摘要
旨在研究猪C1QTNF3基因可变剪接体的特性及miR-101通过C1QTNF3基因促进猪脂肪SV细胞成脂分化的机制。本研究采集3头30日龄健康马身猪仔公猪心、肝、脾、肺、肾、胃、股二头肌、腰大肌、皮下脂肪和背部脂肪组织样品,首先利用RT-PCR技术和生物信息学方法对C1QTNF3基因的不同转录本进行扩增和生物学特性分析,采用qRT-PCR技术检测C1QTNF3基因不同转录本在猪组织中的表达变化。随后,利用生物信息学预测发现,C1QTNF3上游的调控因子是miR-101,采用双荧光素酶报告试验验证miR-101对C1QTNF3的调控作用。最后,利用油红O染色和qRT-PCR技术检测过表达miR-101对猪脂肪SV细胞成脂分化的影响。基因克隆得到猪C1QTNF3存在两个转录本C1QTNF3和C1QTNF3-1,其中C1QTNF3-1为新鉴定的转录本;测序分析显示,与C1QTNF3相比,C1QTNF3-1的第1外显子区域缺失了219个碱基,少编码73个氨基酸。进化树分析显示,猪C1QTNF3和C1QTNF3-1蛋白序列与人和马来亚穿山甲等物种的亲缘关系较近。C1QTNF3和C1QTNF3-1在猪各组织中均有表达,且在各组织中C1QTNF3-1的表达量均极显著高于C1QTNF3(P<0.01)。过表达miR-101极显著下调C1QTNF33′UTR区域的荧光素酶活性(P<0.01)和C1QTNF3 mRNA的表达(P<0.01),同时增加了脂肪细胞成脂分化关键基因PPARγ、C/EBPβ、SREBP-1c和FABP4的mRNA表达量(P<0.01)。本试验成功克隆了猪C1QTNF3基因的两个可变剪接体,其中C1QTNF3-1在猪不同组织中均高表达,推测该转录本为基因发挥功能的主要亚型。并深入研究了C1QTNF3基因的上游调控因子,揭示了miR-101靶向C1QTNF3促进成脂分化的机制,丰富了C1QTNF3的生物学作用和调控网络。
The study aimed to explore the alternative splicing isoforms of pig C1QTNF3 gene and mechanism of miR-101 targeting C1QTNF3 expression in promoting porcine adipose-derived stromal vascular cells(ASVC)adipogenesis.The heart,liver,spleen,lung,kidney,stomach,biceps femoris,psoas magnus,abdominal fat and back fat were obtained from 330-day-old healthy Mashen male piglets.The full-length coding sequence(CDS)of C1QTNF3 transcripts were cloned by RT-PCR,the biological characteristics of C1QTNF3 protein were analyzed by bioinformatics methods.The expression patterns of C1QTNF3 transcripts in porcine tissues were detected by qRT-PCR.Bioinformatics prediction revealed that miR-101 was the upstream regulatory factor of C1QTNF3.The effect of miR-101 on C1QTNF3 was verified by dual luciferase reporter assay.The effect of overexpression of miR-101 on adipogenesis of porcine ASVC was detected by Oil Red O staining and qRT-PCR.Two transcripts of C1QTNF3 gene,C1QTNF3 and C1QTNF3-1,were successfully obtained in this study.C1QTNF3-1 was a newly identified transcript,which lacked 219 bp(73 amino acids)in the first exon compared with C1QTNF3.The phylogenetic tree analysis showed that the porcine C1QTNF3 and C1QTNF3-1 proteins sequence were highly related to Homo sapiens and Manis javanica.C1QTNF3 and C1QTNF3-1 were expressed in all porcine tissues,the expression level of C1QTNF3-1 was significantly higher than that of C1QTNF3(P<0.01).Overexpression of miR-101 significantly reduced the relative luciferase activity of C1QTNF33′UTR(P<0.01)and mRNA expression of C1QTNF3(P<0.01);the mRNA expression of key genes for adipogenesis,PPARγ,C/EBPβ,SREBP-1c and FABP4 were significantly increased(P<0.01).In this study,two transcripts of pig C1QTNF3 gene were successfully cloned.The expression of C1QTNF3-1 was significantly higher than C1QTNF3 in different porcine tissues,suggesting it was the predominant transcript.In addition,miR-101 was identified as the upstream regulatory factor of C1QTNF3.By reducing C1QTNF3 gene expression,miR-101 promot
作者
杨阳
张雪莲
张万锋
李文霞
李文新
蔡春波
路畅
高鹏飞
郭晓红
李步高
曹果清
YANG Yang;ZHANG Xuelian;ZHANG Wanfeng;LI Wenxia;LI Wenxin;CAI Chunbo;LU Chang;GAO Pengfei;GUO Xiaohong;LI Bugao;CAO Guoqing(College of Animal Science, Shanxi Agricultural University, Taigu 030801, China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2021年第11期3042-3052,共11页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
三晋学者支持计划专项经费资助(2016,2017)
国家自然科学基金(31872336)
山西省“1331”工程(J201811301)
山西省青年基金(201901D211369)
山西省优秀博士奖励(SXYBKY2019030)
山西农业大学科技创新(2020BQ01)。
