以hsa_circ_0001461和CDK6为核心的RNA调控网络对软骨细胞衰老的作用机制研究

How to restrain chondrocyte senescence is the key to maintain the homeostasis of chondrocytes, and cyclin-dependent kinase 6 (CDK6) may interfere with chondrocyte senescence by activating the inflammatory pathway, upregulating the expression of P16INK4A and the level of ROS. Hsa_circ_0001461 is correlated with CDK6, and it may participate in the regulation of the chondrocyte senescence through acting as the ceRNA of miR-320c and CDK6. In the pre-experiment, we found that miR-320c and hsa_circ_0001461 showed a "mirror symmetry" relationship during the late stage of chondrogenic differentiation in human bone marrow mesenchymal stem cells (BMSCs). We transfected human BMSCs and SW1353 cell lines, with hsa_circ_0001461 siRNA, and the qPCR results showed that hsa_circ_0001461 was inhibited, while the expression of ACAN, COL2A1 and SOX9 mRNA were upregulated. The result was in accordance with the tendency of gene expression produced when miRNA-320c was overexpressed in our previous study. The purpose of this research was to elucidate the RNA regulatory network with hsa_circ_000146/CDK6 by in vitro and in vivo experiments and to take advantage of the regulatory network to delay the degeneration of articular cartilage by regulating the inhibition of chondrocyte senescence.

如何抑止软骨细胞衰老是维持软骨细胞稳态的关键，而CDK6可能通过激活炎症通路、诱导P16INK4A表达和上调ROS水平干预细胞衰老，从而影响骨关节炎发展进程。hsa_circ_0001461与CDK6有着相关性，有可能通过与miR-320c、CDK6构成的ceRNA调控环参与软骨细胞衰老的调控。预实验中我们发现在hBMSCs成软骨分化过程中后期miR-320c和hsa_circ_0001461的表达呈现此消彼长的“镜像对称”关系，在hBMSCs和SW1353细胞系中通过转染siRNA抑制hsa_circ_0001461可上调ACAN、COL2A1、SOX9 mRNA表达，与我们既往研究中过表达miRNA-320c时产生的基因表达变化趋势一致。本研究旨在通过体内外实验阐明以hsa_circ_0001461和CDK6为核心的RNA调控网络，并利用其调节抑制和阻止软骨细胞衰老，从而延缓关节退变。

按照项目计划从表观遗传学的角度研究间充质干细胞成软骨分化及软骨退变相关的以hsa_circ_0001461和CDK6为核心的RNA调控网络对软骨细胞衰老的作用机制研究的内源性竞争RNA在成软骨分化及软骨退变中的调控网络。研究发现并阐明了miR-490-5p的抑制剂可通过靶向PITPNM1继而抑制PI3K/AKT信号通路从而促进干细胞成软骨分化及减缓OA进展的崭新机制。同时，我们研究发现退变及增生的OA软骨中CDK6均高表达，而间充质干细胞成软骨分化及软骨退变密切相关的miR-320c可调节CDK6的表达。研究发现miR-320c的下游靶点分别通过靶向CDK6、β-catenin来抑制 NF-κB及Wnt信号通路从而调控间充质干细胞成软骨分化及与软骨退变。本研究阐述调控OA软骨进展的作用机制，为阻遏软骨退变的靶向治疗提供参考。