黃生輝, 李建華, 張瑋瓊, 劉貴旺, 許錦煌, 鄭沛中, 黃建榮 , △
(1中山大學(xué)孫逸仙紀(jì)念醫(yī)院骨科,廣東 廣州 510235; 2廣州醫(yī)科大學(xué)生理教研室 ,廣東 廣州 511436; 3增城市人民醫(yī)院骨科,廣東 廣州 511300)
沉默TREM-2對(duì)類風(fēng)濕關(guān)節(jié)炎成纖維樣滑膜細(xì)胞遷移和侵襲的影響*
黃生輝1,李建華2,張瑋瓊3,劉貴旺1,許錦煌3,鄭沛中3,黃建榮1, 3△
(1中山大學(xué)孫逸仙紀(jì)念醫(yī)院骨科,廣東 廣州 510235;2廣州醫(yī)科大學(xué)生理教研室 ,廣東 廣州 511436;3增城市人民醫(yī)院骨科,廣東 廣州 511300)
[摘要]目的: 應(yīng)用RNA干擾(RNAi)技術(shù)沉默類風(fēng)濕關(guān)節(jié)炎成纖維樣滑膜細(xì)胞(RA-FLS)髓樣細(xì)胞觸發(fā)受體2(TREM-2)的表達(dá),探討TREM-2基因沉默對(duì)RA-FLS遷移和侵襲能力的影響及其相關(guān)機(jī)制。方法: 向RA-FLS轉(zhuǎn)染特異性的TREM-2 siRNA,利用RT-PCR法和Western blot法檢測(cè)沉默效果;CCK-8法檢測(cè)各組細(xì)胞生長(zhǎng)情況;Transwell小室測(cè)定細(xì)胞的遷移和侵襲能力;ELISA法檢測(cè)細(xì)胞MMP-2和MMP-9的分泌水平;Western blot法分析沉默TREM-2基因?qū)?xì)胞PI3K/AKT通路的影響。結(jié)果: TREM-2 siRNA能顯著降低RA-FLS中TREM2 mRNA和蛋白的表達(dá)(P<0.05)。沉默TREM-2基因后,各時(shí)點(diǎn)各組細(xì)胞的活力未見(jiàn)明顯差異;特異性干擾組RA-FLS的遷移細(xì)胞數(shù)目與空白組和control siRNA組相比明顯增多(P<0.05);TREM-2 siRNA組侵襲細(xì)胞數(shù)目相比空白組和control siRNA組明顯增多(P<0.05);TREM-2 siRNA干擾后RA-FLS分泌的MMP-2顯著增加(P<0.05)而MMP-9未見(jiàn)明顯變化;特異性轉(zhuǎn)染后的RA-FLS相對(duì)于對(duì)照組PI3K/AKT的磷酸化水平顯著增強(qiáng)(P<0.05)。結(jié)論: TREM-2可能通過(guò)調(diào)節(jié)PI3K/AKT通路的活化對(duì)RA-FLS的遷移和侵襲能力發(fā)揮著重要作用。
[關(guān)鍵詞]髓樣細(xì)胞觸發(fā)受體2; 類風(fēng)濕關(guān)節(jié)炎; 滑膜細(xì)胞; 細(xì)胞遷移; 細(xì)胞侵襲; PI3K/AKT通路
類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis,RA)是一種進(jìn)展性自身免疫性疾病,主要累及關(guān)節(jié),以滑膜增殖以及炎癥細(xì)胞浸潤(rùn)為特征,最終導(dǎo)致組織破壞與殘疾。滑膜成纖維細(xì)胞(fibroblast-like synoviocytes,F(xiàn)LSs)在RA的發(fā)生、發(fā)展中發(fā)揮著極為重要作用,關(guān)節(jié)滑膜襯里層增生的RA-FLS可直接黏附到軟骨表面,伴隨著新形成的血管共同形成有侵襲能力的血管翳侵襲周圍的軟骨和骨組織。造成細(xì)胞外基質(zhì)和軟骨組織破壞的最重要因素是RA-FLS分泌大量的基質(zhì)金屬蛋白酶(metalloproteinases,MMPs)到滑膜液中。
髓樣細(xì)胞觸發(fā)受體2(triggering receptor expressed on myeloid cells-2,TREM-2)是新近發(fā)現(xiàn)的免疫模式受體,主要高表達(dá)于巨噬細(xì)胞、樹(shù)突狀細(xì)胞、小膠質(zhì)細(xì)胞、成纖維細(xì)胞、某些腫瘤細(xì)胞等細(xì)胞中,已有的研究表明TREM-2參與了細(xì)胞的免疫、吞噬、炎癥調(diào)節(jié)等過(guò)程,TREM-2的表達(dá)異常參與了多種疾病病理過(guò)程。Crotti等[1]證實(shí)在在急性類風(fēng)濕關(guān)節(jié)炎的病人滑膜組織的RA-FLS中TREM2的表達(dá)明顯升高。本課題組前期的實(shí)驗(yàn)結(jié)果顯示在類風(fēng)濕關(guān)節(jié)炎模型大鼠滑膜組織FLS 中TREM-2表達(dá)明顯升高[2]。但TREM-2在RA-FLS遷移和侵襲中所起到作用以及其機(jī)制尚缺乏相關(guān)報(bào)道。本文通過(guò)RNA干擾技術(shù)對(duì)該問(wèn)題進(jìn)行了初步探討,以期進(jìn)一步闡明RA病理機(jī)制。
材料和方法
1siRNA沉默RA-FLS細(xì)胞TREM-2基因
人RA-FLS購(gòu)于Cell Applications,用含10%胎牛血清的高糖DMEM培養(yǎng)基(Gibco),37 ℃、5% CO2培養(yǎng)箱中培養(yǎng),每2~3 d換液1次,細(xì)胞覆蓋率>80%時(shí)用胰酶消化傳代,取第3~7代細(xì)胞用于實(shí)驗(yàn)。將RA-FLS 以5×107/L的密度接種于6 孔板,每孔2 mL完全培養(yǎng)基,當(dāng)細(xì)胞融合度達(dá)到50%~70%時(shí)采用脂質(zhì)體介導(dǎo)TREM-2 siRNA 轉(zhuǎn)染細(xì)胞。設(shè)置RA-FLS 轉(zhuǎn)染TREM-2 siRNA組(TREM-2 si-RNA)、RA-FLS空白對(duì)照組(unmanipulated control)和RA-FLS 轉(zhuǎn)染非特異性 siRNA 組(control siRNA)。其中特異性小分子TREM-2 siRNA 序列的正義鏈為5’-GCCUCUUGGAAGGAGAAAUTT-3’,反義鏈為5’-AUUUCUCCUUCCAAGAGGCTT-3’。陰性對(duì)照siRNA序列的正義鏈為5’-UUCUCCGAACGUGUCAGGUTT-3’,反義鏈為5’-ACGUGACACGUUCGG AGAATT-3’。在轉(zhuǎn)染6 h 后,先用PBS(Gibco)洗滌細(xì)胞3 次,洗脫殘留在培養(yǎng)基或細(xì)胞表面的FAM-siRNA,以減少背景干擾。熒光顯微鏡觀察,成功轉(zhuǎn)染的細(xì)胞可看到FAM 的淡綠色熒光散在分布于細(xì)胞質(zhì)。
2RT-PCR反應(yīng)檢測(cè)TREM-2 mRNA 的表達(dá)水平
用Trizol 試劑(Invitrogen)提取總RNA,逆轉(zhuǎn)錄成cDNA,然后進(jìn)行PCR擴(kuò)增。TREM-2上游引物序列為 5’-GTCTTGCCCCTATGACTCCA-3’,下游引物序列為5’-CTGGTAGAGACCCGCATCAT-3’;內(nèi)參照采用GAPDH, 上游引物序列為5’-GAAGGTCGGAGTCAACGG-3’,下游引物序列為5’-GGAAGATGGTGATGGGATT-3’。取RT-PCR 產(chǎn)物5 μL與1 μL 6×loading buffer混合,在2%的瓊脂糖(BIOWEST)凝膠中電泳,然后用凝膠成像系統(tǒng)進(jìn)行拍照,用ImageJ軟件進(jìn)行分析。
3Western blot分析相關(guān)蛋白表達(dá)情況
轉(zhuǎn)染48 h 后收集各組細(xì)胞提取總蛋白,采用BCA 法對(duì)蛋白定量后,各組取25 μg蛋白加入上樣緩沖液后混勻,95 ℃水浴變性5 min。80 V濃縮膠,120 V 分離膠電泳分離蛋白質(zhì),280 mA 1 h 將蛋白質(zhì)轉(zhuǎn)移至PVDF 膜(Millipore)上。5%脫脂奶粉封閉后加入Ⅰ抗,4 ℃搖床孵育過(guò)夜,TBST 洗膜3次,加入辣根過(guò)氧化物酶標(biāo)記的Ⅱ抗室溫孵育1 h,TBST 洗膜處理3次,于凝膠自動(dòng)成像系統(tǒng)曝光,用ImageJ軟件進(jìn)行分析。
4CCK-8 法檢測(cè)細(xì)胞生長(zhǎng)情況
將處在對(duì)數(shù)生長(zhǎng)期的RA-FLS以1.0×107/L 的密度接種于96 孔板,每孔100 μL,細(xì)胞融合度達(dá)到30%時(shí)按以上方法進(jìn)行轉(zhuǎn)染。實(shí)驗(yàn)分組同前,轉(zhuǎn)染24 h后換液, 并于轉(zhuǎn)染12、24、36、48、60和72 h后, 每孔加入CCK-8 液10 μL, 37 ℃繼續(xù)孵育4 h,用分光光度計(jì)490 nm 波長(zhǎng)處檢測(cè)吸光度A值。
5Transwell 細(xì)胞遷移實(shí)驗(yàn)
在24孔板中放入 8 μm 的Boyden 小室(Corning),準(zhǔn)備好無(wú)血清細(xì)胞懸液,計(jì)數(shù)板計(jì)數(shù)后在上室中準(zhǔn)確加入1.0× 108/L 的細(xì)胞懸液100 μL,下室加入含10% 胎牛血清的培養(yǎng)基500 μL 后,置培養(yǎng)箱中遷移24 h。將上室取出,PBS清洗3 遍,加入甲醇(Sigma)200 μL 固定細(xì)胞30 min,0.1%結(jié)晶紫(Sigma)染色20 min。將結(jié)晶紫吸出,用棉簽輕輕擦拭膜內(nèi)表面,用PBS清洗3遍,放在高倍鏡下(×100)觀察 ,每張膜隨機(jī)取8 個(gè)視野計(jì)數(shù),每個(gè)標(biāo)本重復(fù)3 次,取其平均值。
6Transwell 細(xì)胞侵襲實(shí)驗(yàn)
在24孔板中放入 8 μm 的Boyden 小室(Corning)并鋪好Matrigel膠(Corning),準(zhǔn)備好細(xì)胞懸液,計(jì)數(shù)板計(jì)數(shù)后在上室中準(zhǔn)確加入1.0× 108/L 的無(wú)血清細(xì)胞懸液100 μL,下室加入含10% 胎牛血清的培養(yǎng)基500 μL 后,置培養(yǎng)箱中遷移48 h。將上室取出,PBS清洗3 遍,加入甲醇 200 μL 固定細(xì)胞30 min,0.1%結(jié)晶紫染色20 min。將結(jié)晶紫吸出,用棉簽輕輕擦拭膜內(nèi)表面,用PBS清洗3遍,放在高倍鏡下(×100)觀察 ,每張膜隨機(jī)取8 個(gè)視野計(jì)數(shù),每個(gè)標(biāo)本重復(fù)3 次,取其平均值。
7ELISA實(shí)驗(yàn)
轉(zhuǎn)染24 h 后,取上述各組上清液,用ELISA試劑盒(欣博盛)檢測(cè)MMP-2和MMP-9 的濃度,每個(gè)標(biāo)本重復(fù)3 次,取其平均值。
8統(tǒng)計(jì)學(xué)處理
所有數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示,多個(gè)樣本均數(shù)比較采用單因素方差分析(one-way ANOVA),兩兩比較使用Bonferroni法。由SPSS 17.0 統(tǒng)計(jì)軟件完成,以P<0.05 為差異有統(tǒng)計(jì)學(xué)意義。
結(jié)果
1轉(zhuǎn)染siRNAs對(duì)RA-FLS TREM-2 mRNA和蛋白表達(dá)的影響
RT-PCR結(jié)果顯示,TREM-2 siRNA對(duì)RA-FLS的mRNA干擾效率為空白對(duì)照組的(79.0±3.4)% (P<0.05),為陰性對(duì)照組的(80.3±3.6)% (P<0.05),空白對(duì)照組和陰性對(duì)照組之間的TREM-2的mRNA表達(dá)未見(jiàn)明顯差異。Western blot檢測(cè)結(jié)果顯示TREM-2 siRNA組的TREM-2蛋白表達(dá)水平明顯降低,沉默效率為空白對(duì)照組的(78.6±3.7)% (P<0.05),為陰性對(duì)照組(79.7±7.6)% (P<0.05),空白對(duì)照組和陰性對(duì)照組之間的TREM-2蛋白表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義,見(jiàn)圖1。
Figure 1.TREM-2 expression at mRNA and protein levels in RA-FLS 24 h after transfection detected by RT-PCR and Western blot. Mean±SD.n=3.*P<0.05vsTREM-2 siRNA.
圖1RT-PCR和Western blot檢測(cè)轉(zhuǎn)染24 h后RA-FLS中TREM-2 mRNA和蛋白的表達(dá)
2沉默TREM-2后對(duì)RA-FLS的增殖的影響
轉(zhuǎn)染TREM-2 siRNA組、空白對(duì)照組和陰性對(duì)照組3組RA-FLS的細(xì)胞活力差異不顯著,見(jiàn)圖2。
Figure 2.CCK-8 analysis of cell growth curves in RA-FLS treated with siRNA. Mean±SD.n=3.
圖2CCK-8法檢測(cè)siRNA 轉(zhuǎn)染后RA-FLS的生長(zhǎng)曲線
3沉默TREM-2后對(duì)RA-FLS遷移和侵襲的影響
100 倍顯微鏡下觀察,遷移實(shí)驗(yàn)可見(jiàn)TREM-2 siRNA 組的穿膜細(xì)胞明顯多于對(duì)照,TREM-2 siRNA 組相對(duì)于空白對(duì)照組和NC-siRNA 組, 遷移細(xì)胞數(shù)目分別增加了95.68%和81.66%(P<0.05)。侵襲實(shí)驗(yàn)結(jié)果顯示TREM-2 siRNA 組的穿膜細(xì)胞也明顯多于對(duì)照,TREM-2 siRNA 組相對(duì)于空白對(duì)照組和NC-siRNA 組,侵襲細(xì)胞數(shù)目分別增加了74.99%和63.05%(P<0.05),見(jiàn)圖3。
4沉默TREM-2后對(duì)RA-FLS分泌MMP-2和MMP-9的影響
ELISA結(jié)果顯示,TREM-2 siRNA 組的MMP-2分泌水平相對(duì)于空白對(duì)照組和NC-siRNA 組,分別增高了70.97%和74.06%(P<0.05)。而TREM-2 siRNA 組MMP-9的分泌水平相對(duì)于空白對(duì)照組和NC-siRNA 組,差異無(wú)統(tǒng)計(jì)學(xué)意義,見(jiàn)圖4。
Figure 3.Transwell analysis of the migration and invasion of RA-FLS treated with siRNA (crystal violet staining, ×100). Mean±SD.n=3.*P<0.05vsTREM-2 si RNA.
圖3Transwell檢測(cè)siRNA 轉(zhuǎn)染后RA-FLS的 遷移和侵襲情況
5沉默TREM-2后對(duì)RA-FLS中 PI3K/AKT通路的影響
Western blot結(jié)果顯示,TREM-2 siRNA 組AKT相對(duì)磷酸化水平與空白對(duì)照組和NC-siRNA 組比較分別增高了1.33倍和1.22倍(P<0.05),見(jiàn)圖5。
討論
最新的研究表明,RA的主要病理機(jī)制是滑膜增生、炎細(xì)胞浸潤(rùn)、大量血管翳形成最終導(dǎo)致軟骨和骨組織破壞,其中滑膜細(xì)胞在RA的發(fā)生發(fā)展中起到了重要的作用[3]。有研究報(bào)道表明FLS 侵襲能力強(qiáng)的RA患者Sharp評(píng)分更高且更容易形成影像學(xué)可見(jiàn)的骨關(guān)節(jié)破壞[4];Lefevre 等[5]研究發(fā)現(xiàn)RA-FLS除了在關(guān)節(jié)腔內(nèi)發(fā)生遷移和侵蝕外,還可以進(jìn)行長(zhǎng)距離遷移以及侵蝕,對(duì)正常的關(guān)節(jié)組織起到了破壞作用。因此,調(diào)節(jié)RA-FLS遷移和侵襲的激活可能成為一種重要的RA治療策略。MMPs作為一類鋅依賴性肽鏈內(nèi)切酶,在降解基膜和細(xì)胞外基質(zhì)中發(fā)揮著重要作用,其中RA-FLS遷移和侵襲功能與MMP-2和MMP-9的高表達(dá)有密切的關(guān)系[6], MMP-2和MMP-9 可以作為骨侵蝕的重要指標(biāo)。此外RA患者血清和滑液中MMP-2和MMP-9顯著增高,其水平和C反應(yīng)蛋白等病情活動(dòng)指標(biāo)有明顯相關(guān)性。
Figure 4.ELISA analysis of MMP-2 and MMP-9 released by RA-FLS treated with siRNA. Mean±SD.n=3.*P<0.05vsTREM-2 siRNA.
圖4ELISA檢測(cè)siRNA 轉(zhuǎn)染后RA-FLS分泌MMP-2和MMP-9情況
Figure 5.Western blot analysis of PI3K/AKT pathway activation in RA-FLS treated with siRNA for 48 h. Mean±SD.n=3.*P<0.05vsTREM-2 siRNA.
圖5Western blot檢測(cè)轉(zhuǎn)染48 h后RA-FLS中PI3K/AKT通路活化水平
作為免疫受體超家族中的一員,TREM-2是一種重要的受體膜蛋白,近年的研究表明當(dāng)受到配體刺激時(shí)TREM-2可以與DNA活化蛋白12(DNA activating protein-12,DAP12)偶聯(lián),進(jìn)一步激活免疫受體酪氨酸激活基序(immunoreceptor tyrosine-based activation motif,ITAM)等下游傳遞信號(hào)發(fā)揮生理學(xué)效應(yīng)[7]。TREM-2在不同細(xì)胞中表現(xiàn)出不同的生理功能: TREM-2能夠增強(qiáng)巨噬細(xì)胞分化、吞噬病原體以及負(fù)性調(diào)控炎癥反應(yīng)[8];TREM-2能促進(jìn)樹(shù)突狀細(xì)胞的成熟、負(fù)性調(diào)控Toll樣受體介導(dǎo)的生理功能[9]; TREM-2能夠促進(jìn)破骨細(xì)胞的分化、參與骨組織吸收和塑性;最近Ito等[10]研究表明TREM-2能夠促進(jìn)脂肪細(xì)胞肥大從而誘導(dǎo)肥胖。但由于發(fā)現(xiàn)較晚且尚未得到公認(rèn)的天然配體, TREM-2的研究尚處于初步階段。雖然已經(jīng)證實(shí)TREM-2在急性活動(dòng)期RA-FLS中高表達(dá),但TREM-2是否介導(dǎo)FLS的功能異常尚缺乏相關(guān)研究。
短鏈干擾RNA能夠高度特異性降解同源相應(yīng)的mRNA靶序列,成功阻斷相關(guān)基因的表達(dá)。以siRNA為基礎(chǔ)的基因沉默技術(shù)廣泛應(yīng)用于功能基因組學(xué)研究、基因治療、藥物篩選等領(lǐng)域。本研究首先利用該技術(shù)干擾RA-FLS中TREM-2 mRNA及蛋白的表達(dá),RA-FLS的增殖水平并未受到影響。隨后的Transwell遷移和侵襲實(shí)驗(yàn)表明,干擾TREM-2能夠增強(qiáng)RA-FLS的遷移和侵襲能力。之后的ELISA實(shí)驗(yàn)結(jié)果證實(shí),沉默TREM-2后RA-FLS分泌MMP-2水平顯著提高,而MMP-9的分泌水平變化不大,說(shuō)明TREM-2介導(dǎo)的RA-FLS遷移與侵襲能力的改變可能與MMP-2有關(guān),而與MMP-9關(guān)系不大,其機(jī)制尚不清楚。由于PI3K/AKT通路在包括RA-FLS在內(nèi)的多種細(xì)胞的遷移和侵襲中發(fā)揮著重要的作用[11],且已有的研究表明TREM-2的功能與PI3K/AKT通路有密切關(guān)系[12-13],因此我們推測(cè)TREM-2在RA-FLS中很可能也是通過(guò)PI3K/AKT通路起作用,我們進(jìn)一步驗(yàn)證沉默TREM-2后是否影響RA-FLS中PI3K/AKT通路的活化水平,Western blot實(shí)驗(yàn)結(jié)果顯示,與對(duì)照組相比特異性干擾組RA-FLS的磷酸化水平顯著提高,表明TREM-2調(diào)節(jié)了RA-FLS中PI3K/AKT的活化水平,但究竟是否通過(guò)經(jīng)典的TREM-2/DAP12偶聯(lián)受體進(jìn)一步激活下游的ITAM等下游受體,影響了PI3K/AKT通路的活化水平,以及PI3K/AKT是否對(duì)TREM-2的轉(zhuǎn)錄和表達(dá)有反饋性的影響,仍需要進(jìn)一步研究。
總之,TREM-2能夠負(fù)性調(diào)節(jié)RA-FLS的MMP-2分泌水平以及其遷移和侵襲的能力,但并沒(méi)有改變其增殖能力,這說(shuō)明TREM-2在RA的關(guān)節(jié)局部病變中可能起到了一定的保護(hù)作用。這些調(diào)控很可能和PI3K/AKT通路有密切的關(guān)系。本實(shí)驗(yàn)研究對(duì)進(jìn)一步認(rèn)識(shí)RA關(guān)節(jié)破壞的機(jī)制以及發(fā)現(xiàn)RA治療中可能的新靶點(diǎn)具有重要意義。
[參考文獻(xiàn)]
[1]Crotti TN, Dharmapatni AA, Alias E, et al. The immunoreceptor tyrosine-based activation motif (ITAM)-related factors are increased in synovial tissue and vasculature of rheumatoid arthritic joints[J]. Arthritis Res Ther, 2012, 14(6):R245.
[2]葉培,李建華,許錦煌,等. 類風(fēng)濕關(guān)節(jié)炎模型大鼠滑膜組織中證實(shí)有髓樣細(xì)胞誘發(fā)受體2的表達(dá)[J]. 中國(guó)組織工程研究, 2015, 19(18):2807-2813.
[3]Noss EH, Brenner MB. The role and therapeutic implications of fibroblast-like synoviocytes in inflammation and cartilage erosion in rheumatoid arthritis[J]. Immunol Rev, 2008, 223(1):252-270.
[4]劉志昌,肖游君,勞敏曦,等. 抗環(huán)瓜氨酸肽抗體與類風(fēng)濕關(guān)節(jié)炎患者成纖維樣滑膜細(xì)胞遷移和侵襲能力的相關(guān)研究[J]. 中國(guó)病理生理雜志, 2013,29(12):2277-2281.
[5]Lefevre S, Knedla A, Tennie C, et al. Synovial fibroblasts spread rheumatoid arthritis to unaffected joints[J]. Nat Med, 2009, 15(12):1414-1420.
[6]Yuan H, Yang P, Zhou D, et al. Knockdown of sphingosine kinase 1 inhibits the migration and invasion of human rheumatoid arthritis fibroblast-like synoviocytes by down-regulating the PI3K/AKT activation and MMP-2/9 productioninvitro[J]. Mol Biol Rep, 2014, 41(8):5157-5165.
[7]Li G, Zhang Y, Qian Y, et al. Interleukin-17A promotes rheumatoid arthritis synoviocytes migration and invasion under hypoxia by increasing MMP2 and MMP9 expression through NF-κB/HIF-1α pathway[J]. Mol Immunol, 2013, 53(3):227-236.
[8]Paradowska-Gorycka A, Jurkowska M. Structure, expression pattern and biological activity of molecular complex TREM-2/DAP12[J]. Hum Immunol, 2013, 74(6):730-737.
[9]Chen Q, Zhang K, Jin Y, et al. Triggering receptor expressed on myeloid cells-2 protects against polymicrobial sepsis by enhancing bacterial clearance[J]. Am J Respir Crit Care Med, 2013, 188(2):201-212.
[10]Ito H, Hamerman JA. TREM-2, triggering receptor expressed on myeloid cell-2, negatively regulates TLR responses in dendritic cells[J]. Eur J Immunol, 2012, 42(1):176-185.
[11]Park M, Yi JW, Kim EM, et al. Triggering receptor expressed on myeloid cells 2 (TREM2) promotes adipogenesis and diet-induced obesity[J]. Diabetes, 2015, 64(1):117-127.
[12]Peng Q, Malhotra S, Torchia JA, et al. TREM2- and DAP12-dependent activation of PI3K requires DAP10 and is inhibited by SHIP1[J]. Sci Signal, 2010, 3(122):ra38.
[13]Turnbull IR, Colonna M. Activating and inhibitory functions of DAP12[J]. Nat Rev Immunol, 2007, 7(2):155-161.
(責(zé)任編輯: 陳妙玲, 羅森)
Effects of TREM-2 silencing on migration and invasion abilities of rheumatoid arthritis fibroblast-like synoviocytes
HUANG Sheng-hui1, LI Jian-hua2, ZHANG Wei-qiong3, LIU Gui-wang1, XU Jin-huang3, ZHENG Pei-zhong3, HUANG Jian-rong1, 3
(1DepartmentofOrthopaedics,SunYat-senMemorialHospital,SunYat-senUniversity,Guangzhou510235,China;2DepartmentofPhysiology,GuangzhouMedicalUniversity,Guangzhou511436,China;3DepartmentofOrthopaedics,ZengchengPeople’sHospital,Guangzhou511300,China.E-mail:guke16@163.com)
[ABSTRACT]AIM: To investigate the effects of triggering receptor expressed on myeloid cells-2 (TREM-2) silencing on migration and invasion abilities of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). METHODS: Small interference RNA (siRNA) specifically targeting TREM-2 gene was transfected into RA-FLS. The interference efficiency of TREM-2 siRNA on the production of TREM-2 mRNA and protein was determined by RT-PCR and Western blot. The cell activity was assessed by CCK-8 assay. The migration and invasion abilities of RA-FLS were determined by Transwell assay. The releases of MMP-2 and MMP-9 in RA-FLS were analyzed by ELISA. The influence of TREM-2 on PI3K/AKT signal pathway was measured by Western blot. RESULTS: TREM-2 siRNA significantly decreased the mRNA and protein expression of TREM-2. No difference of cell activity between TREM-2 siRNA group and control group was observed. Transwell migration assay showed that RA-FLS through the Transwell membrane in TREM-2 siRNA group were more than the blank control group and the NC-siRNA group. In Transwell invasion assay, RA-FLS through the Transwell membrane in TREM-2 siRNA group were more than the blank control group and the NC-siRNA group. After transfected with TREM-2 siRNA, the MMP-2 secretion and phosphorylation of AKT increased significantly, while the MMP-9 secretion was not changed. CONCLUSION: TREM-2 may play an important role in the migration and invasion of RA-FLS through regulating the activation of PI3K/AKT signal pathway.
[KEY WORDS]Triggering receptor expressed on myeloid cells-2; Rheumatoid arthritis; Synoviocytes; Cell migration; Cell invasion; PI3K/AKT pathway
[文章編號(hào)]1000- 4718(2016)01- 0134- 06
[收稿日期]2015- 07- 24[修回日期] 2015- 09- 10
*[基金項(xiàng)目]廣東省科技基金資助項(xiàng)目(No. 2010B031600184);廣州市科技和信息化局重點(diǎn)項(xiàng)目(No. 11C31120789);廣東省自然科學(xué)基金資助項(xiàng)目(No. S2012010010629; No. S2013010013768)
通訊作者△Tel: 020-82725271; E-mail: guke16@163.com
[中圖分類號(hào)]R363
[文獻(xiàn)標(biāo)志碼]A
doi:10.3969/j.issn.1000- 4718.2016.01.023