干扰lncbMD对牛骨骼肌卫星细胞增殖分化的影响

doi:10.11843/j.issn.0366-6964.2023.03.015

Abstract

Abstract: The study aimed to study the effects of long chain non coding RNA (lncRNA) on the proliferation and differentiation of bovine skeletal muscle satellite cells. In this study, the primary skeletal muscle satellite cells of Luxi cattle fetus were isolated and cultured in vitro as experimental materials, which were induced into muscle differentiation in vitro. The specific expression lncRNA in muscle tissue screened by high-throughput sequencing in the early stage was used as the target, and the full-length amplification was carried out through RACE technology, named lncbMD, and the bioinformatics analysis and subcellular localization of lncbMD were carried out. The cells at the 1st, 2nd and 3rd day of proliferation and differentiation were collected, and RNA (4 replicates in each group) or protein (3 replicates in each group) was extracted. The expression of lncbMD in different stages of bovine skeletal muscle satellite cell differentiation was detected by qRT-PCR. Interfering RNA was designed and synthesized according to the sequence of lncbMD, and transfected into bovine skeletal muscle satellite cells to interfere with the expression of lncbMD, EdU test was used to detect the cell proliferation, qRT-PCR was used to detect the interference effect of lncbMD, the mRNA expression level of lncbMD, the mRNA expression level of proliferation markers Pax7, Cyclin D1, and differentiation markers MyoG and MyHC, and the growth of cells was observed by fluorescence inversion microscope. The results showed that the full-length sequence of lncbMD was 1 902 nt, which was located on chromosome 23 of bovine genome and had no coding potential. It was an unreported lncRNA. The expression level of mRNA in nucleus of bovine skeletal muscle satellite cells and myotubes was significantly different before and after myogenic differentiation (P<0.01). After interfering with lncbMD, the positive rate of EdU cells increased significantly (P<0.05), the mRNA expression level of proliferation marker Pax7 increased significantly compared with the control group (P<0.05), and the mRNA expression level of differentiation marker MyoG and MyHC had no significant variation compared with the control group (P>0.05). In this study, we found a new lncRNA: lncbMD, which mainly exists in the nucleus of bovine skeletal muscle satellite cells and myotubes. Further research results showed that interfering with lncbMD could promote the proliferation of bovine skeletal muscle satellite cells, but had no significant effect on the differentiation of bovine skeletal muscle satellite cells.

Key words: bovine, skeletal muscle, lncRNA, cell proliferation

CLC Number:

S823.2

YANG Guang, XU Jing, LI Xin, HU Debao, GUO Yiwen, DING Xiangbin, GUO Hong, ZHANG Linlin. Effect of Interfering lncbMD on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1015-1025.

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Effect of Interfering lncbMD on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells

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