The Molecular Mechanism of circRNA-11228/miR-103/INSIG1 Pathway Regulating Milk Fat Synthesis in Bovine Mammary Epithelial Cells

Abstract

Milk, known for its high content of short- and medium-chain fatty acids and unsaturated fatty acids, has attracted substantial attention due to its nutritional and health value. The regulation of fatty acid metabolism by non-coding RNAs has become a subject of growing attention, particularly in relation to fatty acid production at the transcriptional/epigenetic and post-transcriptional levels. This study established the circRNA-11228/miR-103/INSIG1 (insulin-inducible gene) regulatory network using methods such as qRT-PCR, dual luciferase reporting, and Western blot, with INSIG1 serving as the starting point. The experimental validation of circRNA-11228’s impact on cholesterol levels, lipid droplet secretion, and unsaturated fatty acid content was conducted using various assays, including triglycerides, cholesterol, oil red O, andEdU(5-ethynyl-2’-deoxyuridine) in bovine mammary epithelial cells (BMECs). Furthermore, the transfection of mimics and inhibitors synthesized from miR-103 into BMECs confirmed that miR-103 can promote cholesterol synthesis and lipid droplet secretion. Conversely, the INSIG1 gene was found to inhibit cholesterol synthesis and lipid droplet secretion. The “remediation” experiment validated the ability of miR-103 to alleviate the cellular effect of circRNA-11228. Taken together, our findings indicate that the binding of circRNA-11228 to miR-103 inhibits the expression of the target gene INSIG, thereby regulating milk fat production in BMECs. This study offers novel insights into producing high-quality milk and new ways to improve the dietary composition of residents.