Study On Expression Characteristics And Regulation Mechanism Of Cholesterol Synthesis Pathway Related Genes In Chicken Liver

The main organ for cholesterol synthesis is the liver.Especially during the laying period,the liver synthesizes a large amount of cholesterol every day,and the cholesterol is encapsulated by VLDL,and then transported to the developing oocyte to form egg yolk.Although the process of chicken liver cholesterol synthesis has received a lot of attention,so far there has been no systematic study on this issue.This study used Lushi green shell hens as research objects.In this study,the mechanism of regulation in hepatic cholesterol synthesis in chicken laying period was systematically studied by using modern molecular biology methods.The molecular regulation mechanism of liver cholesterol synthesis in chicken laying period was preliminarily clarified,which enriched the theory of chicken lipid metabolism regulation and provided a new idea for improving chicken production performance and improving meat quality.In this study,based on the analysis of RNA-seq analysis between the early stage of egg production(20 weeks old)and the stage of peak egg production(30 weeks old)in the liver tissue in Lushi green shell hen.Firstly,the expression changes of genes which related to liver cholesterol synthesis were analyzed and a new gene involved in chicken liver cholesterol synthesis was identified;Secondly,The CDS of this new gene was subsequently cloned,and its characteristics and functions were studied.Thirdly,the estrogen-regulated genes were identified by integration analysis of the existing ChIP-seq database,estrogen-treated chicken liver tissue RNA-seq database and cholesterol synthesis pathway-related genes,and verified by in vivo and in vitro experiments.Fourth,integrating and analyzing estrogen-treated chicken liver tissue miRNA-seq data with chicken liver cholesterol synthesis pathway-related genes,screening for miRNA-regulated target genes.The dual luciferase reporter system was used to verity its targeting relationship.The effect of miRNA on intracellular cholesterol synthesis was analyzed by miRNA's overexpression.Finally,animal models and cell models were used to study the effects of different nutrient levels on cholesterol synthesis in chicken liver.The specific research results are as follows:Part ? Identification of chicken cholesterol synthesis related genesThere is no systematic study on the molecular pathway of cholesterol synthesis in laying hens so far.Referring to the mammalian cholesterol synthesis pathway-related genes.Combined with the RNA-seq data of the liver of the Lushi green-shelled hens in the early stage of laying(20 weeks old)and the peak of laying(30 weeks old),quantitative PCR was used to analysis of key genes(including HMGCR,MVK,PMVK,MVD,IDI1,FDPS,FDFT1,SQLE,LSS,CYP51,GGPS1,LBR,MSMO1,NSDHL,HSD17B7,SC5D,DHCR7,DHCR24)in the cholesterol synthesis pathway.Compared with the pre-laying stage(20 weeks old),these genes were significantly increased in the liver at the peak of egg production.Among them,the expression level of HMGCR which be related to cholesterol synthesis rate-limiting increased with the progress of sexual maturation and it was consistent with the trend of chicken egg production rate curve.In addition,the EBP gene which exist in the mammalian cholesterol synthesis pathway is not present in the chicken Liver RNA-seq data of Lushi green shell hens during pre-laying(20 weeks old)and peak egg production(30 weeks old).By comparing genomics methods,a new gene which be related to cholesterol synthesis in chicken was discovered,the gene is named EBP-like.Part ? The role of EBP-like gene in cholesterol synthesis in chicken liverThe EBP-encoded protein is an important enzyme in the cholesterol synthesis pathway and can catalyze the conversion of the 8-ene isomer to the 7-ene isomer.However,there is no EBP gene in the chicken liver tissue transcriptome database.Using the human EBP protein sequence to query the chicken-related genes,it was found that there is a similar EBP genes in the chicken genome,named EBP-like gene.The chicken EBP-like gene sequence was firstly cloned,and the functional domain of chicken EBPlike protein was analyzed by online software SMART.The evolutionary tree of chicken EBP-like gene was constructed.Then,the EBP-like gene in chicken liver was analyzed in different tissues(lung,liver,spleen,kidney,glandular stomach,pectoral muscle,pancreas,duodenum,leg muscles,ovary)and different developmental stages(5,15,20,30,35 and 50 weeks old)by qRT-PCR.The method of overexpressing and interfering genes was used to analyze the role of EBP-like gene in cholesterol synthesis in chicken liver.The results showed that chicken EBP-like has a typical domain which is similar with human EBP.Chicken EBP-like gene is relatively conserved during species evolution.Chicken EBP-like gene is widely expressed in different tissues,but the expression abundance in liver tissue is highest,while the expression level of EBP-like gene in other tissues is very low.Overexpression of chicken EBP-like gene has no effect on the expression of upstream gene HMGCR and downstream genes SC5D,DHCR7 and DHCR24 in cholesterol synthesis pathway.(P<0.05),and the intracellular cholesterol content between different groups had no significant effect(P>0.05),but interference with chicken EBP-like gene can lead to a significant decrease in the expression levels of downstream genes such as SC5D,DHCR24,DHCR7 and intracellular cholesterol(P<0.05).Part ? Effects of estrogen on cholesterol synthesis in chicken liverIt is generally believed that liver cholesterol metabolism in chicken laying is regulated by estrogen.After 12 hours of estrogen treatment(17?-estradiol/body weight=2mg/kg),the cholesterol content in the serum of the Lushi green shell hens was significantly increased(P<0.05).Compared with the control group,the expression levels of HMGCR,CYP51A,SQLE,NSDHL,EBP-like,MVD,MSMO1 LSS.HSD17B7,FDPS,FDFT1,DHCR24 and DHCR7 were significantly increased in the estrogentreated group(P<0.05).The expression levels of SC5D,PMVK,MVK,IDI1 and LBR genes were not significantly different(P>0.05),but the expression level of GGPS1 gene was significantly down-regulated(P<0.05).The ERE sequence prediction was performed on the sequence of 5000 bp upstream of the transcription initiation site of the up-regulated gene after estrogen treatment.It was found that 10 genes(including HMGCR,CYP51,DHCR7,DHCR24,EBP-like,FDPS,SQLE,HSD17B7,LSS and MVD)were existed ERE sequence.An atypical ERE locus was combined with the previous ChIP-seq(estrogen receptor alpha chromatin immunoprecipitation)database of the subject group to conclude that estrogen directly regulates the expression of the SQLE gene through the ERE site.Further in vivo and in vitro experiments show that estrogen regulates the expression of the SQLE gene through the ERa receptor.Part ? Estrogen regulates cholesterol synthesis through miRNAAfter estrogen treatment of chicken,13 genes related to cholesterol synthesis in the liver are up-regulated,but only one of the genes is determined to be directly regulated by estrogen,and other genes may be regulated by estrogen through miRNA,thereby regulating cholesterol synthesis.Bioinformatics methods were used to predict miRNAs which be related to cholesterol synthesis.The miRNA-seq database of estrogen-treated chicken liver tissue obtained in the previous experiment in our group was used to analyze the miRNA-related target genes.Combined analysis of the above data and the genes related to cholesterol synthesis,identifying the possible cholesterol synthesis genes which be regulated by estrogen through miRNA.Two key enzymes on the cholesterol synthesis gene pathway were selected for miRNA-mRNA targeting relationship validation.One is the rate-limiting enzyme in the cholesterol synthesis pathway,and the other is the last enzyme in the cholesterol synthesis pathway.Further use of dual fluorescence rate enzyme reporter system to verify miRNA-mRNA targeting relationships.MiRNA overexpression experiments were performed in chicken liver primary cells.The results showed that estrogen can significantly down-regulate the expression of miR-27b-3p and miR-219b3p(P<0.05)' the sequence of the 3'UTR of HMGCR gene and the miR-27b-3p binding site are highly conserved in different species;The sequence in the 3'UTR of the DHCR7 gene is not conserved with the miR-219b binding site in different species.The expression of miR-27b-3p was negatively correlated with HMGCR in the liver of laying hens at different ages(5 weeks old,15 weeks old,20 weeks old and 30 weeks old).,the same as miR-219b-3p and DHCR7 gene.Analysis of different tissue expression profiles(lung,liver,spleen,kidney,glandular stomach,pectoral muscle,pancreas,duodenum,leg muscles,ovary)showed that HMGCR/DHCR7 and miR-27b-3p,miR219b-3p were widely expressed in tissues,but the expression abundance of HMGCR and DHCR7 genes is highest in the liver,and the expression abundance of miR-27b-3p and miR-219b-3p is the lowest in the liver.The miR-27b-3p mimics and HMGCR 3'UTR wild-type recombinant vectors co-transfected DF1 cells can decreased luciferase activity,and miR-27b-3p mimics significantly decreased intracellular HMGCR gene expression(P<0.05).There was no significant difference in internal cholesterol levels(P>0.05),This suggests that estrogen may mediate HMGCR expression by regulating the expression of miR-27b-3p;the reason for the insignificant difference in intracellular cholesterol levels may be due to the possible presence of other target genes in miR-27b3p,which have a multi-faceted regulation of cholesterol levels.The miR-219b-3p mimics and DHCR7 3'UTR wild-type recombinant vectors co-transfected DF1 cells reduced luciferase activity,and miR-219b-3p mimics significantly decreased intracellular DHCR7 gene expression(P<0.05).The internal cholesterol level was significantly decreased(P<0.05).This suggests that estrogen may mediate DHCR7 expression by regulating the expression of miR-219b-3p,thereby reducing intracellular cholesterol levels.Part ? Effects of oil on cholesterol synthesisTaking Lushi green shell hens as the research object.The original basal diets were separately added 0%and 4%of soybean oil and was fed chicken for 30 days,and serum and liver tissues were collected for analysis.The results showed that the serum cholesterol level of the 4%soybean oil group was not significantly with the control group(P>0.05).There was no significant difference in liver index between the different groups(P>0.05).Hepatic HE staining showed that the liver cells were tightly arranged,the distribution was relatively uniform,the nucleus was in the middle,and no lesions were found.There was no change in the expression of cholesterol synthesis gene between the control group and the 4%soybean oil group(P<0.05).After 0.1 mmol/L oleic acid treatment of chicken embryo liver cells,the cholesterol content decreased slightly,but the difference was not significant(P<0.05).Cholesterol synthesis genes HMGCR,MVD,FDPS,FDFT1,MVK,PMVK,IDI1,LBR,SC5D,SQLE,LSS,CYP51A1,MSMO1,NSDHL,HSD17B7,EBP-like,DHCR7,DHCR24 were significantly up-regulated compared to the control group(P<0.05),the expression level of GGPS1 gene was not significantly different from the control group(P>0.05).It is indicated that oleic acid has an effect on the synthesis of cholesterol.With the suited concentration of oleic acid can promote the rise of genes involved in the cholesterol synthesis pathway.In summary,cholesterol synthesis in laying hens is regulated in many ways.Estrogen can regulate the synthesis of SQLE gene through estrogen receptor ER?,which affects cholesterol synthesis.Estrogen can also mediate the expression of miR27b-3p and miR-219b-3p,thereby affecting the expression of target genes HMGCR and DHCR7,ultimately affecting cholesterol synthesis;Dietary soybean oil can affect cholesterol synthesis related genes,thereby affecting cholesterol synthesis.This study enriched the theory of lipid metabolism regulation in chicken liver,and laid a theoretical foundation for further utilization of various control methods to improve the performance of chickens and improve the quality of meat and eggs.