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南京醫(yī)科大學(xué)附屬南京醫(yī)院(南京市第一醫(yī)院) 1.消化科； 2.中心實(shí)驗(yàn)室，江蘇 南京 210006； 3.解放軍南京總醫(yī)院急診科

【Abstract】ObjectiveTo investigate whether Rebamipide plays a protective role on Aspirin-induced injury through inhibiting the expression of miR-877-5p in human gastric epithelial cells (GES-1).MethodsCultured GES-1 cells were divided into control group, Aspirin injured group and different concentrations (0.25, 0.5, 1.0 mmol/L) Rebamipide plus Aspirin groups. The expression of miR-877-5p was detected by qRT-PCR. The Aspirin treated cells were transfected with miR-877-5p inhibitors. The combination of Rebamipide and Aspirin treated cells were transfected with miR-8877-5p mimics. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry. The targeted genes of miR-877-5p were predicted by miRNA target databases, the GO and KEGG pathway were analyzed by DAVID database.ResultsqRT-PCR showed that the expression of miR-877-5p in Aspirin group was the highest than others. The expressions of miR-877-5p in different concentrations (0.25, 0.5, 1.0 mmol/L) of Rebamipide plus Aspirin groups were 4.28±0.25, 2.45±0.28 and 1.47±0.17. The expression of miR-877-5p was gradually decreased with the increase of concentration of Rebamipide. The CCK-8 assay and flow cytometry showed that miR-877-5p mimics transfection blocked proliferations and promoted apoptosis in combination of Rebamipide and Aspirin treated cells, while miR-877-5p inhibitors promoted proliferation and inhibited apoptosis in Aspirin treated cells. The pathways of miR-877-5p targeted genes were almost focused on cAMP signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, and so on.ConclusionRebamipide alleviates Aspirin-induced injury through inhibiting the expression of miR-877-5p in human gastric epithelial cells.

【Keywords】 Rebamipide; Aspirin; Gastric mucosa damage; miR-877-5p

近年來，隨著阿司匹林在心腦血管疾病中的廣泛應(yīng)用，其相關(guān)不良反應(yīng)尤其是胃黏膜損傷的發(fā)生率逐年上升。阿司匹林相關(guān)胃黏膜損傷的臨床表現(xiàn)可以無(wú)癥狀或有消化不良、出血、穿孔等。瑞巴派特是臨床上常用的胃黏膜保護(hù)劑，廣泛應(yīng)用于胃黏膜損傷和潰瘍的防治中，尤其在阿司匹林相關(guān)胃黏膜損傷的防治中效果突出[2-4]。然而，瑞巴派特對(duì)胃黏膜保護(hù)作用的機(jī)制尚未完全明確，近年來研究[5-6]發(fā)現(xiàn)，miRNAs參與了細(xì)胞發(fā)育、增殖、分化和凋亡等重要過程，這為研究瑞巴派特對(duì)胃黏膜保護(hù)作用機(jī)制提供了新的研究方向。

有研究[7]發(fā)現(xiàn)，NSAIDs類藥物引起的細(xì)胞損傷中miR-877-5p明顯上調(diào)，而我們經(jīng)生物信息學(xué)初步分析發(fā)現(xiàn)，其可能參與細(xì)胞的增殖和凋亡調(diào)控，因此推測(cè)NSAIDs藥物阿司匹林對(duì)胃黏膜的損傷作用可能與miR-877-5p有關(guān)，而胃黏膜保護(hù)劑瑞巴派特也可能是通過調(diào)控該miRNA表達(dá)發(fā)揮保護(hù)性作用。因此，本研究將從阿司匹林和瑞巴派特對(duì)miR-877-5p表達(dá)的影響和miR-877-5p對(duì)人胃黏膜上皮細(xì)胞GES-1增殖、凋亡的影響兩方面入手，闡明瑞巴派特防治阿司匹林相關(guān)胃黏膜損傷的作用機(jī)制，為瑞巴派特的臨床應(yīng)用提供更多的理論基礎(chǔ)。

1 材料與方法

1.1材料人胃黏膜上皮細(xì)胞株GES-1由南京市第一醫(yī)院中心實(shí)驗(yàn)室保存。瑞巴派特(上海阿拉丁生物技術(shù)有限公司)，阿司匹林(美國(guó)Sigma公司)，脂質(zhì)體轉(zhuǎn)染試劑 Lipofectamine 2000(美國(guó) Invitrogen)，miR-877-5p mimics和miRNA negative control(NC)、miR-877-5p inhibitor和miRNA inhibitor negative control(INC)、實(shí)時(shí)熒光定量試劑盒(上海吉瑪)，細(xì)胞總RNA提取試劑Trizol、CCK-8試劑盒、Annexin V-FITC/PI雙染細(xì)胞凋亡檢測(cè)試劑盒(南京翼飛雪生物科技有限公司)。

1.2 方法

1.2.1 細(xì)胞培養(yǎng)：GES-1細(xì)胞用含質(zhì)量濃度為100 g/L胎牛血清的DMEM高糖培養(yǎng)基，在37 ℃、體積分?jǐn)?shù)為5%的CO2條件下常規(guī)培養(yǎng)傳代。

1.2.2 實(shí)驗(yàn)分組：研究瑞巴派特對(duì)細(xì)胞中miR-877-5p表達(dá)的影響，實(shí)驗(yàn)分為5組：不加藥物干預(yù)的空白對(duì)照組(NC組)；加入2.21 mmol/L濃度的阿司匹林培養(yǎng)液(即IC10濃度)處理24 h設(shè)為阿司匹林損傷組[8]；加入不同濃度(0.25、0.5、1.0 mmol/L)瑞巴派特預(yù)處理2 h，再加入IC10濃度的阿司匹林培養(yǎng)液處理24 h，設(shè)為瑞巴派特保護(hù)組。實(shí)驗(yàn)重復(fù)3次。

研究miR-877-5p對(duì)GES-1細(xì)胞增殖和凋亡的影響，實(shí)驗(yàn)分為4組：1、2組均用終濃度為0.5 mmol/L瑞巴派特聯(lián)合阿司匹林處理24 h后分別加入miR-877-5p mimics、NC進(jìn)行轉(zhuǎn)染；3、4組均用阿司匹林處理24 h后分別加入miR-877-5p inhibitor、INC進(jìn)行轉(zhuǎn)染。轉(zhuǎn)染后進(jìn)行細(xì)胞凋亡實(shí)驗(yàn)和細(xì)胞增殖實(shí)驗(yàn)。

1.2.3 細(xì)胞轉(zhuǎn)染：取對(duì)數(shù)生長(zhǎng)期接種于細(xì)胞培養(yǎng)板中培養(yǎng)24 h，待細(xì)胞達(dá)70%～80%融合時(shí)進(jìn)行轉(zhuǎn)染。按照Lipofectamine 2000說明書進(jìn)行轉(zhuǎn)染，將miR-877-5p mimics、NC、inhibitor和INC分別轉(zhuǎn)染至細(xì)胞中。6 h后更換新鮮培養(yǎng)基，48 h后收集細(xì)胞或進(jìn)行相關(guān)實(shí)驗(yàn)，通過實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(qRT-PCR)驗(yàn)證轉(zhuǎn)染是否成功。

1.2.4 qRT-PCR：收集細(xì)胞，提取總RNA，測(cè)定RNA濃度并按照說明書進(jìn)行逆轉(zhuǎn)錄，將逆轉(zhuǎn)錄產(chǎn)物cDNA加入反應(yīng)體系中進(jìn)行qPCR反應(yīng)，檢測(cè)各組細(xì)胞間miRNA表達(dá)的差異。基因相對(duì)表達(dá)量用2-ΔΔCt表示。實(shí)驗(yàn)重復(fù)3次，取均值。

1.2.5 細(xì)胞增殖檢測(cè)：細(xì)胞鋪于96孔板中，分別繼續(xù)培養(yǎng)24 h、48 h和72 h，每孔加入10 μl CCK-8，37 ℃孵育2 h，以450 nm為測(cè)定波長(zhǎng)，在酶標(biāo)儀上檢測(cè)各孔密度值(OD值)。OD值可反映細(xì)胞增殖情況，每組設(shè)4個(gè)復(fù)孔，實(shí)驗(yàn)重復(fù)3次，取均值。

1.2.6 細(xì)胞凋亡檢測(cè)：各組細(xì)胞轉(zhuǎn)染48 h后，收集細(xì)胞，PBS洗滌2遍，依次加入500 μl結(jié)合緩沖液及Annexin V FITC和PI各5 μl，混勻后室溫避光孵育15 min，上流式細(xì)胞儀檢測(cè)。實(shí)驗(yàn)重復(fù)3次，取均值。

1.2.7 miR-877-5p靶蛋白預(yù)測(cè)：通過miRNA靶基因在線預(yù)測(cè)軟件(Targetscan、miRWalk、miRanda)，取3個(gè)數(shù)據(jù)庫(kù)預(yù)測(cè)的交集靶基因進(jìn)行GO功能富集分析和KEGG信號(hào)通路富集分析。

2 結(jié)果

2.1miR-877-5p在阿司匹林損傷組和瑞巴派特保護(hù)組中的表達(dá)情況qRT-PCR檢測(cè)miR-877-5p表達(dá)量，設(shè)空白對(duì)照組表達(dá)量為1，阿司匹林損傷組為6.43±0.48,0.25 mmol/L瑞巴派特保護(hù)組為4.28±0.25,0.5 mmol/L瑞巴派特保護(hù)組為2.45±0.28,1.0 mmol/L瑞巴派特保護(hù)組為1.47±0.17,阿司匹林損傷組中miR-877-5p表達(dá)高于其他保護(hù)組(P<0.001)。不同濃度的瑞巴派特保護(hù)組中，miR-877-5p表達(dá)量依次降低，呈濃度依賴性(F=71.87,P<0.01)。以上結(jié)果說明,瑞巴派特能夠抑制GES-1細(xì)胞中miR-877-5p的表達(dá)。

2.2轉(zhuǎn)染miR-877-5pmimics和inhibitor的效果分析qRT-PCR結(jié)果顯示，miR-877-5p mimics組miR-877-5p的表達(dá)是NC組的(92.77±8.00)倍，差異有統(tǒng)計(jì)學(xué)意義(P<0.001)；miR-877-5p inhibitor組miR-877-5p的表達(dá)是INC組的(0.55±0.02)倍，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。以上結(jié)果說明轉(zhuǎn)染成功。

2.3miR-877-5p對(duì)人胃黏膜上皮細(xì)胞GES-1增殖的影響細(xì)胞增殖結(jié)果顯示，轉(zhuǎn)染miR-877-5p mimics組的細(xì)胞增殖能力較NC組顯著降低(P<0.01)，第3天抑制增殖效果最明顯[(0.683±0.035)vs(1.112±0.039),P<0.001]。轉(zhuǎn)染miR-877-5p inhibitor組的細(xì)胞增殖能力較INC組明顯增強(qiáng)(P<0.05)(見表1)。以上結(jié)果提示，miR-877-5p對(duì)人胃黏膜上皮細(xì)胞的增殖具有抑制作用。

表1 CCK-8法檢測(cè)miR-877-5p對(duì)人胃黏膜上皮細(xì)胞GES-1增殖的影響Tab 1 Analysis of proliferation in miR-877-5p transfected GES-1 cells by CCK-8

注：與NC組相比，*P<0.05，**P<0.001；與INC組相比，#P<0.05。

2.4miR-877-5p對(duì)人胃黏膜上皮細(xì)胞GES-1凋亡的影響流式細(xì)胞凋亡實(shí)驗(yàn)結(jié)果顯示，NC組、miR-877-5p mimics組、INC組和miR-877-5p inhibitor組細(xì)胞凋亡率分別為(8.93±0.74)%、(24.87±3.35)%、(13.4±0.96)%、(5.87±0.53)%。與NC組比較，miR-877-5p mimics處理的細(xì)胞凋亡率明顯增加，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。與INC組比較，miR-877-5p inhibitor處理的細(xì)胞凋亡率降低，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)(見圖1)。以上結(jié)果表明，上調(diào)細(xì)胞內(nèi)miR-877-5p的表達(dá)可促進(jìn)人胃黏膜上皮細(xì)胞的凋亡，而下調(diào)其表達(dá)，可抑制細(xì)胞的凋亡。

圖1 流式細(xì)胞術(shù)檢測(cè)miR-877-5p對(duì)人胃黏膜上皮細(xì)胞凋亡的影響

A：NC組；B：miR-877-5p mimic組；C：INC組；D：miR-877-5p inhibitor組

Fig1AnalysisofapoptosisinmiR-877-5ptransfectedGES-1cellsbyflowcytometry

A: NC group; B: miR-877-5p mimic group; C: INC group; D: miR-877-5p inhibitor group

2.5miR-877-5p靶基因預(yù)測(cè)和功能分析應(yīng)用Targetscan、miRWalk和miRanda 3個(gè)數(shù)據(jù)庫(kù)進(jìn)行靶基因預(yù)測(cè)并取交集基因，共發(fā)現(xiàn)交集基因有945個(gè)。對(duì)這些靶基因富集進(jìn)行GO功能富集分析發(fā)現(xiàn)，miR-877-5p靶基因主要參與了金屬離子結(jié)合、轉(zhuǎn)錄調(diào)節(jié)、細(xì)胞內(nèi)蛋白修飾、蛋白定位等生物功能(P<0.05)。對(duì)這些靶基因進(jìn)行KEGG通路分析發(fā)現(xiàn)，共有65個(gè)明顯富集的通路(P<0.05)，其中富集指數(shù)較高的通路有cAMP信號(hào)通路、鈣離子信號(hào)通路、PI3K-Akt信號(hào)通路、MAPK信號(hào)通路等(見圖2)。

3 討論

miRNA是近年來生命科學(xué)領(lǐng)域研究的一個(gè)熱點(diǎn)，它是一類廣泛存在于真核生物體內(nèi)，長(zhǎng)度在19～25個(gè)核苷酸的內(nèi)源性非編碼RNA。它能通過干擾mRNA翻譯過程即堿基互補(bǔ)配對(duì)方式來調(diào)節(jié)靶基因的表達(dá)[9]，對(duì)體內(nèi)細(xì)胞的增殖、分化、損傷、凋亡等方面發(fā)揮重要作用。隨著近幾年研究的不斷深入，細(xì)胞損傷及凋亡與miRNA的聯(lián)系越來越清晰。多項(xiàng)研究[10-14]發(fā)現(xiàn)，阿司匹林可通過調(diào)節(jié)多種特異性miRNA的表達(dá)，在多種疾病如先兆子癇、胃腸道腫瘤等中發(fā)揮作用。既往研究發(fā)現(xiàn)，miR-877-5p參與多種細(xì)胞凋亡的過程[15-18]，在多種藥物引起的肝損傷中[19-22]，miR-877-5p表達(dá)上調(diào)且參與肝損傷過程。

注：-lg(P-value)：條形圖長(zhǎng)度，表示富集指數(shù)。

圖2miR-877-5p靶基因富集的GO分析和KEGG通路分析

A：GO分類中分子功能領(lǐng)域(MF)；B：GO分類中細(xì)胞成分領(lǐng)域(CC)；C：GO分類中生物過程領(lǐng)域(BP)；D：KEGG通路分析結(jié)果

Fig2Top10significantfunctionalGOtermsandKEGGpathwaysofmiR-877-5ptargetgenes

A: the field of molecular function (MF) in GO classification; B: the field of cellular components (CC) in GO classification; C: the biological processes (BP) of GO classification; D: the analysis results of KEGG pathway

既往研究[23-29]發(fā)現(xiàn),瑞巴派特主要通過促進(jìn)胃黏液的分泌，促進(jìn)前列腺素E(postaglandin E2, PGE2)的產(chǎn)生，抑制炎癥細(xì)胞和炎癥因子的產(chǎn)生，拮抗氧化應(yīng)激等發(fā)揮胃黏膜保護(hù)作用。有研究[30-31]報(bào)道,瑞巴派特可通過改善胃腸道黏膜屏障來預(yù)防阿司匹林引起的胃黏膜損傷。但其是否通過改變miRNA發(fā)揮胃黏膜保護(hù)作用，目前尚無(wú)報(bào)道。

本研究發(fā)現(xiàn),阿司匹林損傷的胃黏膜上皮細(xì)胞株GES-1中miR-877-5p表達(dá)明顯升高，而經(jīng)瑞巴派特預(yù)處理后miR-877-5p的表達(dá)降低，說明miR-877-5p參與瑞巴派特保護(hù)阿司匹林所致胃黏膜損傷的過程。為進(jìn)一步探討miR-877-5p的作用，我們向阿司匹林損傷組的GES-1細(xì)胞中轉(zhuǎn)染miR-877-5p inhibitor，發(fā)現(xiàn)細(xì)胞增殖能力增強(qiáng)，細(xì)胞凋亡減少；用miR-877-5p mimics轉(zhuǎn)染瑞巴派特保護(hù)組的GES-1細(xì)胞，細(xì)胞增殖受到抑制，凋亡增加，這些結(jié)果提示，miR-877-5p對(duì)GES-1細(xì)胞具有抑制增殖促進(jìn)凋亡的作用。通過生物信息學(xué)分析發(fā)現(xiàn)，miR-877-5p可能參與細(xì)胞生長(zhǎng)、增殖和凋亡等生物行為的調(diào)控過程。以上實(shí)驗(yàn)說明，瑞巴派特通過抑制GES-1細(xì)胞中阿司匹林所誘導(dǎo)的miR-877-5p的表達(dá)，發(fā)揮胃黏膜保護(hù)作用。目前已知miRNA對(duì)細(xì)胞功能活動(dòng)的影響往往是通過在轉(zhuǎn)錄后水平下調(diào)某些特定的功能基因，即靶基因的表達(dá)實(shí)現(xiàn)的[9]。本課題組下一步將在靶基因水平和蛋白水平上驗(yàn)證miR-877-5p的靶基因在GES-1細(xì)胞中的功能，進(jìn)一步證實(shí)miR-877-5p發(fā)揮的具體作用機(jī)制。

本研究證實(shí)了瑞巴派特發(fā)揮胃黏膜保護(hù)作用與miR-877-5p相關(guān)，通過抑制GES-1細(xì)胞中miR-877-5p的表達(dá)，減輕阿司匹林引起的胃黏膜上皮細(xì)胞的損傷。

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doi:10.3969/j.issn.1006-5709.2018.06.016