家禽骨骼系统具有多种功能,不仅提供结构完整性,而且还保持钙和磷等必需矿物质的平衡。然而,近年来,在肉鸡的选择性育种中忽略了骨骼特性的考虑,导致骨骼系统适应不足,无法应对体重的快速增加。因此,这导致跛行和骨骼疾病,如胫骨软骨发育不良(TD),显著影响肉鸡的生产性能。越来越多的证据表明microRNAs(miRNA)在分化过程中起着至关重要的作用,地层,和软骨疾病。然而,miRNA介导的鸡TD形成的分子机制仍然知之甚少。本研究旨在探讨miRNA在鸡TD形成过程中的生物学功能及其调控机制。通过对健康组和TD组胫骨软骨的转录组测序,发现miR-206a-3p在TD软骨中高度表达。通过miR-206a-3p模拟物和miR-206a-3p抑制剂的转染试验探讨miR-206a-3p的功能。在这项研究中,我们利用qRT-PCR,CCK-8,EdU,westernblot,和流式细胞术检测增殖,分化,软骨细胞凋亡。结果表明,miR-206a-3p抑制TD软骨细胞的增殖和分化,同时促进其程序性细胞死亡。此外,通过生物合成和双荧光素酶测定,确定BMP6是miR-206a-3p的直接靶基因。这一发现得到了拯救实验的进一步支持,所述拯救实验证实了BMP6参与由miR-206a-3p控制的调节途径。我们的结果表明miR-206a-3p可以通过靶基因BMP-6抑制鸡TD软骨细胞的增殖和分化,促进凋亡,并抑制Smad2/3信号通路。
The poultry skeletal system serves multiple functions, not only providing structural integrity but also maintaining the balance of essential minerals such as calcium and phosphorus. However, in recent years, the consideration of skeletal traits has been overlooked in the selective breeding of broilers, resulting in an inadequate adaptation of the skeletal system to cope with the rapid increase in body weight. Consequently, this leads to lameness and bone diseases such as tibial dyschondroplasia (TD), which significantly impact the production performance of broilers. Accumulating evidence has shown that microRNAs (miRNA) play a crucial role in the differentiation, formation, and disease of cartilage. However, the miRNA-mediated molecular mechanism underlying chicken TD formation is still poorly understood. The objective of this study was to investigate the biological function and regulatory mechanism of miRNA in chicken TD formation. Based on transcriptome sequencing of tibial cartilage in the healthy group and TD group, miR-206a-3p was found to be highly expressed in TD cartilage. The function of miR-206a-3p was explored through the transfection test of miR-206a-3p mimics and miR-206a-3p inhibitor. In this study, we utilized qRT-PCR, CCK-8, EdU, western blot, and flow cytometry to detect the proliferation, differentiation, and apoptosis of chondrocytes. The results revealed that miR-206a-3p suppressed the proliferation and differentiation of TD chondrocytes while promoting their programmed cell death. Furthermore, through biosynthesis and dual luciferase assays, it was determined that BMP6 was the direct target gene of miR-206a-3p. This finding was further supported by rescue experiments which confirmed the involvement of BMP6 in the regulatory pathway governed by miR-206a-3p. Our results suggest that miR-206a-3p can inhibits the proliferation and differentiation promote apoptosis through the target gene BMP-6 and suppressing the Smad2/3 signaling pathway in chicken TD chondrocytes.