| Background:The liver is the largest organ of material metabolism,energy metabolism,and detoxification.Recently,liver diseases have become one of the most common human diseases,such as viral hepatitis,alcoholic or non-alcoholic fatty liver disease,cirrhosis,as well as liver cancer resulting in heave family and social-economic burden.Therefore,an in-depth understanding of liver pathophysiology helps to provide a theoretical basis for the treatment of liver-related diseases.The liver originates from the endoderm and differentiates into hepatoblasts through the stages of the liver diverticulum and liver bud.The hepatoblasts proliferate and differentiate into hepatocytes and cholangiocytes.The postnatal liver also experiences hepatocyte proliferation and functional maturation in 2 to 3 weeks after birth.The liver is an organ with strong regenerate ability.Almost all the hepatocytes in a mature liver are in quiescent;however,when liver injury leads to the loss of hepatocytes,a large proportion of the remaining hepatocytes are activated to enter into the cell cycle and proliferate rapidly and synchronously,which compensatively restores the liver to its original cell number,liver mass,and function.Abnormal liver regeneration is usually associated with liver functional decompensation,fatty liver,cirrhosis,and even hepatocellular carcinoma(HCC).Both liver development and liver regeneration are involved in the proliferation of hepatocytes,which are precisely regulated by a great number of genes and signaling pathways.Any dysregulation in these genes or signaling pathways can result in aberrant liver physiology,histology,and function.As the most common and conservative RNA modification in eukaryotes,N6methyladenine(m~6A)has become a research hotspot in recent years.m~6A refers to the methylation of the nitrogen atom at position 6 of RNA adenine.Similar to DNA methylation,m~6A modification is also dynamically reversible.Methyltransferase,also known as the Writer,catalyzes the formation of m~6A;demethylase,also known as the Eraser,removes m~6A;and Reader,the m~6A binding protein,guides the interaction between m~6A methylated RNA and other functional proteins,thus regulating RNA splicing,exporting,stabilization and translation.m~6A modification regulates almost every stage of RNA metabolism.Mettl3,Mettl14,and Wtap are the major methyltransferases of m~6A.They combine to form the methyltransferase complex and catalyze the modification of RNA by m~6A.m~6A modification mainly affects the stability and/or translation of the key transcripts,increases or suppresses the protein expression level,thus participates in the regulation of related signaling pathways and plays a role in biological processes.However,there are still some controversies about the biological function of m~6A.For example,Mettl3 mediated m~6A modification can promote or inhibit the self-renewal and differentiation of mouse embryonic stem cells(ESCs);Mettl3 or Mettl14mediated m~6A modification can promote or inhibit the growth and migration of HCC cell lines,respectively.Recently,more and more proteins have been found to be related to m~6A modification,which also indicates the complexity of m~6A modification.The research on the function of m~6A modification is diverse but contradictory.During the process of liver development and liver regeneration,the expression of a large number of genes has changed,and the function of m~6A modification which regulates gene expression in liver development and liver regeneration is still unknown.Therefore,in this study we specifically and respectively knocked out the m~6A methyltransferases Mettl3,Mettl14,and Wtap in hepatocytes and explored their roles in mouse liver development and regeneration,as well as the underlying molecular mechanisms.Methods:Part one:(1)Hepatocyte-specific Mettl3,Mettl14,or Wtap knockout mice(refers to Mettl3-KO,Mettl14-KO,and Wtap-KO,respectively)were constructed by Albumin Cre/Lox P system.(2)The survival and liver histological morphology of the knockout mice were observed;the glucose and lipid metabolism was detected by oil red O and PAS staining,respectively;the proliferation markers Ki67 and p H3S10 were detected by immunohistochemistry staining.(3)Transcriptome sequencing and m~6A sequencing were performed to detect the change of m~6A modification map and transcriptome in KO mice,and the target genes related to liver development phenotype were screened.Part two:(1)We performed 70%partial hepatectomy(PHx)in wild-type mice to establish a liver regeneration model.The changes of m~6A methyltransferase in the process of liver regeneration were detected by q PCR and Western blot,and the changes of m~6A modification map during liver regeneration were described by m~6A sequencing.(2)The 70%PHx liver regeneration model of knockout mice was established.The liver remodeling was observed by measuring liver weight to body weight ratio and H&E staining;the proliferation indexes Brd U and Ki67 were detected by immunohistochemistry staining;the expression of cell cycle markers was detected by western blot;(3)m~6A sequencing was performed to detect the changes of m~6A modification in knockout mice during liver regeneration.Part three:(1)The morphology of endoplasmic reticulum in Mettl14-KO mice was observed by transmission electron microscope.The expression of endoplasmic reticulum stress markers,apoptosis and proliferation markers were detected by western blot.(2)The si RNA was used to knockdown Mettl14 in normal mouse hepatocyte line AML12,the endoplasmic reticulum stress,apoptosis and proliferation were verified in vitro.(3)q PCR and RIP-q PCR were performed to explore the stability and translation efficiency in Mettl14-KO mice hepatocytes.(4)The chemical chaperone tauroursodeoxycholate(TUDCA)was used to alleviate endoplasmic reticulum stress,and hepatocyte necrosis and proliferation indexes were detected.Results:Part One:(1)Mettl3,Mettl14,or Wtap hepatocyte-specific knockout mice were successfully constructed.In these transgenic mice,Mettl3,Mettl14 or Wtap were efficiently and stably knocked out,respectively.(2)Loss of Mettl3 or m Mettl14 did not affect liver development in mice.(3)Wtap knockout mice died soon after birth with aberrant liver development in Wtap-KO mice.(4)Transcriptome and m~6A sequencing showed that the loss of Wtap in the liver decreased the expression of hepatic nuclear factor 4α(HNF4α)in an m~6A-dependent way.Part two:(1)The expression of Mettl3 and Mettl14 peaked at 3 h and 6 h after PHx during liver regeneration,respectively.While the expression of Wtap remained unchanged.m~6A sequencing showed that the dynamic changes of m~6A modification during liver regeneration,and suggested that the increased level of m~6A modification may play a role in the early phase of liver regeneration.(2)Mettl3 knockout did not affect liver regeneration in mice;however,loss of Mettl14 inhibited hepatocyte proliferation and arrested hepatocytes in G0/G1 phase;Also,Mettl14 knockout resulted in hepatocyte necrosis in the regenerating liver.(3)The results of m~6A sequencing showed that loss of Mettl14 resulted in the decrease of m~6A modification level of most genes,and the genes with decreased m~6A modification level were mainly enriched in the transcripts of polypeptide-chain processing proteins.Part three:(1)Loss of Mettl14 led to excessive endoplasmic reticulum stress in regenerating hepatocytes;Mettl14 knockdown also led to excessive endoplasmic reticulum stress in vitro,thereby inhibiting proliferation and promoting apoptosis.(2)The deletion of Mettl14 decreased the m~6A level on the transcripts of polypeptide-chain processing proteins,such as Hsp90b1,Lman1,Stt3a,Erp29,and P4hb,therefore promoted the degradation and inhibited translation efficiency.(3)Hepatocyte necrosis in Mettl14-KO mice during liver regeneration was effectively reduced after TUDCA injection.Conclusions:In the process of liver development,compared with Mettl3 and Mettl14,loss of Wtap down-regulated the expression of HNF4α,which is the core factor of the liver,resulting in the loss of hepatocyte metabolism and maturation.Wtap mediated m~6A is an indispensable regulatory factor in the liver development of weight.During liver regeneration,loss of Mettl14,rather than Mettl3,disrupted hepatocyte proliferation and promoted hepatocyte necrosis.Mechanically,Mettl14 mediated m~6A promotes the stability and translation of the transcripts of polypeptide-chain processing proteins,thus maintains the homeostasis of the endoplasmic reticulum,and promotes normal liver regeneration. |
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