 47(06): 1659-1667. (Wei Y F, Li F X, Ma W G, etal.2020. Cloning, bioinformatics analysis and tissue expression study of the CDS region of canine IGF-1 gene in Kunming[J]. China Animal Husbandry and Veterinary Medicine, 47(06): 1659-1667.) [10] 汪忠艳, 杜炳旺 . 2004. 类胰岛素生长因子的研究进展[J]. 畜禽业, (11): 62-64. (Wang Z Y, Du B W. 2004. Research progress of insulin-like growth factor[J]. Livestock and Poultry Industry, (11): 62-64.) [11] 祝梦琦 .2020. IGF1 和 FSH 对鸡小黄卵泡颗粒细胞功能的影响[D]. 硕士学位论文, 西北农林科技大学, 导师: 魏泽辉, pp. 33-44. (Zhu M Q.2020. Effects of IGF1 and FSH on the function of granulosa cells of chicken small yellow follicle[D]. Thesis for M.S., Northwest A&F University, Supervisor: Wei Z H, pp. 33-44.) [12] 朱晓锋 .2020. 从江香猪 GHR、IGF-1 基因对成肌细胞增殖的影响研究[D]. 硕士学位论文, 贵州大学, 导师: 许厚强, pp. 64-66. (Zhu X F.2020. Effects of GHR and IGF-1 genes on the proliferation of myoblasts in Congjiangxiang pigs[D]. Thesis for M. S., Guizhou University, Supervisor: Xu H Q, pp. 64-66.) [13] 朱晓锋, 许厚强, 陈伟, 等 . 2019. 从江香猪 IGF-1 和 IGF-2 基因的克隆, 表达及生物信息学分析[J]. 农业生物技术学报, 27(8): 1382-1391. (Zhu X F, Xu H Q, Chen W, et al.2019. Cloning, expression and bioinformatics analysis of IGF-1 and IGF-2 genes in Congjiangxiang pigs[J]. Chinese Journal of Agricultural Biotechnology, 27(8): 1382-1391.) [14] 张雅丽, 刘理想, 孙大明, 等 . 2021. 基于胰岛素样生长因子-Ⅰ信号通路体外研究丁酸钠促进羔羊瘤胃上皮细胞增殖的机理[J]. 动物营养学报 , 33(03): 1687-1698. (Zhang Y L, Liu L L, Sun D M, et al.2021. In vitro study on the mechanism of sodium butyrate to promote the proliferation of lamb rumen epithelial cellsbased on insulin-like growth factor-Ⅰ signaling path‐way[J]. Chinese Journal of Animal Nutrition, 33(03): 1687-1698 .) [15] Ichikawa T, Nakao K, Hamasaki K, et al. 2007. Role of growth hormone, insulin-like growth factor 1 and insulin-like growth factor-binding protein 3 in development national, 1(2): 287-294. [16] Jones J I, Clemmons D R.1995. Insulin-like growth factors and their binding proteins: Biological actions[J]. Endocrine Reviews, 16(1): 3-34. [17] Kineman R D, Rio-Moreno M D, André Sarmento-Cabral.2018. Understanding the tissue-specific roles of IGF1/ IGF1R in regulating metabolism using the Cre/loxP system[J]. Journal of Molecular Endocrinology, 61(1): T187-T198. [18] Lai Z, Wu F, Li M, et al.2020. Tissue expression profile, polymorphism of IGF1 gene and its effect on body sizetraits of Dezhou donkey[J]. Gene, 766(3): 145118. [19] Norbury C, Nurse P.1992. Animal cell cycles and their control[J]. Annual Review of Biochemistry, 61(1): 441-468. [20] Rinderknecht E, Humbel R E.1978. The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin[J]. Journal of Biological Chemistry, 253(8): 2769-2776. [21] Salmon W D, Daughaday W H.1957. A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro[J]. Journal of Laboratory and Clinical Medicine, 49(6): 825-36. [22] Sterle J A, Cantley T C, Lamberson W R, et al.1995. Effects of recombinant porcine somatotropin on placental size, fetal growth, and IGF-I and IGF-II concentrations in pigs[J]. Journal of Animal Science, 73(10): 2980-2985. [23] Susann Blüher, Jürgen Kratzsch, Kiess W.2005. Insulin-like growth factor I, growth hormone and insulin in white adipose tissue[J]. Best Practice & Research Clinical Endocrinology & Metabolism, 19(4):577-587. [24] Wetering J, Elfring R H, Oosterlaken-Dijksterhuis M A, et al.1997. Perinatal expression of IGFBPs in rat lung and its hormonal regulation in fetal lung explants[J]. American Journal of Physiology, 273(6): L1174-L1181. [25] Wei W, Wang G, Cheng Y, et al.2019. A miR-511-binding site SNP in the 3′ UTR of IGF-1 gene is associated with proliferation and apoptosis of PK-15 cells[J]. In Vitro Cellular & Developmental Biology-Animal, 55(5): 323-330. [26] Wang W J, Guo Y Q, Xie K J, et al.2021. A functional variant in the promoter region of IGF1 gene is associated with chicken abdominal fat deposition -ScienceDirect[J]. Domestic Animal Endocrinology, 75: 106584. [27] You H, Zheng H, Murray S A, et al.2002. IGF‐1 induces Pin1 expression in promoting cell cycle S phase entry[J]. Journal of Cellular Biochemistry, 84(2): 211-216. [28] Yang Z Q, Zhang H L, Duan C C, et al.2017. IGF1 regulatesRUNX1 expression via IRS1/2: Implications for antler chondrocyte differentiation[J]. Cell Cycle, 16(6): 522-532.