长非编码RNA SNAI3-AS1调控人软骨细胞C28/I2增殖能力及退变的机制研究

Mechanism of Long Non-coding RNA SNAI3-AS1 on Proliferation and Degeneration of Human Chondrocytes

目的:为了探究长非编码RNA SNAI3-AS1(LncRNA SNAI3-AS1,即SNAI3-AS1)在骨性关节炎(osteoarthritis,OA)进展中的作用与机制。方法:通过全转录组测序筛选出在OA中差异表达的lncRNA SNAI3-AS1,并通过实时荧光定量PCR(qRT-PCR)检测SNAI3-AS1在软骨细胞退变模型中的表达情况。在软骨细胞C28/I2中分别转染SNAI3-AS1特异性si RNA或真核过表达质粒,分别敲低或过表达SNAI3-AS1,通过MTT、平板克隆形成和EdU掺入实验检测细胞增殖活力,Western Blot检测炎症和细胞外基质蛋白的表达情况。通过生物信息学网站预测SNAI3-AS1相互作用的miRNA和下游靶基因,并通过双荧光素酶报告基因和RIP实验进行验证。结果:相较于正常软骨细胞,SNAI3-AS1的表达水平在OA中显著下调。敲低正常软骨细胞中SNAI3-AS1的表达后,软骨细胞的增殖能力减弱并促进了软骨细胞的退变,而在OA模型的软骨细胞中过表达SNAI3-AS1后,软骨细胞的增殖活力加强并抑制了软骨细胞的退变。在机制上,SNAI3-AS1可充当竞争性内源性RNA(ceRNA),经海绵吸附miR-2278间接上调PRELP,发挥促进软骨细胞增殖和抑制其退变的作用。结论:LncRNA SNAI3-AS1通过LncRNA SNAI3-AS1/miR-2278/PRELP轴参与骨性关节炎的发生发展过程。

Objective:In order to explore the function and influence of long non-coding RNA SNAI3-AS1(LncRNA SNAI3-AS1,SNAI3-AS1)in the progression of osteoarthritis(OA).Methods:LncRNA SNAI3-AS1 was screened by whole-transcriptome sequencing,and its expression in cell model of chondrocyte degeneration was detected by real-time quantitative fluorescence polymerase chain reaction(qRT-PCR).Transfecting SNAI3-AS1-specific si-RNA or eukaryotic overexpression plasmid in chondrocytes to knock down or overexpress SNAI3-AS1 separately.The proliferation activity of chondrocytes was detected by MTT assay,plate colony assay and EdU assay,and the expression of inflammatory and extracellular matrix proteins was detected by Western Blot.The lncRNA-miRNA and miRNA-mRNA interactions were predicted by bioinformatics website,and verified by dual luciferase reporter gene and RIP assay.Results:Compared with normal human chondrocytes,the expression level of SNAI3-AS1 was significantly down-regulated in OA.And knockdown of SNAI3-AS1 in chondrocytes can reduce chondrocyte proliferation and promote chondrocyte degeneration,while overexpression of SNAI3-AS1 in chondrocytes of OA model can enhance chondrocyte proliferation and inhibit chondrocyte degeneration.In terms of mechanism,SNAI3-AS1 can act as a competitive endogenous RNA(ceRNA),which indirectly up-regulates PRELP by sponging miR-2278,and plays a role in promoting chondrocyte proliferation and inhibiting its degeneration.Conclusion:LncRNA SNAI3-AS1 is involved in the occurrence and development of osteoarthritis through the lncRNA SNAI3-AS1/miR-2278/PRELP axis.