lncRNA-UDC/miR-411/MMP-13轴在骨关节炎软骨细胞退变中的作用

Degeneration of chondrocytes is a hallmark biomarker of osteoarthritis (OA), but the specific mechanism is still not completely clear. Our previous studies have confirmed that miR-411 regulates chondrocyte degeneration by targeting matrix metallopeptidase 13(MMP-13).And we screened out the lncRNA-UDC, which was upregulated in the degenerative chondrocytes, and found that the expression of miR-411 and lncRNA-UDC was correlated.LncRNA is rich in miRNA binding sites and act as miRNA "sponges" in cells, so we speculated that lncRNA-UDC could cancel the inhibition effect of miR-411 on its target gene MMP-13 by binding to it, which can increase the expression of MMP-13 and regulate the proliferation and apoptosis of chondrocytes.In order to elucidate the functional mechanism of lncRNA-UDC in OA, this project is intended to determine the effect of miR-411-MMP-13 on chondrocyte degeneration in animals first, and then verify that lncRNA-UDC can be used as miR-411 competitive ce RNA. Finally, the mechanism of lncRNA-UDC/miR-411/MMP-13 axis regulating chondrocyte degeneration was clarified by using DMM and conditioned miR-411 gene knockout mice.The project is expected to provide a new target for exploring gene targeting for the treatment of OA cartilage degeneration.

软骨细胞退变是骨关节炎(osteoarthritis,OA)发生发展的主要原因，但具体机制不明确。课题组前期发现miR-411靶向抑制MMP-13调控软骨细胞退变，并筛选出在退变软骨细胞高表达的lncRNA-UDC，且发现miR-411与lncRNA-UDC的表达具有相关性。因此我们提出lncRNA-UDC通过调控miR-411，解除其对MMP-13的抑制，从而调控软骨细胞退变的假说。为阐明lncRNA-UDC在OA中的作用，本项目拟首先在动物体内明确miR-411/MMP-13的作用，再验证lncRNA-UDC是否可作为miR-411的ceRNA起作用，最后利用DMM小鼠OA模型和条件性miR-411基因敲除鼠，过表达及和沉默lncRNA-UDC，明确lncRNA-UDC/miR-411/MMP-13轴调控软骨细胞退变的机制。预期研究结果可为探索基因靶向治疗OA软骨退变提供新的作用靶点。

背景：关节软骨退变是骨关节炎发病的根本，也是启动其他病理进程的首要因素。近年来研究发现，lncRNA在软骨发育及骨关节炎病理进程中起到了重要的调控作用。主要研究内容：lncRNA-UDC在正常和退变软骨中的筛选，研究lncRNA-UDC与miR-411的调控作用，体内实验研究miR-411在骨关节炎发生、发展中的作用，最后明确lncRNA-UDC在人软骨细胞水平和动物体内的功能研究。重要结果及关键数据：通过基因芯片检测出骨关节炎患者组和NC正常组的软骨组织中lncRNAs和mRNAs存在差异表达。GO分析和Pathway分析对骨关节炎患者中lncRNAs和mRNAs的共表达网络进行了预测，表明lncRNAs在骨关节炎的病理过程中发挥重要作用，提示差异表达的lncRNAs可作为骨关节炎诊断和治疗的新型生物标记物；FISH荧光结果显示，lncRNA-UDC主要在细胞质中表达，免疫荧光证实，与对照组比，骨关节炎组及si-NC组COLL II蛋白表达下降，与si-NC组相比，si-lnc UDC上调COLL II表达；流式检测证实，与NC+NC-i组比，NC+miR-i组表达上调，与si-Lnc UDC+NC-i组相比，si-Lnc UDC+miR-i上调。分别使用lncRNA和miR-411拉取复合物，PCR检测lncRNA-UDC和miR-411，上述研究结果表明二者存在互作。细胞实验证实，与NC(OE)组相比，lncRNA-UDC过表达组促进了软骨细胞的增殖；与空白对照组相比， NC(OE)+IL-1β组抑制了细胞增殖；与NC(OE)+IL-1β组相比，shlncRNA-UDC+IL-1β组抑制了细胞增殖。HE染色结果显示： UDC过表达腺病毒组大鼠软骨组织结构层次模糊，细胞数目更少，炎症细胞浸润程度更加严重；甲苯胺蓝染色结果如图所示， UDC过表达腺病毒组软骨浅层颜色变浅，软骨细胞排列欠有序，软骨细胞数目更少，层次尚清。两种不同的染色方法表明UDC过表达进一步促进骨关节炎模型中软骨组织损伤。科学意义：明确差异表达的lncRNA-UDC功能，揭示lncRNA-UDC/miR-411/ MMP-13通路在骨关节炎关节软骨凋亡、自噬以及进一步导致软骨退变中的作用及其相关机制，为临床骨关节炎的诊断、修复提供重要的理论支持和参考。