Expression Analyses of m6A Methylase Genes in Bovine Adipogenesis

doi:10.13560/j.cnki.biotech.bull.1985.2021-1231

Abstract

Abstract:

The aim of this study is to investigate the mRNA expressions of relevant genes,including METTL3,METTL14,WTAP for m⁶A methyltransferase,and FTO and ALKBH5 for m⁶A demethylaes,in bovine various tissues and in the proliferation and differentiation of preadipocytes,aiming to provide a reference for clarifying the role and mechanism of m⁶A modification in adipogenesis. Real-time quantitative PCR（RT-qPCR）was used to detect the mRNA expressions of m⁶A methylases in the various tissues of newborn and adult Qingchuan beef cattle. Then,CCK-8 treatment and oil-red O staining were adapted to detect the growth curves of the preadipocytes cells and lipid droplet formation during adipogenic differentiation. The 5 genes expressed lowly in skeletal muscles,and highly in rest tissues,while the mRNA expressions of them in the adipose tissue of adult cattle was significantly higher than that of newborn cattle（P < 0.05）. The isolated preadipocytes had a good growth state and adipogenic differentiation was accordance with the general pattern. The expressions of METTL3,METTL14 and WTAP decreased in the early stage of bovine preadipocytes proliferation,then increased and remained at low level. The expression of FTO showed a slow upward trend. The temporal expression of ALKBH5 increased in the early stage,decreased in the metaphase,and increased continuously after the day 4（P < 0.01）. The expression patterns of METTL3,METTL14 and FTO were similar in the adipogenic differentiation of bovine preadipocytes,which decreased in the early stage of differentiation,and highly expressed in the metaphase,then decreased. The expressions of WTAP and ALKBH5 increased with the progress of differentiation. Our results indicate that m⁶A modification plays a potentially important role in fat development and deposition of Qinchuan cattle,and m⁶A methylases may regulate the lipogenesis of bovine preadipocytes in vitro.

Key words: N⁶-methyladenosine（m⁶A）, m⁶A methylases, cattle, preadipocytes, cell proliferation, adipogenic differentiation

YANG Xin-ran, WANG Jian-fang, MA Xin-hao, ZAN Lin-sen. Expression Analyses of m⁶A Methylase Genes in Bovine Adipogenesis[J]. Biotechnology Bulletin, 2022, 38(7): 70-79.

Figures/Tables 6

Table 1 Primers for RT-qPCR

基因名称 Gene name	引物序列 Primer sequence（5'-3'）	产物长度 Product length/bp
METTL3	F：TCGAAAGCTGCACTTCAGAC	199
METTL3	R：TCCAACGCTCTGTGTAAGGG	199
METTL14	F：TGACATCAGAGAACTGACACCC	198
METTL14	R：AGGTCCAATCCTTCCCCAGA	198
WTAP	F：GCCTGGAAGTTTACGCCTGA	176
WTAP	R：TCCTGACTGCTTTTAAGCTCCT	176
FTO	F：AGCAGCGTACAACGTCACTT	193
FTO	R：AGGGTCGTCCTCACTTTCCT	193
ALKBH5	F：TACTTCTTCGGCGAGGGCTA	191
ALKBH5	R：TGGTAGTCGTTGATGACGGC	191
PPARγ	F：TGAAGAGCCTTCCAACTCCC	117
PPARγ	R：GTCCTCCGGAAGAAACCCTTG	117
C/EBPα	F：ATCTGCGAACACGAGACG	73
C/EBPα	R：CCAGGAACTCGTCGTTGAA	73
C/EBPβ	F：TTCCTCTCCGACCTCTTCTC	79
C/EBPβ	R：CCAGACTCACGTAGCCGTACT	79
FABP4	F：TGAGATTTCCTTCAAATTGGG	101
FABP4	R：CTTGTACCAGAGCACCTTCATC	101
GAPDH	F：AGTTCAACGGCACAGTCAAGG	124
GAPDH	R：ACCACATACTCAGCACCAGCA	124
β-actin	F：CATCGGCAATGAGCGGTTCC	147
β-actin	R：ACCGTGTTGGCGTAGAGGTC	147

Fig. 1 Expressions of m6A methylation-related genes in different tissues of Qinchuan beef cattle A-E：Tissue mRNA expression profiles of METTL3（A）,METTL14（B）,WTAP（C）,FTO（D）and ALKBH5（E）in newborn Qinchuan beef cattle. F-J：Tissue mRNA expression profiles of METTL3（F）,METTL14（G）,WTAP（H）,FTO（I）and ALKBH5（J）in adult Qinchuan beef cattle. K：Comparison of expression levels of METTL3,METTL14,WTAP,FTO and ALKBH5 in the adipose tissues of newborn and adult Qinchuan beef cattle. A-J：*P < 0.05,** P < 0.01,other tissues versus heart;# P < 0.05,##P < 0.01,fat versus skeletal muscles;no superscripts indicate no significant difference（P > 0.05）. K：*P < 0.05,** P < 0.01

Fig. 2 Morphology and growth curve of preadipocytes from Qinchuan beef cattle in vitroA：Phenotypic observation of preadipocytes proliferation in Qinchuan beef cattle at different growth points（scale bar：200 μm）. B：Growth curve of preadipocytes in Qinchuan beef cattle

Fig. 3 Detection of preadipocytes adipogenic differentiation in Qinchuan beef cattle A：Observation of lipid droplet formation on day 3,6,9 and 12 of culture in differentiation medium after the induction of adipogenic differentiation（scale bar：20 μm）;B：Relative mRNA expression of adipogenesis-related genes（PPARγ,C/EBPα,C/EBPβ and FABP4）during bovine preadipocytes differentiation. Different capital letters indicate very significant differences（P < 0.01）,different lowercase letters indicate significant differences（P < 0.05）,and the same letters indicate no significant differences（P > 0.05）. The same below

Fig. 4 Temporal expression profile of m6A methylation-related genes during preadipocytes proliferation

Fig. 5 Temporal expression profile of m6A methylation-related genes during preadipocytes differentiation

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Expression Analyses of m⁶A Methylase Genes in Bovine Adipogenesis

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