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

摘要/Abstract

摘要： 旨在研究长链非编码RNA(lncRNA)对牛骨骼肌卫星细胞增殖和分化的影响。本研究以体外分离培养的鲁西黄牛胎牛原代骨骼肌卫星细胞为试验材料，体外诱导成肌分化，以前期高通量测序筛选的肌肉组织特异性表达lncRNA为靶标，通过RACE技术对其进行全长扩增，命名为lncbMD，对lncbMD进行生物信息学分析和亚细胞定位。收取增殖期和分化期第1、2、3天的细胞，提取 RNA(每组设置4个重复)或蛋白质(每组设置3个重复)，通过qRT-PCR检测lncbMD在牛骨骼肌卫星细胞分化进程中不同时期的表达情况。根据lncbMD的序列设计合成干扰RNA，转染牛骨骼肌卫星细胞干扰lncbMD的表达，利用EdU试验检测细胞增殖情况，qRT-PCR技术检测lncbMD干扰效果、lncbMD的mRNA表达水平，增殖标志因子Pax7、Cyclin D1以及分化标志因子MyoG、MyHC的mRNA表达水平，荧光倒置显微镜观察细胞生长情况。结果显示，lncbMD全长序列1 902 nt，位于牛基因组23号染色体上，不具有编码潜能，是一条未报道的lncRNA。在牛骨骼肌卫星细胞与肌管中均主要存在于细胞核，在牛骨骼肌卫星细胞成肌分化前后的mRNA表达水平有极显著差异(P<0.01)。干扰lncbMD后，EdU细胞阳性率显著上升(P<0.05)，增殖标志因子Pax7的mRNA表达水平与对照组相比显著上升(P<0.05)，分化标志因子MyoG、MyHC的mRNA表达水平与对照组相比无显著差异(P>0.05)。本研究发现一条新的lncRNA：lncbMD，其主要存在于牛骨骼肌卫星细胞和肌管的细胞核中，进一步研究结果表明干扰lncbMD后能够促进牛骨骼肌卫星细胞的增殖，对牛骨骼肌卫星细胞的分化没有显著影响。

关键词: 牛, 骨骼肌, lncRNA, 细胞增殖

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

中图分类号:

S823.2

杨光, 徐景, 李新, 胡德宝, 郭益文, 丁向彬, 郭宏, 张林林. 干扰lncbMD对牛骨骼肌卫星细胞增殖分化的影响[J]. 畜牧兽医学报, 2023, 54(3): 1015-1025.

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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