LncRNA TCONS＿00010987 Competitively Binds MiR-323 To Target LEPR To Regulate Proliferation And Differentiation Of Porcine Preadipocytes

Studying and clarifying the molecular genetic mechanism of pig fat metabolism has important guiding significance for understanding the mechanism of fat deposition in pigs,accelerating the genetic improvement of pig breeds and producing high quality pigs.In order to study the effect of lncRNA and miRNA competitive binding target genes on the proliferation and differentiation of porcine preadipocytes,combined with the previous.sequencing results of the group,we first found the lncRNA TCONS＿00010987 with the targeting relationship of LEPR through cis mechanism,and then used Bioinformatics software analysis found that miR-323,which may be targeted to bind to lncRNA TCONS＿00010987.The expression patterns of the three genes in the Anqing Six-end-white and Large White pigs and expression profiles during porcine preadipocytes differentiation were analyzed by real-time quantitative PCR,and LEPR protein levels between two pigs were observed by Western Blots.Subsequently,the lncRNA TCONS＿00010987 and miR-323 competitively bind to LEPR were verified by dual luciferase reporter.Finally,siRNA technology was used to silence lncRNA TCONS 00010987,LEPR mRNA,biosynthesis miR-323 mimics and inhibitor,respectively,to infect the primary preadipocytes of pigs,respectively.Real-time qPCR was conducted to detected the adipogenetic and lipolytic markers.The expression levels of the gene,and the proliferation of porcine preadipocytes was detected by MTT assay and the mRNA levels of Cyclin D and p21 were detected.The main findings are as follows:(1)The qPCR analysis revealed that the backfat expression levels of lncRNA TCONS＿00010987 and LEPR in Anqing Six-end-white pigs were significantly higher than that in Large White pigs(P<0.01),the backfat expression levels of miR-323 in Anqing Sixend-white pigs were significantly lower than that in Large White pigs(P<0.05).The results of western blots showed that LEPR protein level in the backfat tissues of Anqing Six-endwhite pigs were also greatly higher than that in Large White pigs(P<0.01).(2)The results of dual luciferase assay showed that miR-323 has a targeting relationship with lncRNA TCONS＿00010987,and there is also a targeting relationship between miR323 and LEPR.(3)Proliferation:Interfering with lncRNA TCONS 00010987 and LEPR and adding miR323 mimics,the proliferation level of porcine preadipocytes were significantly inhibited,the target gene LEPR expression levels were significantly down-regulated,the proliferation key gene Cyclin D mRMA was significantly down-regulated,and p21 mRMA was significantly up-regulated.(4)Differentiation:Interfering with lncRNA TCONS＿00010987 and LEPR and adding miR-323 mimics,the target gene LEPR expression level was significantly down-regulated,the adipogenic genes PPAR-γ and C/EBP-α were significantly down-regulated,and the lipolysis genes ATGL and HSL were significantly up-regulated.In summary,lncRNA TCONS＿00010987 competitively binds to miR-323 to regulate LEPR to promote proliferation and differentiation of porcine preadipocytes.