Effects Of LncRNA TCONS＿00056481 In Regulating The Proliferation And Differentiation Of Porcine Preadipocytes

The levels of the proliferation and differentiation of muscle cells and fat cells are closely related to the growth and development of animal bodies,and are also key factors affecting meat quality.Long-chain non-coding RNA(lncRNA)can participate in many important biological processes with micro RNA(mi RNAs)sponges.The new lncRNA TCONS＿00056481 was obtained by the previous sequencing result of our research group.And we found the lncRNA TCONS＿00056481 has a potential targeting relationship with IRS1 through cis prediction mechanism.On these basis,in order to study the effect of lncRNA competitively binding mi RNA to regulate target genes on the proliferation and differentiation of porcine preadipocytes,first,through bioinformatics analysis,we found mi R-660 has a potential targeting relationship with lncRNA TCONS＿00056481 and IRS1,respecitively.Secondly,real-time fluorescence quantitative PCR(qPCR)technology was used to detect the tissue expression profiles of lncRNA TCONS＿00056481,mi R-660 and IRS1 in the heart,liver,spleen,lung,kidney,longissimus dorsi and back fat in Anqing Sixend-white and Large White pigs.Western Blots were used to detect the protein expression of IRS1 in the back fat tissues of Anqing Six-end-white and Large White pigs.The dual luciferase reporter system was used to verify the potential targeting relationship between lncRNA TCONS＿00056481 and mi R-660,and between mi R-660 and IRS1,respeictively.Finally,siRNA technology was used to silence lncRNA TCONS＿00056481and IRS1 respectively,biosynthesis of mi R-660 mimics and mi R-660 inhibitor,to infect porcine preadipocytes respectively,and to detect the proliferation levels of porcine preadipocytes and the expression levels of Cyclin D and p21.After induction of differentiation,the expression levels of adipogenic marker genes PPAR-γ and C/EBP-α were detected.The main findings are as follows:(1)qPCR results showed that the expression levels of lncRNA TCONS＿00056481 and IRS1 in the back fat tissue of Anqing Six-end-white pigs were significantly higher than that of Large White pigs(P <0.01),while the expression level of mi R-660 in the back fat tissue of Anqing Six-end-white pigs was extremely lower than that of Large White pig(P <0.01);Western Blots results showed that the expression level of IRS1 protein in the back fat tissue of Anqing Six-end-white pig was significantly higher than that of Large White pig(P<0.01).(2)The dual luciferase verification results show that mi R-660 has a direct targeting relationship with lncRNA TCONS＿00056481,and mi R-660 also has a direct targeting relationship with IRS1.(3)Proliferation: Interfering with lncRNA TCONS＿00056481 and IRS1 and adding mi R-660 mimics,the proliferation of porcine preadipocytes was inhibited,the expression level of target gene IRS1 were significantly down-regulated,the expression levels of key proliferation gene Cyclin D were significantly down-regulated,and p21 expression levels were significantly up-regulated.(4)Differentiation: Interfering with lncRNA TCONS＿00056481 and IRS1 and adding mi R-660 mimics,the expression levels of target gene IRS1 were significantly downregulated,and the expression levels of lipogenic genes PPAR-γ and C/EBP-α were significantly down-regulated.In summary,lncRNA TCONS＿00056481 competitively binds with mi R-660 to regulate IRS1 to promote the proliferation and differentiation of porcine preadipocytes.