TGF? Induces Cell Cycle Arrest By Rewriting RNA M~6A Epitranscriptome

TGF?signaling pathway is critical for cell division,differentiation,and apoptosis.Its aberrant regulation may result in severe diseases like cancer.However,the association of m~6A modification with TGF?signaling pathway remains elusive.Through pre-experiment,we find that the total m~6A level of breast cancer cell which exhibits estrogen receptor positive is up-regulated after TGF?treatment.By further detecting the expression level of methyltransferases using western blot,we find that the expression of WTAP and METTL3 are increased.The RNA-Seq data indicate that TGF?rewriting the transcriptome of MCF7 cells,the up-regulated RNA enrich in cell cycle pathway,suggesting that TGF?influences cell cycle.To investigate the related function of m~6A modification in TGF?signaling pathway,we carry out RNA methylation sequencing(MeRIP-Seq).The traditional MeRIP-Seq requires hundreds of micrograms of total RNA to extract mRNA.We first optimize the traditional MeRIP-Seq method to remove ribosomal RNA with single stranded DNA probes and to determine the optimal conditions for RNA fragmentation,thereby reducing the total RNA consumption.The optimized MeRIP-Seq shows that the method can detect m~6A site in transcriptome accurately.By using the optimized MeRIP-Seq method,we map the m~6A profile of MCF7 cells induced by TGF?.Data showed that in TGF?-treated MCF7cells,m~6A epitranscriptome is reprogramming after TGF?treatment,we identify a subset of genes with up-regulated m~6A modification,which enrich in the TGF?signaling pathway.Western blot result shows that the phosphorylation level of core transcription factor SMAD2 and SMAD3 of TGF?signaling pathway is impaired after knockdown of WTAP which is methyltransferase,suggesting that m~6A is involved in the activity of TGF?signaling pathway.After TGF?treatment,the m~6A modification level and the mRNA level of Jun B,a cell cycle inhibitor,are both increased,the western blot result shows that JunB is positively regulated by m~6A directly.In addition,after knockdown of WTAP,the cell cycle markers such as CDK4 and p21 no long changes with TGF?treatment.At the cellular level,flow cytometry shows that the cell cycle arrest of breast cancer cells MCF7caused by TGF?rescue after knocking down WTAP.CCK8 result demonstrates that in MCF7 cells of WTAP knockdown,cell proliferation inhibition caused by TGF?is impaired.It suggests that WTAP is an important factor causing breast cancer cells MCF7 proliferation inhibition.From the analysis result on the TCGA database,low WTAP expression is associated with poor prognosis of breast cancer patients.Collectively,by using an optimized MeRIP-Seq method,we preliminarily investigate the molecular mechanism of TGF?-induced breast cancer cell cycle arrest.The results show that in vitro stimulation of TGF?can cause breast cancer cell cycle arrest.The mechanism of cell cycle arrest caused by TGF?in vitro is mainly methyltransferases WTAP-dependent,WTAP causes cell cycle arrest in breast cancer cells by mediating the rewriting of the m~6A epitranscriptome,which will cause proliferation inhibition.Our results provide new insight for explaining the mechanism of cell cycle arrest caused by TGF?.