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

doi:10.7668/hbnxb.20193739

Abstract

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

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.

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Figures/Tables 5

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

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.

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.

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).

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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