牛circMYH8的鉴定及其调控牛肌原代细胞增殖的功能探究

Identification of Bovine circMYH8 and Its Function in Regulating the Proliferation of Primary Bovine Muscle Cells

旨在鉴定牛circMYH8以及探究其对牛肌原代细胞增殖的调控作用。设计合成circMYH8的divergent primer及convergent primer 2组引物,分别以牛背最长肌cDNA、RNase R处理的牛背最长肌cDNA以及牛背最长肌gDNA为模板进行PCR扩增,通过测序及琼脂糖凝胶电泳进行环状RNA鉴定;对circMYH8进行不同发育时期骨骼肌RT-qPCR分析、核质分离试验以及RNase R耐受性试验;构建及合成circMYH8的过表达载体及干扰片段,并转染至牛肌原代细胞中,通过RT-qPCR、Western Blot、EdU、流式细胞术等手段探究了circMYH8对牛肌原代细胞增殖表型的影响。结果表明,测序及琼脂糖凝胶电泳证实circMYH8为环状RNA;RT-qPCR分析显示,circMYH8在牛骨骼肌发育早期高表达,核质分离试验结果显示,circMYH8在细胞核和细胞质中都有表达,RNase R耐受性试验表明,circMYH8的稳定性高于其线性转录物;过表达circMYH8抑制了增殖标志基因的表达,EdU活性降低,而抑制circMYH8促进了增殖标志基因的表达,EdU活性上升,S期细胞比例上升。结果表明,牛circMYH8能显著抑制牛肌原代细胞增殖。

The aim of this study was to identify bovine circMYH8 and to explore its regulation effect on the proliferation of primary bovine muscle cells.Two sets of primers,namely divergent primer and convergent primer,were designed and synthesized from circMYH8.The cDNA of bovine longissimus dorsi,the cDNA of bovine longissimus dorsi treated with RNase R and the gDNA of bovine longissimus dorsi were amplified by PCR,respectively,and circMYH8 was identified by sequencing and agarose gel electrophoresis;RT-qPCR analysis of skeletal muscle at different developmental stages,nucleo-plasmic separation,and RNase R tolerance tests were performed on circMYH8;the overexpression vector and interference fragment of circMYH8 were constructed and synthesized,which were then transfected into primary bovine muscle cells,respectively,and RT-qPCR,Western Blot,EdU and flow cytometry were performed to investigate the effects of circMYH8 on the proliferative phenotype of primary bovine muscle cells.The results of sequencing and agarose gel electrophoresis showed that circMYH8 was real circRNA,and RT-qPCR analysis revealed that circMYH8 was overexpressed in early skeletal muscle development;the results of nucleo-plasmic separation showed that circMYH8 was expressed in both nucleus and cytoplasm,and RNase R tolerance test verified that circMYH8 was more stable than its linear transcript;overexpression of circMYH8 inhibited the expression of proliferation marker genes and activity of EdU,and inhibition of circMYH8 promoted the expression of proliferating marker genes,with the increased activity of EdU and the proportion of S phase cells.These results indicated that bovine circMYH8 could significantly inhibit the proliferation of primary bovine muscle cells.