METTL3 Regulates The Pluripotency Of Porcine Pluripotent Stem Cells

Mammalian RNAs have more than 100 post-transcriptional modifications,among which the methylation modification(N~6-methyladenosine,m6A)of adenine is the most common modification form of eukaryotic mRNA.It is shown that m6A influences the transcriptional level of pluripotent factors by regulating the stability of mRNA,and can regulate the eporcineenetic state and cellular reprogramming of mouse pluripotent stem cells.METTL3 is an important component of m6A methyltransferase complex,which plays an important role in the process of cell reprogramming,embryonic stem cells and induced pluripotent stem cells.The purpose of this study was to reveal the expression pattern of porcine METTL3,to construct the overexpression and knockdown vectors of METTL3,and to study the regulation effect of its expression changes on the pluripotent genes of porcine stem cells.By adding m6A methylation inhibitor cycloleucine in the culture medium of porcines stem cells,it was found that the results were consistent with that of interfering METTL3 expression.In addition,piPSCs cultured in medium containing 10 mmol/L cycloleucine for 48 h exhibited the similar result as that interfering METTL3.These findings set the stage for the optimization of piPS culture condition and further study on the roles of m~6A in piPSCs.The main results obtained in this study are as follows:1.Expression profile and cloning of porcine METTL3We detected the expression profile of METTL3 in different porcine tissues and organs(stomach,skin,heart,fat,muscle,small intestine,colon,uterus,liver,lung,spleen,kidney,brain),somatic cells(PEF,PK-15),pluripotent stem cells(DOX-iPS,PS11,PS23)by RT-PCR.The results showed that METTL3 was widely expressed in different porcine tissues,organs and cells.To identify the function of METTL3,we cloned a 1 859-bp coding sequence of METTL3,constructed the overexpression vector pEGFP-METTL3,detected the expression of EGFP-METTL3 fusion protein by western blot,and immunofluorescence staining result showed that the EGFP-METTL3 fusion protein was located in the nucleus.2.METTL3 regulates the pluripotency of porcine pluripotent stem cellsAfter cloning the coding sequence of porcine METTL3,we synthesized a shRNA against METTL3,and constructed the overexpression and knockdown vectors of METTL3.We used these vectors to transfect HEK293T cells,and infected porcine pluripotent stem cells.The results showed that when overexpressing METTL3 in piPSCs,the cell morphology did not change significantly,the activity of alkaline phosphatase was decreased,and the expression levels of the pluripotent genes(OCT4 and LIN28A)were not increased.After knocking down the expression of METTL3,porcine pluripotent stem cells became na?ve-like morphology,and the activity of alkaline phosphatase was increased,and the expression levels of pluripotent genes(NANOG,OCT4 and LIN28A)were significantly elevated.3.Cycloleucine regulates the pluripotency of porcine pluripotent stem cellsBy adding 10 mmol/L and 20 mmol/L cycloleucine respectively in DOX-iPS cells medium,the results showed that DOX-iPS colony morphology cultured in 20 mmol/L cycloleucine medium disappeared gradually and grew slowly.Therefore,we prepared 2 mmol/L,4 mmol/L,and 10 mmol/L cycloleucine mediums respectively to culture DOX-iPS cells 24 h and 48 h.According to the results,cells morphology cultured for 24 h and 48 h had not been a significant change,but the alkaline phosphatase activity of cells in 10 mmol/L for 48 h was better than the other groups,and the expression levels of pluripotent genes(NANOG,OCT4 and LIN28A)were much higher than the other groups.This indicated that the addition of a certain concentration(10mmol/L)of cycloleucine can significantly improve the expression levels of porcine endogenous pluripotent genes.To sum up,this study revealed the expression pattern of porcine METTL3,cloned coding sequence of porcine METTL3 gene,constructed the overexpression and knockdown vectors of porcine METTL3,confirmed the regulation effect of METTL3 on pluripotent genes in porcine pluripotent stem cells.This meaned that knocking down the expression of METTL3 or adding10 mmol/L cycloleucine in piPSCs medium can significantly improve the expression levels of porcine endogenous pluripotent stem cells genes.