CircARID1A regulates mouse skeletal muscle regeneration by functioning as a sponge of miR-6368.

The noncoding RNAs play important role in growth and development of mammalian skeletal muscle. Recent work has shown that circRNAs are abundant in skeletal muscle tissue, with significant changes in their expression patterns during muscle development and aging. We identified a novel circRNA called circARID1A that is highly expressed in mice skeletal muscle compare to its linear transcript. Experiments shown that circARID1A significantly inhibited the process of C2C12 cell proliferation and promoted its differentiation. Interactions between circRNA and miRNA were screened by miRNA gene chip sequencing. The results indicated that circARID1A can sponge miR-6368, which was further verified by miRNA sensor and other experiments. Besides, miR-6368 is a commonly expressed miRNA that regulates the expression of several target genes including Tlr4. A mouse model of skeletal muscle injury was successfully established to explore the role of circARID1A in skeletal muscle development and regeneration in vivo. Moreover, we found the overexpression of circARID1A significantly promoted the regeneration of skeletal muscle. The results of our study suggest that circARID1A may regulate skeletal muscle cell development and regeneration by sponging miR-6368.

MiR-204-5p promotes lipid synthesis in mammary epithelial cells by targeting SIRT1.

OBJECTIVES: Understanding the molecular mechanisms of lipid synthesis in the mammary gland is crucial for regulating the level and composition of lipids in milk. This study aimed to investigate the functional and molecular mechanisms of miR-204-5p in mammary epithelial cells to provide a theoretical basis for milk lipid synthesis. METHODS: Real-time quantitative PCR was performed to detect the transcriptional levels of miR-204-5p and related mRNA abundance in mammary epithelial cells. Western blotting was conducted to determine protein expression. Cell proliferation was assessed by Cell Counting Kit-8. A dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-204-5p and SIRT1. siRNA and overexpression plasmids were transfected into mouse HC11 mammary epithelial cells. RESULTS: The abundance of miR-204-5p was much higher in lactating mouse mammary glands than in other tissues, which indicated that miR-204-5p may be involved in regulating milk production. MiR-204-5p affected the expression of ß-casein and milk lipid synthesis in HC11 mouse mammary epithelial cells but did not influence the proliferation of HC11 cells. Overexpression of miR-204-5p significantly increased the number of Oil Red O+ cells, triglyceride accumulation and the expression of markers associated with lipid synthesis, including FASN and PPARγ, whereas inhibition of miR-204-5p had the opposite effect. miR-204-5p promotes lipid synthesis by negatively regulating SIRT1. Overexpression of SIRT1 can repress the promotion of miR-204-5p on lipid synthesis. CONCLUSION: Our findings showed that miR-204-5p can promote the synthesis of milk lipids in mammary epithelial cells by targeting SIRT1.

Prolactin-Responsive Circular RNA circHIPK3 Promotes Proliferation of Mammary Epithelial Cells from Dairy Cow.

The highly expressed circHIPK3 is a circular RNA that has been previously reported to regulate the growth of human cells. In this study, we found an increased expression of circHIPK3 in bovine mammary epithelial cells treated with prolactin (PRL) in high-throughput sequencing data. Thus, we further investigated the effect of circHIPK3 on the proliferation and differentiation of mammary epithelial cells. We used qRT-PCR/Cell Counting Kit-8 (CCK-8) and a Western blotting analysis to evaluate the effects on cell proliferation. We found that circHIPK3 promotes the proliferation of mammary epithelial cells. The STAT5 signaling pathway was previously associated with the prolactin response and when the STAT5 was suppressed, the expression of circHIPK3 decreased. The results suggest that the response to prolactin and the associated STAT5 signaling pathway affect the expression of circHIPK3, which subsequently affects the proliferation of mammary epithelial cells in dairy cows.