干扰lnc721对牛骨骼肌卫星细胞增殖与分化的影响

Effects of Interference lnc721 on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells

【目的】探究对牛骨骼肌发育具有调控作用的长链非编码RNA(long non-coding RNA,lncRNA),研究其在牛骨骼肌卫星细胞增殖期和分化期的表达量变化,为牛肌肉发育相关机制方面的研究提供参考依据。【方法】选取实验室前期高通量测序获得的1条lncRNA(lnc721),通过NCBI数据库和CPC网站分析其生物学信息并预测其编码能力,通过核质分离试验确定其亚细胞定位。设计并合成lnc721的siRNA转染牛骨骼肌卫星细胞,通过成熟的体外成肌细胞诱导分化模型培养牛骨骼肌卫星细胞并进行诱导分化。分别采用EdU试验、实时荧光定量PCR和Western blotting分析lnc721在细胞发育不同时期表达量的变化情况。【结果】lnc721位于牛全基因组的18号染色体上,其蛋白编码能力为―1.33129,主要定位于细胞质内。在干扰lnc721表达量后发现,EdU阳性细胞比率极显著上升,细胞增殖标志因子Pax 7和Ki-67基因的mRNA表达水平极显著上调(P<0.01);Western blotting结果进一步证明,干扰lnc721后极显著促进了Pax7蛋白的表达(P<0.01),在细胞分化期干扰lnc721表达后,细胞分化标志因子MyHC基因的mRNA和蛋白表达水平均极显著下调(P<0.01)。【结论】干扰lnc721表达可促进牛骨骼肌卫星细胞的增殖并抑制成肌分化进程。

【Objective】This experiment was aimed to explore the long non-coding RNA(lncRNA)that played a regulatory role in bovine skeletal muscle development,and study its effect on the expression of bovine skeletal muscle satellite cells in proliferation and differentiation,so as to provide reference for the study of bovine muscle development related mechanisms.【Method】A lncRNA obtained by high-throughput sequencing in the previous test was named lnc721.The biological information of lnc721 was analyzed using NCBI database,the coding ability and subcellular localization of lnc721 were determined by CPC website and nucleo-cytoplasmic separation experiment,respectively.The designed and synthesized lnc721 siRNA was transfected into bovine skeletal muscle satellite cells,and the bovine skeletal muscle satellite cells were cultured and differentiated through a mature in vitro myoblast induced differentiation model.The expression changes of lnc721 in different stages of cell development were analyzed in detail by EdU experiment,Real-time quantitative PCR and Western blotting,respectively.【Result】lnc721 was located on chromosome 18 of the whole bovine genome,and its protein coding capacity was―1.33129,which was mainly located in cytoplasm.After interference with lnc721 expression,the EdU positive cellular rates was extremely significantly increased,and the mRNA expression of cell proliferation marker genes Pax 7 and Ki-67 were extremely significantly up-regulated(P<0.01).Western blotting results further proved that interference with lnc721 extremely significantly promoted the expression of Pax7 protein(P<0.01).The mRNA and protein expression of the cell differentiation marker MyHC gene were extremely significantly down-regulated after interfering with the expression of lnc721 at the cell differentiation stage(P<0.01).【Conclusion】Interference with lnc721 could promote the proliferation of bovine skeletal muscle satellite cells and inhibit the process of myoblast differentiation.