Bilogical Function And Expression Hepatic Regulation Mechanism Of MTTPL Gene In Lipid Metabolism

In mammals,VLDL assembly was performed in conjunction with molecules such as Apolipoprotein B(Apo B)and Microsomal Triglyceride Transfer Protein(MTTP),MTTP is the limiting factor of VLDL assembly and secretion.However,in chickens,significant changes in VLDL levels were not associated with MTTP expression levels,suggesting that MTTP is not a key factor in VLDL assembly and secretion in chicken liver.Therefore,the mechanism of VLDL assembly and secretion in chicken liver remains unclear.In our previous study,we found that there is a microsomal triglyceride transporter(MTTP-Like,MTTPL)in chicken genome.This gene has similar nucleotide sequences with mammalian MTTP gene,and its corresponding protein has conserved motif and functional domain with mammalian MTTP gene.Moreover,the expression level of MTTPL in chicken liver is significantly positively correlated with VLDL concentration,and is regulated by estrogen in a dose-dependent manner.However,the role of chicken MTTPL in liver lipid metabolism and its regulatory mechanism remain unclear.In this study,the chicken MTTPL promoter region sequence deletion expression vector was constructed,and the core regulatory region of the MTTPL promoter was identified by bioinformatics analysis combined with dual luciferase reporting detection system.Overexpression,interference and dual luciferase tests were used to identify key transcription factors that bind to core regulatory regions and are regulated by estrogen.The full length sequence of MTTPL gene 3′UTR was cloned by 3′RACE technique,and the miRNA binding to MTTPL gene 3′UTR was identified by bioinformatics analysis combined with dual luciferase reporting system.Overexpression assay combined with estrogen treatment demonstrated the mechanism of miRNA targeting regulation of MTTPL expression.Then,the adeno-associated virus interference vector of MTTPL gene was constructed,and the expression of MTTPL was induced by exogenous estrogen.The role of chicken MTTPL in liver lipid metabolism was further verified through the strategy of function acquisition and deletion.Based on the above three studies,the role of chicken MTTPL gene in liver lipid metabolism and its expression regulation mechanism were systematically clarified.The main results are as follows:1.The results showed that-236bp~+1bp was the core regulatory region of the MTTPL promoter,and the transcription factors PPARα and RXRα that might bind to the MTTPL promoter were screened.Overexpression of PPARα and RXRα significantly increased the expression level of MTTPL m RNA,while interference with PPARα and RXRαsignificantly decreased the expression level of MTTPL.The double luciferase reporting assay system verified that overexpression of PPARα enhanced luciferase activity in the promoter core region,while overexpression of RXRα had no effect on the luciferase activity in the promoter core region.Both PPARα and RXRα are positively regulated by estrogen.In conclusion,PPARα directly regulates the expression of MTTPL gene by binding to the core regulatory region of MTTPL gene promoter,while RXRα may indirectly regulate the expression of MTTPL through other ways.2.First,the full length sequence of MTTPL 3’UTR was 1555 bp by RACE technique,and 166 bp unknown sequence was obtained.Subsequently,bioinformatics was used to predict and screen miRNA,miR-7439-3p,which was targeted to interact with MTTPL gene,and the targeting relationship between MTTPL and miR-7439-3p was verified by dual luciferase reporting system.miR-7439-3p was expressed in liver,heart,ovary,kidney,pancreas,spleen,duodenum,pectoral muscle and leg muscle of Lu’s green shell laying hens.In liver,the expression level at 10 w was significantly higher than that at30 w during laying peak(P<0.05).When miR-7439-3p was overexpressed in LMH cell lines,the expression level of MTTPL was significantly decreased,and miR-7439-3p was negatively regulated by estrogen,which was contrary to the expression trend of MTTPL and consistent with the expected results.Overexpression of miR-7439-3p also significantly decreased the expressions of fatty acid,TC synthesisation-related genes and lipid transporter genes,and significantly increased the TG synthesisation-related genes,thus reducing the contents of TC and VLDL and increasing the contents of TG in cells.3.At the individual level,the interference of MTTPL in chicken liver significantly reduced the contents of TC,TG and VLDL secreted into plasma.HE/ oil red staining tests on liver sections showed that the accumulation of liver lipids increased significantly,and the inflammatory response of liver was increased to a certain extent,which verified the important role of MTTPL in lipid transport.In summary,this study explored the regulatory mechanism of MTTPL expression at the transcriptional and post-transcriptional levels respectively,and verified the role of MTTPL in lipid metabolism at the individual level.