隋小芳,于淑倩, 王鳳玲,蘇亞楠,黃佳濱,范巧菊,陳尚君,費(fèi)秀斌*

(1.佳木斯大學(xué)附屬第一醫(yī)院 老年病科，黑龍江 佳木斯154002;2.安徽省太和縣人民醫(yī)院 老年病科; 3.佳木斯大學(xué)附屬第一醫(yī)院 科研科)

過表達(dá)miR-338-3p對(duì)炎癥信號(hào)通路的影響

隋小芳1,于淑倩2, 王鳳玲1,蘇亞楠3,黃佳濱1,范巧菊1,陳尚君1,費(fèi)秀斌1*

(1.佳木斯大學(xué)附屬第一醫(yī)院 老年病科，黑龍江 佳木斯154002;2.安徽省太和縣人民醫(yī)院 老年病科; 3.佳木斯大學(xué)附屬第一醫(yī)院 科研科)

目的研究過表達(dá)miR-338-3p在內(nèi)皮細(xì)胞系EOMA中對(duì)炎癥信號(hào)通路的影響。方法將EOMA細(xì)胞分為四組即對(duì)照組、miR-338-3p過表達(dá)組、TNF-α處理組、miR-338-3p與TNF-α共處理組。用Real time PCR檢測miR-338-3p及黏附分子VCAM-1和ICAM-1mRNA表達(dá)水平，Western blot分析ERK/p38 MAPK信號(hào)通路活性。結(jié)果與對(duì)照組比較，miR-338-3p過表達(dá)組的黏附分子VCAM-1及ICAM-1 mRNA表達(dá)水平和ERK/p38信號(hào)通路活性明顯降低，而TNF-α處理組的黏附分子VCAM-1及ICAM-1 mRNA表達(dá)水平和ERK/p38信號(hào)通路活性明顯升高；miR-338-3p與TNF-α共處理組與TNF-α處理組比較，黏附分子VCAM-1及ICAM-1 mRNA表達(dá)水平和ERK/p38信號(hào)通路活性明顯降低；miR-338-3p與TNF-α共處理組與miR-338-3p過表達(dá)組比較，黏附分子表達(dá)水平及ERK/p38信號(hào)通路活性不再升高。結(jié)論過表達(dá)miR-338-3p抑制ERK/p38信號(hào)通路活性，降低黏附分子VCAM-1和ICAM-1 mRNA表達(dá)水平；過表達(dá)miR-338-3p能夠逆轉(zhuǎn)TNF-α對(duì)ERK/p38 通路的激活和TNF-α對(duì)黏附分子VCAM-1及ICAM-1mRNA表達(dá)的促進(jìn)作用。

miR-338-3p；內(nèi)皮細(xì)胞；EOMA；TNF-α；炎癥

(ChinJLabDiagn,2017,21:1991)

動(dòng)脈粥樣硬化是心血管疾病最常見的原因。研究發(fā)現(xiàn)，microRNAs(miRNAs)參與調(diào)控動(dòng)脈粥樣硬化斑塊形成、心肌缺血/再灌注及心律失常等[1]。miR-338-3p，作為miRNA的一員，已證明參與腫瘤細(xì)胞的增值、分化與遷徙[2]，前期實(shí)驗(yàn)證實(shí)TNF-α及低表達(dá)miR-338-3p能夠激活炎癥信號(hào)通路活性。因此，本課題將進(jìn)一步探討過表達(dá)miR-338-3p對(duì)內(nèi)皮細(xì)胞炎癥信號(hào)通路的影響，為靶向治療動(dòng)脈硬化提供理論基礎(chǔ)。

1 材料與方法

1.1材料

EOMA細(xì)胞(小鼠血管瘤內(nèi)皮細(xì)胞珠，購于中國醫(yī)學(xué)科學(xué)院細(xì)胞庫)；H-DMEM 培養(yǎng)基(美國Invitrogen公司)；FBS胎牛血清(美國Hyclone公司)；TNF-α (美國Epitomics公司)；HRP標(biāo)記的羊抗兔IgG相關(guān)抗原抗體(北京中杉金橋生物技術(shù)有限公司)；miR-338-3p過表達(dá)腺病毒載體(AD-338-3p mimics)(購于上海吉?jiǎng)P科技有限公司)；Rabbit GAPDH Antibody(美國CST)；磷酸化絲裂原活化蛋白激酶(p-ERK)抗體、ERK抗體、磷酸化p38絲裂原活化蛋白激酶抗體及p38抗體均購于美國CST。

1.2細(xì)胞培養(yǎng)

EOMA細(xì)胞用含10% 胎牛血清的高糖培養(yǎng)基于37℃、含5% CO2的培養(yǎng)箱中孵育。每兩天更換一次培養(yǎng)液，細(xì)胞達(dá)80%-90%豐度時(shí)進(jìn)行傳代或用于實(shí)驗(yàn)。

1.3構(gòu)建EOMA過表達(dá)miR-338-3p模型及炎癥模型

將EOMA細(xì)胞接種于六孔板中，隨機(jī)分為四組：對(duì)照組(NC，陰性對(duì)照病毒感染EOMA細(xì)胞48 h)；過表達(dá)組(338M，過表達(dá)miR-338-3p腺病毒感染EOMA細(xì)胞48 h)；TNF-α處理組(NC+TNF-α，感染陰性對(duì)照病毒24 h后再給予TNF-α 20 ng/ml共處理24 h)；miR-338-3p與TNF-α共處理組(338M+TNF-α，過表達(dá)miR-338-3p感染EOMA細(xì)胞24 h后再給予TNF-α 20 ng/ml 處理24 h)。

1.4miR-338-3p和VCAM-1及ICAM-1mRNA表達(dá)檢測

收集上述細(xì)胞，用RNAVzol萃取細(xì)胞總RNA并純化，將RNA逆轉(zhuǎn)錄cDNA，用SYBR Green熒光定量試劑盒和實(shí)時(shí)熒光定量PCR(Real time PCR)儀檢測miR-338-3p表達(dá)和VCAM-1及ICAM-1 mRNA 表達(dá)。結(jié)果用2-ΔΔct計(jì)算相對(duì)表達(dá)量。miR-338-3p引物設(shè)計(jì)如下：

引物名稱反轉(zhuǎn)錄引物序列5′?3′miR?338?3pGTCGTATCCAGTGCAGGGTCCGAGGTAT?TCGCACTGGATACGACCAACAAU6GTCGTATCCAGTGCAGGGTCCGAGGTAT?TCGCACTGGATACGACAAATATG

引物名稱PCR引物序列5′?3′miR?338?3pGCGTCCAGCATCAGTGATTU6GCGCTCGTGAAGCGTTCReverseGTGCAGGGTCCGAGGT

VCAM-1和ICAM-1及內(nèi)參18S的引物設(shè)計(jì)如下：

引物名稱PCR引物序列5′?3′VCAM?1ForwardCACTTGTGGAAATGTGCCCGVCAM?1ReverseTCACACTCGTATATGCCGGCICAM?1ForwardTTTTCAGCTCCGGTCCTGACICAM?1ReverseCCGCTCAGAAGAACCACCTT18sForwardGGAAGGGCACCACCAGGAGT18sReverseTGCAGCCCCGGACATCTAAG

1.5Westernblot分析ERK/p38MAPK信號(hào)通路蛋白變化

用高效裂解液RIPA裂解1.3中4組細(xì)胞，高速離心取上清液，即獲得細(xì)胞總蛋白。BCA試劑盒檢測蛋白濃度，計(jì)算含15-20 μg蛋白溶液的體積即為上樣量。取出上樣量，加入4×SDS上樣緩沖液至終濃度為1×SDS，上樣前將樣品于沸水浴中煮沸5-10 min。將蛋白轉(zhuǎn)移到活化的PVDF膜上，5%脫脂牛奶封閉2 h，孵一抗過夜，室溫下孵二抗2 h，用凝膠成像儀顯影，分析蛋白條帶灰度值，以GAPDH為內(nèi)參，計(jì)算ERK和p38磷酸化水平即p-ERK和p-p38相對(duì)值。

1.6統(tǒng)計(jì)學(xué)分析

應(yīng)用SPSS 17.0統(tǒng)計(jì)軟件，采用t檢驗(yàn)和單因素方差分析進(jìn)行數(shù)據(jù)處理，以GraphPad Prism 5.0軟件繪制統(tǒng)計(jì)圖，95%可信區(qū)間，Plt;0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1EOMA細(xì)胞中過表達(dá)miR-338-3p抑制ERK/p38MAPK信號(hào)通路活性，降低促炎基因VCAM-1和ICAM-1mRNA表達(dá)水平

miR-338-3p mimics感染EOMA細(xì)胞48 h，與對(duì)照組相比，Real time PCR檢測miR-338-3p表達(dá)水平顯著升高，黏附分子VCAM-1和ICAM-1 mRNA表達(dá)水平明顯降低；Western blot分析ERK蛋白磷酸化水平(p-ERK)及p38蛋白磷酸化水平(p-p38)明顯降低。結(jié)果表明：EOMA細(xì)胞中過表達(dá)miR-338-3p能夠抑制ERK/p38 MAPK信號(hào)通路活性，降低黏附分子VCAM-1和ICAM-1 mRNA水平(圖1)。

2.2過表達(dá)miR-338-3p能夠逆轉(zhuǎn)TNF-α對(duì)EOMA細(xì)胞ERK/p38MAPK信號(hào)通路活性及黏附分子VCAM-1及ICAM-1mRNA的促進(jìn)作用

圖2結(jié)果表明，TNF-α處理EOMA細(xì)胞升高了ERK和p38磷酸化水平及黏附因子表達(dá)水平，單獨(dú)轉(zhuǎn)染miR-338-3p mimics降低了ERK和p38磷酸化水平及黏附分子表達(dá)水平，當(dāng)EOMA細(xì)胞高表達(dá)miR-338-3p再給予TNF-α處理，ERK和p38磷酸化水平及黏附分子表達(dá)水平不再升高。結(jié)果表明，過表達(dá)miR-338-3p能夠逆轉(zhuǎn)TNF-α對(duì)EOMA細(xì)胞的炎癥反應(yīng)。

A.miR-338-3p表達(dá)水平；B.血管內(nèi)皮細(xì)胞黏附分子VCAM-1表達(dá)水平；C.細(xì)胞間黏附分子ICAM-1表達(dá)水平；D.Western blot檢測ERK/p38蛋白磷酸化水平(n=3,*Plt;0.05,**Plt;0.01，***Plt;0.001)

圖1miR-338-3p過表達(dá)腺病毒抑制EOMA細(xì)胞炎癥反應(yīng)

A.血管內(nèi)皮細(xì)胞黏附分子VCAM-1表達(dá)水平；B.細(xì)胞間黏附分子ICAM-1表達(dá)水平；C.Western blot檢測ERK/p38蛋白磷酸化水平(n=3,*Plt;0.05,**Plt;0.01，** *Plt;0.001)

圖2miR-338-3p高表達(dá)腺病毒逆轉(zhuǎn)TNF-α誘導(dǎo)的內(nèi)皮細(xì)胞炎癥反應(yīng)

3 討論

內(nèi)皮細(xì)胞功能紊亂是動(dòng)脈硬化形成和斑塊破裂的開始[3]，且在動(dòng)脈粥樣硬化慢性血管炎癥發(fā)病機(jī)制中有著至關(guān)重要的作用[4,5]。內(nèi)皮細(xì)胞損傷后，細(xì)胞表面黏附分子VCAM-1和ICAM-1表達(dá)增加，促進(jìn)循環(huán)血液中的單核細(xì)胞聚集到血管壁內(nèi)膜，這些單核細(xì)胞成熟以后變成炎癥巨噬細(xì)胞，分泌促炎因子，進(jìn)一步促進(jìn)內(nèi)皮細(xì)胞激活從而形成一種正反饋使白細(xì)胞持續(xù)聚集[6]。NF-кB和MAPK等促炎信號(hào)通路的激活，能夠激活內(nèi)皮細(xì)胞并產(chǎn)生血管炎癥[6]。促動(dòng)脈硬化細(xì)胞因子如TNF-α、IL-1和IL-6由巨噬細(xì)胞、自然殺傷細(xì)胞和平滑肌細(xì)胞分泌[7]。其中TNF-α和IL-1主要由p38 MAPK和NF-кB信號(hào)通路介導(dǎo)，通過促進(jìn)細(xì)胞因子和黏附分子表達(dá)影響動(dòng)脈硬化形成[8]。

miRNAs是一類重要的內(nèi)源性非編碼小RNA,平均約18-24 個(gè)核苷酸分子,通過綁定靶mRNA 非翻譯區(qū)(3′UTR)負(fù)性調(diào)控基因表達(dá)[9]。越來越多的研究表明miRNAs參與調(diào)控不同生物學(xué)進(jìn)程，如細(xì)胞分化、增生、增長和凋亡等[10]。另外，miRNAs還可通過直接結(jié)合靶基因3′UTR及調(diào)控炎性信號(hào)通路參與調(diào)控動(dòng)脈硬化的形成，如miR-17-3p結(jié)合靶基因ICAM-1和miR-31結(jié)合靶基因E選擇素[11]，miR-181b通過調(diào)控NF-кB信號(hào)通路抑制內(nèi)皮細(xì)胞炎癥反應(yīng)[12]，miR-146通過抑制促炎通路NF-кB和MAPK 信號(hào)通路抑制內(nèi)皮細(xì)胞激活[6]。miR-338位于17號(hào)染色體上，以miR-338-3p及miR-338-5p兩種成熟體形式存在[13]，miR-338-3p通過其宿主基因AATK，參與調(diào)控腫瘤細(xì)胞增值、分化、凋亡及遷徙等[14-17]，還有研究發(fā)現(xiàn)miR-338-3p參與調(diào)控β細(xì)胞維持血糖穩(wěn)態(tài)[18]，并且與糖尿病心肌病關(guān)系密切[19]。然而miR-338-3p在內(nèi)皮細(xì)胞炎癥中的作用研究較少。

在之前的研究中發(fā)現(xiàn)TNF-α 20ng/ml能夠降低miR-338-3p表達(dá)水平，說明miR-338-3p參與內(nèi)皮損傷炎癥反應(yīng)，并且低表達(dá)miR-338-3p能夠激活NF-кB/MAPK信號(hào)通路活性誘導(dǎo)內(nèi)皮細(xì)胞炎癥反應(yīng)。為了進(jìn)一步驗(yàn)證miR-338-3p對(duì)炎癥信號(hào)通路的作用，我們在EOMA細(xì)胞中過表達(dá)miR-338-3p及其陰性對(duì)照，結(jié)果表明過表達(dá)miR-338-3p能夠抑制ERK/p38 MAPK信號(hào)通路活性，降低促炎基因VCAM-1和ICAM-1 mRNA表達(dá)，說明過表達(dá)miR-338-3p抑制內(nèi)皮細(xì)胞炎癥通路活性。本實(shí)驗(yàn)中我們還發(fā)現(xiàn)，EOMA細(xì)胞增強(qiáng)miR-338-3p 表達(dá)后再用TNF-α處理，能夠逆轉(zhuǎn)TNF-α對(duì)促炎基因VCAM-1及ICAM-1的促進(jìn)作用和對(duì)ERK及p38信號(hào)通路活性激活作用，進(jìn)一步說明TNF-α通過調(diào)節(jié)miR-338-3p表達(dá)而影響炎癥信號(hào)通路活性。

綜上所述，過表達(dá)miR-338-3p不僅能夠抑制內(nèi)皮細(xì)胞炎癥信號(hào)通路活性，并能逆轉(zhuǎn)TNF-α誘導(dǎo)內(nèi)皮損傷對(duì)促炎信號(hào)通路的促進(jìn)作用。在本實(shí)驗(yàn)中我們首次證實(shí)了miR-338-3p在EOMA細(xì)胞中對(duì)促炎信號(hào)通路的抑制作用。因此，研究miR-338-3p在內(nèi)皮細(xì)胞炎癥信號(hào)通路中的調(diào)節(jié)作用對(duì)動(dòng)脈硬化的風(fēng)險(xiǎn)預(yù)測及治療有十分重要的應(yīng)用前景。

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TheeffectofoverexpressionofmiR-338-3pontheinflammatorysignalpathway

SUIXiao-fang1,YUShu-qian2,WANGFeng-ling1,etal.

(1.JiamusiUniversity,FirstAffiliatedHospitalofGeriatric;2.TaiheCountyinAnhuiProvince,People'sHospitalofGeriatrics;3.JiamusiUniversityFirstAffiliatedHospitalScientificResearchDivision,Jiamusi154002,China)

ObjectiveAims Our studyaims toinvestigate the effect of miR-338-3p on the inflammatory signal pathway in endothelial cell line EOMA.MethodsEOMA cells were divided into four groups:control group,miR-338-3p over expression group,TNF-α treatment group,miR-338-3p and TNF-α treatment.ResultsCompared with the control group,miR-338-3p over expression group of adhesion molecule VCAM-1 and ICAM-1 expression level of mRNA and ERK/p38 signaling pathway activity decreased significantly,and TNF-α treatment group of adhesion molecule VCAM-1 and ICAM-1 expression level of mRNA and ERK/p38 signaling pathway activity was significantly increased; miR-338-3p and TNF-α treated group compared with TNF-α treatment,adhesion molecule VCAM-1 and ICAM-1 expression of mRNA was significantly reduced,ERK/p38 pathway activity decreased significantly; miR-338-3p and TNF-α treated group and miR-338-3p overexpression group,adhesion molecule expression and ERK/p38 signaling pathway activity is no longer increasing.ConclusionOverexpression of miR-338-3p inhibited the activation of ERK and p38 signal pathways and reduced the levels of VCAM-1 and ICAM-1.Overexpression of miR-338-3p can reverse the effect of TNF-α on the activation of ERK/p38 pathway and the promotion of the expression of VCAM-1 and ICAM-1.

miR-338-3p;endothelial cell;EOMA;TNF-α;inflammatory response

黑龍江省教育廳科學(xué)技術(shù)研究項(xiàng)目(2016-KYYWF-0593)

*通訊作者

1007-4287(2017)11-1991-05

R392

A

隋小芳(1976-)，女，主任醫(yī)師，碩士研究生導(dǎo)師，主要從事心血管疾病的臨床與基礎(chǔ)研究。

2017-03-14)