Résumé
Circular RNA (circRNA), as a competitive endogenous RNA (ceRNA), plays an importantrole in the regulation of cell differentiation. The purpose of this study was to identify and analyze porcinecircular RNA insulin-like growth factor 1 receptor (circIGF1R), explore its expression patterns, construct a ceRNA regulatory network related to circIGF1R, and explore the regulation of its ectopicexpression on adipogenic differentiation of mouse mesenchymal stem cells (C3H10T1 / 2) effect. Forwardand reverse PCR, Sanger sequencing, RNase R enzyme digestion tests, and qRT-PCR were used toverify that circIGF1R is a circRNA formed by the second exon of insulin-like growth factor 1 receptor(IGF1R). It was expressed in all tissues of pigs, and its expression level increased with age in adiposetissues. miRDB, TargetScan and miRWalk online software were used to predict circIGF1R target genes. RNAhybrid software was used for binding site prediction. DAVID bioinformatics functional analysissoftware was used to perform GO and KEGG enrichment analysis on candidate target genes. Cytoscapesoftware was used to construct the ceRNA network diagram. Based on the gene expression correlation andpredicted target relationship, the GO and KEGG enrichment analysis was drawn and the ceRNA networkwas constructed; the dual luciferase reporter gene test was used, and we found that circIGF1R andFABP4 can bind to ssc (Sus scrofa chromosome) -miR-133a-5p. The circIGF1R overexpression vectorwas successfully constructed and expressed in C3H10T1/ 2 cells. It was found that after overexpression ofcircIGF1R, the expression of key adipogenic regulatory factors CEBPa, CEBPß, FABP4 and PPAR? increased significantly(P<0. 01), and the number of lipid droplets increased significantly. The results ofthis study show that circIGF1R exists in pig adipose tissues, and may positively regulate the adipogenicdifferentiation of C3H10T1/ 2 cells through the ceRNA mechanism, which lays a theoretical foundation forfurther research on circIGF1R regulating the adipogenic differentiation of pig precursor intramuscularadipocytes.
Résumé
The objective of this study was to investigate the expression profile of the myozenin2 (MYOZ2) gene and elucidate its effect on adipogenic differentiation of C3H10T1 / 2 cells and its possible mechanism∙ The longissimus dorsi‚ subcutaneous fat and liver tissue was collected from 180-day-old Mashen pigs‚ 60-day-old ICR mice‚ 35-day-old Ross broiler and 12-month-old Small tail han sheep‚ and the expression profile of the MYOZ2 gene mRNA was detected∙ The results showed that the MYOZ2 gene has similar patterns of tissue expression in examined species‚ with the highest expression level in longissimus dorsi‚ and a small amount of expression in the subcutaneous fat and liver tissue∙ After the MYOZ2 gene was silenced in C3H10T1 / 2 cells‚ qRT-PCR results showed that the expression levels of key adipogenic genes PPARγ and FABP4 were significantly down-regulated compared with the control group (P < 0∙ 01) ; Western Blotting results showed that the PPARγ protein content was significantly decreased (P < 0∙ 05) ; Oil red O staining showed that the number of lipid droplets and the content of triglyceride were significantly decreased after silencing MYOZ2 (P < 0∙ 05) ∙ The expression of fatty acid metabolism related genes SCD‚ FASN‚ SREBP1‚ NR1H3‚ DGAT1‚ PNPLA2‚ HSL‚ CES1‚ CPT1 after MYOZ2 silencing were detected by qRT-PCR∙ The results showed that SCD‚ FASN‚ SREBP1‚ PNPLA2 and HSL were significantly down-regulated (P < 0∙ 01) ‚ NR1H3 was significantly reduced (P < 0∙ 05) ‚ DGAT1 expression was down-regulated but the difference was not significant‚ CES1 and CPT1 were significantly up-regulated (P < 0∙ 05) ∙ The STRING database was used to construct a MYOZ2-related protein interaction network map‚ and it was found that MYOZ2 may affect the adipogenic differentiation through the interaction of titin-cap (TCAP) and PPARγ∙ After silencing TCAP‚ qRT-PCR results showed that compared with the control group‚ the expression of key adipogenic genes PPARγ and FABP4 were significantly up-regulated (P < 0∙ 01) ; Western Blotting results showed that PPARγ protein was significantly increased (P< 0∙ 05) ; Oil red O staining showed that the number of lipid droplets and the content of triglyceride were significantly increased after TCAP silencing (P < 0∙ 05) ∙ qRT-PCR was used to detect the expression of TCAP after silencing MYOZ2‚ and the results showed that the expression of TCAP was significantly increased (P<0∙ 01) ∙ In summary‚ MYOZ2 was highly expressed in longissimus dorsi and lower expressed in subcutaneous fat and liver tissues∙ In addition‚ MYOZ2 may 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‚ thus playing an important role in the process of adipogenic differentiation∙
Résumé
The aim of this study was to explore the regulatory mechanism of Type Ⅲ domain-containing protein5 (FNDC5) on adipogenic differentiation in C3H10T1/2 cells. qRT-PCR and Western blot were used to detect the expression of FNDC5 during adipogenic differentiation of C3H10T1/2 cells. The lentivirus-coated overexpression and interference vector of FNDC5 were constructed and transfected into C3H10T1/2 cells. qRT-PCR was used to detect the expression of the key genes of adipogenic differentiation. Oil red O staining was used to detect the formation of lipid droplets; Western blot was used to detect the content of ERK1/2 and ERK1/2 phosphorylated protein (P-ERK1/2). After 8 days of adipogenic differentiation of C3H10T1/2 cells, the expression of Fndc5 increased significantly. After overexpression of FNDC5 in C3H10T1/2 cells, the expression of key genes for adipogenic differentiation, including peroxisome proliferator-activated receptor-酌 (PPAR酌), CCAAT enhancer binding protein beta (C/EBP茁), fatty acid binding protein 4 (FABP4) and CCAAT enhancer binding protein alpha (C/EBPα), all decreased significantly. The content of lipid droplets and P-ERK1/2 also decreased significantly. On the contrary, after interference of FNDC5 in C3H10T1/2 cells, the expression of key genes for adipogenic differentiation, including PPARγ, C/EBP茁, FABP4 and C/EBPα were significantly increased. Meanwhile, the content of lipid droplets and P-ERK1/2 also increased significantly. This study found that FNDC5 can inhibit the adipogenic differentiation of C3H10T1/2 cells by inhibiting the phosphorylation level of ERK1/2, which can provide reference data for the mechanism of FNDC5 in regulating fat deposition.