Long noncoding RNAs have been identified as important regulators of gene expression and animal development. The expression of natural antisense transcripts (NATs) transcribed in the opposite direction to protein-coding genes is usually positively correlated with the expression of homologous sense genes and is the key factor for expression. Here, we identified a conserved noncoding antisense transcript, CFL1-AS1, that plays an important role in muscle growth and development. CFL1-AS1 overexpression and knockout vectors were constructed and transfected into 293T and C2C12 cells. CFL1-AS1 positively regulated CFL1 gene expression, and the expression of CFL2 was also downregulated when CFL1-AS1 was knocked down. CFL1-AS1 promoted cell proliferation, inhibited apoptosis and participated in autophagy. This study expands the research on NATs in cattle and lays a foundation for the study of the biological function of bovine CFL1 and its natural antisense chain transcript CFL1-AS1 in bovine skeletal muscle development. The discovery of this NAT can provide a reference for subsequent genetic breeding and data on the characteristics and functional mechanisms of NATs.

Long noncoding RNAs have been identified as important regulators of gene expression and animal development. The expression of natural antisense transcripts (NATs) transcribed in the opposite direction to protein-coding genes is usually positively correlated with the expression of homologous sense genes and is the key factor for expression. Here, we identified a conserved noncoding antisense transcript, CFL1-AS1, that plays an important role in muscle growth and development. CFL1-AS1 overexpression and knockout vectors were constructed and transfected into 293T and C2C12 cells. CFL1-AS1 positively regulated CFL1 gene expression, and the expression of CFL2 was also downregulated when CFL1-AS1 was knocked down. CFL1-AS1 promoted cell proliferation, inhibited apoptosis and participated in autophagy. This study expands the research on NATs in cattle and lays a foundation for the study of the biological function of bovine CFL1 and its natural antisense chain transcript CFL1-AS1 in bovine skeletal muscle development. The discovery of this NAT can provide a reference for subsequent genetic breeding and data on the characteristics and functional mechanisms of NATs.

MicroRNA (miRNA) are single-stranded non-coding RNA of approximately 22 nucleotides estimated to regulate 60% of human and animal genes some of which are associated with cell differentiation, signal transduction and immune processes. Streptococcus agalactiae (S. agalactiae)-induced bovine mastitis is associated with up-regulation of miR-122 and down-regulation of erythropoietin (EPO) abundance in mammary gland tissue. TargetScan analysis revealed that EPO is a predicted target gene of miR-122 and this regulatory relationship was verified by dual-luciferase reporter assay. Overexpression of miR-122 in primary bovine mammary cells led to down-regulation of EPO and also the JAK-STAT signalling pathway genes EPOR, JAK2, STAT5A and STAT5B. Accordingly, the mRNA abundance of BCL-2, a downstream gene in the JAK-STAT signalling pathway was significantly down-regulated. Under conditions of miR-122 inhibition, EPO, EPOR, JAK2, STAT5A and STAT5B were up-regulated. These results demonstrated that, through its action on EPO, miR-122 may play an important role in S. agalactiae-induced mastitis by regulating the JAK-STAT signalling pathway.HighlightsUnderstanding the relationship among miR-122, EPO and the JAK-STAT pathway would be helpful in developing new strategies for controlling mastitis. Understanding the relationship among miR-122, EPO and the JAK-STAT pathway would be helpful in developing new strategies for controlling mastitis.

MicroRNA (miRNA) are single-stranded non-coding RNA of approximately 22 nucleotides estimated to regulate 60% of human and animal genes some of which are associated with cell differentiation, signal transduction and immune processes. Streptococcus agalactiae (S. agalactiae)-induced bovine mastitis is associated with up-regulation of miR-122 and down-regulation of erythropoietin (EPO) abundance in mammary gland tissue. TargetScan analysis revealed that EPO is a predicted target gene of miR-122 and this regulatory relationship was verified by dual-luciferase reporter assay. Overexpression of miR-122 in primary bovine mammary cells led to down-regulation of EPO and also the JAK-STAT signalling pathway genes EPOR, JAK2, STAT5A and STAT5B. Accordingly, the mRNA abundance of BCL-2, a downstream gene in the JAK-STAT signalling pathway was significantly down-regulated. Under conditions of miR-122 inhibition, EPO, EPOR, JAK2, STAT5A and STAT5B were up-regulated. These results demonstrated that, through its action on EPO, miR-122 may play an important role in S. agalactiae-induced mastitis by regulating the JAK-STAT signalling pathway.HighlightsUnderstanding the relationship among miR-122, EPO and the JAK-STAT pathway would be helpful in developing new strategies for controlling mastitis. Understanding the relationship among miR-122, EPO and the JAK-STAT pathway would be helpful in developing new strategies for controlling mastitis.

Figure S2. Alignment of full-length of BLV env gene nucleotide sequences (S2-A: 6–769 bp; S2-B: 777–1543 bp) between sequences obtained in this study together with 10 reference sequences and all Chinese sequences available in the GenBank database. Strains identified in this study are in red (cluster into genotype 4) and green (cluster into genotype 6). Numbers above the sequences are nucleotide number indicated by the env gene of AB934282. The countries of the strains are marked with abbreviations in parentheses to the right of the GenBank accession numbers. Dots indicate nucleotides identical to the reference sequences. The mark above the square frames indicate unique mutations for our isolates of BLV genotype 4 (▼) and genotype 6 (▽). The BLV reference strains from GenBank have accession numbers AF933703 (G1), AF257515 (G2), EF065647 (G3), JN695878 (G4), EF065635 (G5), LC080656 (G6), KF801457 (G7), JQ675759 (G8), LC080659 (G9), and LC154066 (G10). The Chinese sequences available in the GenBank database have the accession numbers: MH040198-MH040203, MH040205, MH040207-MH040209, MF574053-MF574068. The seven Chinese strains from this study have the accession numbers MK820044 and MK840875-MK840880. JPN = Japan; CHN = China; BRA = Brazil; ARG = Argentina; USA = United States of America; RUS = Russia; CRC = Costa Rica; PAR = Paraguay; MDA = Moldova; BOL = Bolivia; MYA = Myanmar. (ZIP 5157 kb)

Figure S2. Alignment of full-length of BLV env gene nucleotide sequences (S2-A: 6–769 bp; S2-B: 777–1543 bp) between sequences obtained in this study together with 10 reference sequences and all Chinese sequences available in the GenBank database. Strains identified in this study are in red (cluster into genotype 4) and green (cluster into genotype 6). Numbers above the sequences are nucleotide number indicated by the env gene of AB934282. The countries of the strains are marked with abbreviations in parentheses to the right of the GenBank accession numbers. Dots indicate nucleotides identical to the reference sequences. The mark above the square frames indicate unique mutations for our isolates of BLV genotype 4 (▼) and genotype 6 (▽). The BLV reference strains from GenBank have accession numbers AF933703 (G1), AF257515 (G2), EF065647 (G3), JN695878 (G4), EF065635 (G5), LC080656 (G6), KF801457 (G7), JQ675759 (G8), LC080659 (G9), and LC154066 (G10). The Chinese sequences available in the GenBank database have the accession numbers: MH040198-MH040203, MH040205, MH040207-MH040209, MF574053-MF574068. The seven Chinese strains from this study have the accession numbers MK820044 and MK840875-MK840880. JPN = Japan; CHN = China; BRA = Brazil; ARG = Argentina; USA = United States of America; RUS = Russia; CRC = Costa Rica; PAR = Paraguay; MDA = Moldova; BOL = Bolivia; MYA = Myanmar. (ZIP 5157 kb)