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| 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 |
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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
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Received: 24 September 2021
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Corresponding Authors:
*gzdxxhq@163.com
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