武定鸡肝脏miR-143基因及其脂类代谢靶基因表达规律研究

Study on expression of miR-143 and targeted lipid metabolism genes in livers of Wuding chickens

为了探究miR-143基因与脂类代谢靶基因的关系及其在脂类代谢过程中的调控作用以及对武定鸡肝脏组织脂类代谢的调控机制,试验通过靶基因预测软件microRNA.org(http://www.microrna.org)预测miR-143基因与脂类代谢靶基因的结合位点;选取胚胎期第15天和出生后第1,7,14,28,56天各时期的武定鸡、AA鸡各6只,公母各半,屠宰取肝脏组织,采用实时荧光定量PCR方法检测miR-143基因及与脂类代谢相关靶基因的相对表达量,并进行相关性分析。结果表明:脂类代谢基因ACC、FAS、LPL、C/EBPβ、FABP4/ap2、FAT是miR-143基因的调控靶基因,未发现C/EBPβ基因的结合位点,其他靶基因的结合位点为互补配对的CAUCUC序列。武定鸡肝脏组织中miR-143基因的相对表达量在出生后1~7,14~56天呈上升趋势,出生后7~14天呈下降趋势,第7天相对表达量最高,第14天相对表达量最低;AA鸡miR-143基因相对表达量只有出生后第14,56天时高于武定鸡,其他时期均低于武定鸡。武定鸡ACC、FAT基因在各生长发育期的相对表达量均高于AA鸡;除出生后第7天外,武定鸡FAS基因相对表达量均高于AA鸡;武定鸡LPL基因相对表达量只有在出生后第56天时低于AA鸡;除胚胎期第15天外,武定鸡C/EBPβ基因相对表达量均高于AA鸡;除胚胎期第15天和出生后第56天外,武定鸡FABP4/ap2基因相对表达量均高于AA鸡。武定鸡miR-143基因与ACC、FAS、LPL、FABP4/ap2、C/EBPβ、FAT基因的相关系数分别为-0.200,-0.556,-0.620,-0.360,-0.318,-0.375,呈负相关,与LPL基因的负相关性最强,FAS基因次之;脂类代谢相关的各靶基因之间呈正相关关系,其中FAT基因和FAS基因之间的正相关性最强(r=0.937),ACC基因和FABP4/ap2基因之间的正相关性次之(r=0.932),ACC基因和C/EBPβ基因之间的正相关性位列第3(r=0.923)。说明武定鸡miR-143基因通过负调控主效基因LPL和FAS,以及脂类代谢基因之间的相互作用,来调控肝脏脂类代谢,且miR-143基因与脂类代谢靶基因的相互作用具有发育阶段性特点。

To explore the relationship between the miR-143 gene and the lipid metabolic target gene,the regulatory role of miR-143 gene in lipid metabolism process,and the regulatory mechanism of miR-143 gene on lipid metabolism in liver tissue of Wuding chickens,the binding site of miR-143 and lipid metabolic target genes were predicted by target gene prediction software microRNA.org(http://www.microrna.org).Six Wuding chickens and six AA chickens,half male and half female,were selected from the 15 th day of embryo and the 1 st,7 th,14 th,28 th and 56 th day after birth.Liver tissues were selected from chickens after slaughtered and the relative expression levels of miR-143 and lipid metabolization-related target genes were detected by real-time quantitative PCR for correlation analysis.The results showed that the lipid metabolic gene ACC,FAS,LPL,C/EBPβ,FABP4/ap2,FAT were predicted to be the regulatory target gene of miR-143.No binding sites of the C/EBPβgene were found,and the binding sites of other target genes were complementary paired CAUCUC sequences.The relative expression of miR-143 in liver tissues showed an upward trend from 1 to 7,14 to 56 days after birth,and a downward trend from 7 to 14 days after birth.The relative expression of miR-143 was highest on day 7 and lowest on day 14.The relative expression of miR-143 gene in AA chickens was higher than that in Wuding chickens only at the 14 th and 56 th day after birth,and lower than that in other periods.The relative expression of ACC and FAT genes in Wuding chickens were higher than that of AA chickens.The relative expression of FAS gene in Wuding chickens was higher than that in AA chickens except for the 7 th day after birth.The relative expression of LPL gene in Wuding chickens was lower than that in AA chickens only on the 56 th day after birth.The relative expression level of C/EBPβgene in Wuding chickens was higher than that in AA chickens except the 15 th day of embryo stage.The correlation coefficients between miR-143 gene and the relative expression of ACC,FAS,LPL,FABP4/ap2,C/EBPβ,FAT gene were-0.200,-0.556,-0.620,-0.360,-0.318 and-0.375,respectively,showing negative correlation.The negative correlation with LPL was strongest,followed by FAS.There was a positive correlation among target genes related to lipid metabolism,among which the positive correlation between FAT gene and FAS gene was the strongest(r=0.937),followed by the positive correlation between ACC gene and FABP4/ap2 gene(r=0.932).The positive correlation between ACC gene and C/EBPβgene ranked third(r=0.923).These results indicated that the miR-143 gene regulated liver lipid metabolism by negatively regulating the master genes LPL and FAS genes,as well as the interactions between lipid metabolism genes,and the interaction between miR-143 and lipid metabolism target genes was characterized by developmental stages.