牛circMYH8的鉴定及其调控牛肌原代细胞增殖的功能探究

doi:10.7668/hbnxb.20193739

摘要/Abstract

摘要：

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。

关键词: 牛circMYH8, 骨骼肌, 细胞增殖

Abstract:

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.

Key words: Bovine circMYH8, Skeletal muscle, Cell proliferation

岳炳霖, 杨友抓拉母, 冉宏标, 王会, 蔡欣, 王嘉博, 柴志欣, 彭巍, 舒适, 付长其, 王国文, 钟金城. 牛circMYH8的鉴定及其调控牛肌原代细胞增殖的功能探究[J]. 华北农学报, 2023, 38(4): 197-204. doi: 10.7668/hbnxb.20193739.

YUE Binglin, YANG Youzhualamu, RAN Hongbiao, WANG Hui, CAI Xin, WANG Jiabo, CHAI Zhixin, PENG Wei, SHU Shi, FU Changqi, WANG Guowen, ZHONG Jincheng. Identification of Bovine circMYH8 and Its Function in Regulating the Proliferation of Primary Bovine Muscle Cells[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(4): 197-204. doi: 10.7668/hbnxb.20193739.

http://www.hbnxb.net/CN/Y2023/V38/I4/197

图/表 5

表1 引物信息

Tab.1 Primers information

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
circMYH8-D-F	TAGAGGAAGCAGAGGTGAAT
circMYH8-D-R	CTGACGTTTCAGTTCCTCAATC
circMYH8-C-F	GCACTGGCCTTGGATGAAAC
circMYH8-C-R	CCAGTTCCTTCCTTTTGGCCT
circMYH8-F	GGGGTACCGAGGACCAAGTACGAGACGG
circMYH8-R	CGGGATCCCACTGGGCCACCTCGCTGTT
GAPDH-F	CACTGAGGACCAGGTTGTCT
GAPDH-R	TGTCGTACCAGGAAATGAGC
β-actin-F	GTCATCACCATCGGCAATGAG
β-actin-R	AATGCCGCAGGATTCCATG
CDK2-F	TTTGCTGAGATGGTGACCCG
CDK2-R	TAACTCCTGGCCAAACCACC
CyclinD1-F	CCGTCCATGCGGAAGATC
CyclinD1-R	CAGGAAGCGGTCCAGGTAG
CDK4-F	GTGACGCAGAGTGAACAACAG
CDK4-R	AGGTTGCCTTCTCATTGGTGG
P21-F	AGGGCACGTCTCAGGAGGA
P21-R	CAGTCTGCGTTTGGAGTGGTAG

图1 circMYH8的筛选 A.牛胚胎期与成年期差异表达环状RNA的火山图。筛选阈值为|log₂FC|>1,P<0.01;B.牛胚胎期和成年期差异表达环状RNA的热图,其中包括circMYH8,以及cir cMYH8和线性MYH8在胎牛和成年牛骨骼肌中的表达水平。不同大写字母表示差异极显著(P<0.01)。图2—4同。

Fig.1 Screening of circMYH8 A.Volcano plot of differentially expressed circRNAs for bovine embryo versus adult.Criteria are based on |log₂ FC|>1 and P values<0.01;B.Heatmap of differentially expressed circRNAs identified by comparison of embryonic and adult stages,including circMYH8,and expression of circMYH8 and linear MYH8 in bovine skeletal muscle from embryo and adult cattle.The different uppercase letter indicates that the difference is extremely significant(P<0.01).The same as Fig.2—4.

图2 circMYH8的鉴定 A.circMYH8在牛基因组上的定位,以及Sanger测序证实了circMYH8环化位点的存在;B.PCR扩增和琼脂糖凝胶电泳结果确证了circMYH8的真实性;C.细胞核质定位试验显示circMYH8定位于细胞核和细胞质;D.RNase R的抗性试验结果显示circMYH8比线性MYH8更稳定。

Fig.2 Identification of circMYH8 A.Mapping of circMYH8 in bovine genome,and the head-to-tail splicing of circMYH8 was confirmed by Sanger sequencing;B.PCR and agarose gel electrophoresis assay with divergent or convergent primers indicated the existence of circMYH8;C.Nuclear and cytoplasmic localization experiments revealed circMYH8 was localized in the nucleus and cytoplasm;D.Resistance to RNase R results indicated that circMYH8 was more stable than linear MYH8.

图3 过表达及干扰circMYH8的qRT-PCR及Western Blot结果 A,B.circMYH8在培养24 h的牛肌原代细胞中被成功过表达(A)及抑制(B);C、D.过表达(C)及干扰(D)circMYH8后CDK2、CyclinD1、CDK4及P21的mRNA水平;E.过表达及干扰circMYH8后CDK2、CyclinD1、CDK4及P21的蛋白水平。不同小写字母表示差异显著(P<0.05)。

Fig.3 qRT-PCR and Western Blot results of overexpression and interference with circMYH8 A,B.circMYH8 was successfully overexpressed(A)and inhibited(B)in primary bovine skeletal muscle cells after culturing for 24 h; C, D. mRNA expression levels of CDK2, CyclinD1, CDK4 and P21 after overexpression (C) and interference (D) with circMYH8; E.Protein expression levels of CDK2, CyclinD1, CDK4 and P21 after overexpression and interference with circMYH8; the different lowercase letter indicates that the difference is significant(P<0.05).

图4 过表达及干扰circMYH8的EdU及流式细胞检测结果 A.EdU检测用于评估细胞增殖能力,刻度尺,200 mm;B.流式细胞术检测细胞周期分布。提供的数据来自3个独立的试验。

Fig.4 EdU assays and flow cytometry assay results of overexpression and interference with circMYH8 A.EdU assays were used to measure the ability of proliferation.Scale bars,200 mm;B.The cell cycle distribution was evaluated using a flow cytometry assay.Data presented are from three independent experiments.

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