Abstract
Background
Intramuscular fat content is positively correlated with meat flavor and juiciness. Increasing the intramuscular fat (IMF) content of chickens while increasing their growth rate has become a hot topic in molecular breeding.The group's previous studies showed that miR-128-3p inhibited chicken intramuscular adipocyte differentiation and lipogenesis. However, the regulatory mechanism of miR-128-3p in intramuscular preadipocytes is currently unknown. In this study, we investigated the mechanism of miR-128-3p regulation of chicken intramuscular adipocyte differentiation and deposition.
Methods
RNA-seq was performed to screen for long non-coding RNAs (lncRNAs) that bind to miR-128-3p. Dual luciferase reporter system was used to verify the targeting relationship between miR-128-3p and LincRNA-MSTRG.673.2; nucleoplasmic localization analysis and fluorescence in situ hybridization were used to investigate the localization of LincRNA-MSTRG.673.2 in the cells; a series of experiments such as Q-PCR, Oil O Red staining and triglyceride assay were used to explore the effect of interference with LincRNA-MSTRG.673.2 on the differentiation of intramuscular preadipocytes; co-transfection experiments were used to validate the regulatory patterns of miR-128-3p and LincRNA-MSTRG.673.2 in intramuscular adipocytes.
Results
Transcriptome data analysis of differential LincRNAs indicated that, compared to the NC group, the mimics-treated group had 17 significantly differentially expressed LincRNAs (P < 0.05), including 6 upregulated and 11 downregulated ones; the inhibitor-treated group had 17 differentially expressed LincRNAs (P < 0.05), including 8 upregulated and 9 downregulated ones; and 24 differentially expressed LincRNAs (P < 0.05) were observed when comparing the mimics-treated group to the inhibitor-treated group, with 14 upregulated and 10 downregulated ones. Functional enrichment analysis revealed that DELincRNAs from the overexpression group (M group) and interference group (SI group) were involved in negative regulation of metabolic processes, response to steroid hormones, regulation of actin cytoskeleton. Furthermore, target gene prediction analysis showed that miR-128-3p can target many of the DELincRNAs, such as LincRNA-MSTRG.673.2, LincRNA-MSTRG.39.2, LincRNA-MSTRG.39.3, and LincRNA-MSTRG.14270.2. LincRNA-MSTRG.673.2 was predominantly expressed in cytoplasm of intramuscular adipocytes. Dual luciferase reporter identified the targeting relationship between miR-128-3p and LincRNA-MSTRG.673.2. The results of subsequent functional assays demonstrated that Interfering with MSTRG.673.2 has been shown to inhibit lipid deposition in intramuscular preadipocytes. Transfection experiments have shown that LincR-MSTRG.673.2 can affect the expression of miR-128-3p.
Conclusion
This study found that LincRNA-MSTRG.673.2 promoted chicken intramuscular adipocytes differentiation by down regulating miR-128-3p. The results are noteworthy for improving chicken meat quality, molecular breeding, and lipid metabolism research.