| The fat content determines the quality of pork.Adipocytes are differentiated from the precursor adipocytes and their formation is a complex network process regulated by a variety of transcription factors.Long non-coding RNA(lncRNA)refers to a kind of is mainly composed of Pol Ⅱ transcription and length is more than 200 nt,do not code for or have very weak ability of RNA,have confirmed that the lncRNA can through a variety of ways to participate in regulating fat formation process.At present,preliminary progress has been made in studies on lncRNAs related to adipogenic differentiation,but mostly concentrate in humans and mice,and there are few reports on studies on pigs.In this study,a new long non-coding RNA lnc-88672 and its target gene MYOZ2 were found through the analysis of the transcriptome sequencing data in the previous laboratory.Bioinformatics predicted that this lncRNA might play a role in lipid metabolism.The effect of lnc-88672 on adipogenic differentiation of porcine precursor adipocytes was studied by silences.RNA pull-down technique was used to search for proteins that could bind to lnc-88672.The effect of silencing lnc-88672 on the methylation level of MYOZ2 gene was analyzed by BSP technique.After MYOZ2 was silenced,the effects of MYOZ2 on adipogenic differentiation were investigated.The STRING database was used to construct the MYOZ2-related protein interaction network map to further explore the functional mechanism of MYOZ2-related protein.The research results are as follows:1.NCBI analysis showed that lnc-88672 was located on chromosome 8 and did not have the ability of protein coding.The results of nucleo-cytoplasmic separation showed that lnc-88672 was expressed in both the nucleus and cytoplasm,and its distribution in the nucleus was higher than that in the cytoplasm.A 487 bp sequence of lnc-88672 was obtained by PCR amplification.q RT-PCR results showed that lnc-88672 had the highest expression in longissimus dorsi,and was also expressed in subcutaneous fat,psoas muscle,and biceps femoris muscle.By bioinformatics,it was found that lnc-88672 may be involved in lipid metabolism.2.After silenced lnc-88672 in porcine precursor adipocytes,qRT-PCR results showed that the expressions of PPARγ and FABP4 were significantly decreased(P<0.01),Western Blotting results showed that the protein content of PPARγ was significantly decreased(P<0.05),and oil red O staining showed that the number of lipid droplets was significantly decreased,indicating that silenced lnc-88672 could inhibit adipogenic differentiation.3.Through RNA pull-down technique,it was found that lnc-88672 could bind to AHCY protein and affect the expression of its downstream target protein DNMT1.Using BSP and q RT-PCR,it was found that after silencing lnc-88672,the degree of methylation of MYOZ2 gene was increased and the expression level was decreased.4.Silent MYOZ2 gene can inhibit adipogenic differentiation.When exploring the mechanism of MYOZ2 affecting adipogenic differentiation,it was found that MYOZ2 could play a role through MYOZ2-TCAPPPARγ,and then regulate genes related to fat metabolism.In summary,a new lncRNA lnc-88672 was found in this study,which preliminarily proved that by interacting with AHCY,lnc-88672 affected the expression of its downstream gene DNMT1,thereby changing the degree of methylation of MYOZ2 gene,affecting the expression of MYOZ2,MYOZ2 regulate the expression of key adipogenic genes PPARγ and FABP4 through the interaction of MYOZ2-TCAP-PPARγ,and to further regulate the expression of fatty acid metabolism related genes SCD,FASN,SREBP1,NR1H3,DGAT1,PNPLA2,HSL,CES1 and CPT1,playing an important role in the process of adipogenic differentiation. |
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