LncRNA GAS5通过TIMP-3启动子甲基化促进软骨胶原蛋白降解的实验研究

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Abstract Objective To investigate the basic mechanism of LncRNA GAS5 in osteoarthritis.Methods 14 normal knee cartilage tissues were collected from patients who received internal fixation for tibial plateau fracture in Zhongshan Hospital Wusong Branch，Fudan University from July 2018 to July 2020，including 8 males and 6 females.The age ranged from 19 to 40 years，with an average of (31±5) years.35 OA knee cartilage tissues were collected from patients diagnosed with OA and indicated for knee replacement surgery in Zhongshan Hospital Wusong Branch of Fudan University from July 2018 to July 2020，including 17 males and 18 females.The average age was (70±4) years.The expression of TIMP-3 mRNA was detected by real time quantitative polymerase chain reaction (RT-qPCR).OA cell model was established by treatment with interleukin-1β (10 ng/mL).GSE16464 OA gene chips were analyzed.The expression of TIMP-3 was detected by RT-qPCR and Western blot.CpG islands enriched in TIMP-3 promoter region were detected by chip quantitative polymerase chain reaction，qPCR (qPCR).The target relationship between GAS5 and TIMP-3 was predicted by bioinformatics method，and verified by double luciferase reporter gene detection.Results TIMP-3 mRNA expression in OA patients was significantly lower than that in normal cartilage tissues.The positive rates of TIMP-3 promoter methylation in OA patients and normal cartilage tissue controls were 72.1% and 35.7%，respectively.The expression of LncRNA GAS5 in OA was significantly higher than that in normal cartilage tissues (P<0.05).Blast sequence alignment has binding site between GAS5 sequence and TIMP-3 promoter sequence.Silencing LncRNA GAS5 promoted TIMP-3 expression and inhibited collagen degradation in OA chondrocytes.Double luciferase reporter gene assay confirmed that GAS5 and TIMP-3 have a targeted regulatory relationship.Conclusion TIMP-3 expression is low in OA cartilage，while LncRNA GAS5 expression was up-regulated; LncRNA GAS5 can enrich DNA methyltransferase，inhibit TIMP-3 expression and promote collagen degradation of chondrocytes.

Key words： long non-coding RNA GAS5 osteoarthritis DNA methyltransferases tissue inhibitor of metalloproteinase-3

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