【摘要】：目的探討成軟骨相關(guān)miR-4287對人軟骨細胞聚蛋白多糖酶-1(a disintegrin and metalloproteinase with thrombospondin motif 4,ADAMTS4)調(diào)控作用及其機制。方法取自愿捐贈的膝關(guān)節(jié)正常及骨關(guān)節(jié)炎軟骨組織,采用實時熒光定量PCR檢測miR-4287和ADAMTS4 mRNA表達量;然后分離培養(yǎng)軟骨細胞,取第1代骨關(guān)節(jié)炎細胞,給予IL-1β處理,觀察其對軟骨細胞miR-4287和ADAMTS4 mRNA表達的影響;分別給予MAPK信號通路抑制劑SP600125以及NF-κB信號通路抑制劑SN50預(yù)處理后聯(lián)合IL-1β刺激,觀察IL-1β介導(dǎo)的信號通路對軟骨細胞miR-4287和ADAMTS4 mRNA表達的影響;分別轉(zhuǎn)染miR-4287模擬物及其陰性對照、miR-4287抑制物及其陰性對照,觀察miR-4287調(diào)控軟骨細胞ADAMTS4 mRNA及蛋白的表達。熒光素酶報告實驗驗證miR-4287與ADAMTS4 mRNA 3’非翻譯區(qū)(untranslated region,UTR)的直接結(jié)合效應(yīng)。結(jié)果與正常軟骨組織比較,骨關(guān)節(jié)炎軟骨組織miR-4287相對表達量下降,ADAMTS4 mRNA相對表達量上升,比較差異有統(tǒng)計學(xué)意義(P0.05)。IL-1β下調(diào)軟骨細胞miR-4287表達、上調(diào)ADAMTS4 mRNA表達,與未經(jīng)IL-1β處理的軟骨細胞相比差異均有統(tǒng)計學(xué)意義(P0.05)。經(jīng)IL-1β介導(dǎo)的信號通路抑制劑預(yù)處理后,軟骨細胞miR-4287相對表達量上升,ADAMTS4 mRNA相對表達量降低,與未經(jīng)信號通路抑制劑預(yù)處理細胞相比,差異均有統(tǒng)計學(xué)意義(P0.05)。轉(zhuǎn)染miR-4287模擬物后,軟骨細胞內(nèi)ADAMTS4 mRNA及蛋白表達均降低(P0.05);而轉(zhuǎn)染miR-4287抑制物后,軟骨細胞內(nèi)ADAMTS4 mRNA及蛋白表達均升高(P0.05)。無論結(jié)合位點為野生型或突變型,過表達miR-4287均不能改變報告載體的熒光素酶活性(P0.05)。結(jié)論成軟骨相關(guān)miR-4287可能是一種與軟骨退變相關(guān)的miRNA。miR-4287能調(diào)控人軟骨細胞ADAMTS4表達,但不是通過靶向結(jié)合mRNA 3’UTR的方式發(fā)揮作用,其具體機制有待進一步研究。
[Abstract]:Objective to investigate the regulatory effect of chondrogenic miR-4287 on human chondrocyte polyproteoglycan enzyme 1 (a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4) and its mechanism. Methods the normal and osteoarthritis cartilage tissues of knee joint were collected and the expression of miR-4287 and ADAMTS4 mRNA were detected by real-time fluorescence quantitative PCR, then the chondrocytes were isolated and cultured, and the first generation of osteoarthritis cells were collected and treated with IL-1 尾. The effects of SN50 on the expression of miR-4287 and ADAMTS4 mRNA in chondrocytes were observed, and the effects of IL-1 尾 -mediated signal pathway on miR-4287 and ADAMTS4 mRNA expression in chondrocytes were observed after pretreatment with MAPK signal pathway inhibitor SP600125 and NF- 魏 B signal pathway inhibitor SN50 with IL-1 尾 stimulation. The expression of ADAMTS4 mRNA and protein in chondrocytes was observed by transfection of miR-4287 analogue and its negative control inhibitor miR-4287 and negative control respectively. Luciferase reporting experiments demonstrated the direct binding effect of miR-4287 to ADAMTS4 mRNA 3 'untranslated region (untranslated region,UTR). Results compared with normal chondrocytes, the relative expression of miR-4287 in osteoarthritis cartilage decreased and the relative expression of ADAMTS4 mRNA increased. The difference was statistically significant (P0.05) .IL-1 尾 down-regulated miR-4287 expression in chondrocytes and up-regulated ADAMTS4 mRNA expression. Compared with the chondrocytes without IL-1 尾 treatment, the difference was statistically significant (P0.05). After pretreatment with signal pathway inhibitor mediated by IL-1 尾, the relative expression of miR-4287 in chondrocytes increased and the relative expression of ADAMTS4 mRNA in chondrocytes decreased, compared with the cells without signal pathway inhibitor pretreatment, the difference was statistically significant (P0.05). The expression of ADAMTS4 mRNA and protein in chondrocytes decreased after transfection of miR-4287 mimics (P0.05), while the expression of ADAMTS4 mRNA and protein in chondrocytes increased after transfection of miR-4287 inhibitors (P0.05). Overexpression of miR-4287 could not change the luciferase activity of the reporter vector, regardless of whether the binding site was wild type or mutant type (P0.05). Conclusion chondrogenic miR-4287 may be a kind of miRNA.miR-4287 associated with cartilage degeneration that can regulate the expression of ADAMTS4 in human chondrocytes, but not by targeting mRNA 3'UTR, and its specific mechanism needs further study.
【作者單位】： 中山大學(xué)附屬第一醫(yī)院關(guān)節(jié)外科;貴州醫(yī)科大學(xué)附屬醫(yī)院骨科;
【基金】：國家自然科學(xué)基金資助項目(81572171) 廣東省自然科學(xué)基金資助項目(2014A03031385)~~
【分類號】：R684.3

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