RIPK3基因轉(zhuǎn)染的SH-SY5Y細(xì)胞中HIF-1α基因及其信號(hào)通路相關(guān)基因表達(dá)變化

張國(guó)祿，程世翔，徐忠偉，衣泰龍，廖吉連，涂悅，張賽

(1武警后勤學(xué)院附屬醫(yī)院腦科醫(yī)院*，天津300162；2武警后勤學(xué)院中心實(shí)驗(yàn)室)

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RIPK3基因轉(zhuǎn)染的SH-SY5Y細(xì)胞中HIF-1α基因及其信號(hào)通路相關(guān)基因表達(dá)變化

張國(guó)祿1，程世翔1，徐忠偉2，衣泰龍1，廖吉連1，涂悅1，張賽1

(1武警后勤學(xué)院附屬醫(yī)院腦科醫(yī)院*，天津300162；2武警后勤學(xué)院中心實(shí)驗(yàn)室)

摘要：目的觀察受體相互作用蛋白激酶3(RIPK3)基因轉(zhuǎn)染的神經(jīng)母細(xì)胞瘤細(xì)胞系SH-SY5Y中低氧誘導(dǎo)因子1α(HIF-1α) mRNA及其信號(hào)通路相關(guān)基因表達(dá)變化。方法 構(gòu)建表達(dá)RIPK3基因的pCMV6-AC-GFP質(zhì)粒(重組質(zhì)粒)，培養(yǎng)SH-SY5Y細(xì)胞，分為實(shí)驗(yàn)組及對(duì)照組，分別轉(zhuǎn)染重組質(zhì)粒和空載質(zhì)粒。采用Western blotting法檢測(cè)細(xì)胞中的RIPK3蛋白，分別于培養(yǎng)8、14、20、26、32、38 h后，通過(guò)MTT實(shí)驗(yàn)檢測(cè)細(xì)胞增殖情況(OD值)。采用轉(zhuǎn)錄組測(cè)序技術(shù)(RNAseq)及Ingenuity Pathway Analysis(IPA)軟件檢測(cè)并篩選RIPK3-HIF1α下游信號(hào)通路中的關(guān)鍵基因。采用微滴式數(shù)字PCR(ddPCR)檢測(cè)兩組細(xì)胞中的HIF-1α mRNA。結(jié)果實(shí)驗(yàn)組細(xì)胞中RIPK3蛋白相對(duì)表達(dá)量(0.806±0.097 5)高于對(duì)照組(0.455±0.088 6)，P<0.05。隨培養(yǎng)時(shí)間延長(zhǎng)，實(shí)驗(yàn)組細(xì)胞增殖受到抑制。實(shí)驗(yàn)組細(xì)胞中HIF-1α mRNA相對(duì)表達(dá)量(0.015 43±0.003 47)低于對(duì)照組(0.046 28±0.010 26),P<0.05。在HIF-1α為核心的相互作用關(guān)系網(wǎng)絡(luò)中，篩選出關(guān)鍵分子泛素綴合酶樣蛋白(UBC)、希佩爾-林道蛋白(VHL)、轉(zhuǎn)錄延伸因子B多肽1(TCEB1)、血管內(nèi)皮生長(zhǎng)因子A(VEGFA)。結(jié)論 RIPK3基因轉(zhuǎn)染SH-SY5Y后，細(xì)胞中HIF-1α mRNA表達(dá)下調(diào)，同時(shí)HIF-1α信號(hào)通路相關(guān)基因(UBC、VHL、TCEB1、VEGFA)的表達(dá)水平受到影響。

關(guān)鍵詞：神經(jīng)母細(xì)胞瘤；受體相互作用蛋白激酶3；低氧誘導(dǎo)因子1α；泛素綴合酶樣蛋白；希佩爾-林道蛋白；轉(zhuǎn)錄延伸因子B多肽；血管內(nèi)皮生長(zhǎng)因子A

受體相互作用蛋白激酶3(RIPK3)屬于受體相互作用蛋白家族(RIPs)成員。作為腫瘤壞死因子(TNF)重要的下游效應(yīng)分子，RIPK3廣泛表達(dá)于人體各組織，并被證實(shí)在細(xì)胞增殖、凋亡過(guò)程中發(fā)揮重要作用[1~4]。低氧誘導(dǎo)因子1α(HIF-1α)表達(dá)于所有已知的多細(xì)胞物種[5]，是組織適應(yīng)缺氧環(huán)境的重要轉(zhuǎn)錄因子[6]，在缺血再灌注、腫瘤及黏膜炎癥等進(jìn)程中發(fā)揮作用[7, 8]。本研究建立了穩(wěn)定表達(dá)RIPK3的人神經(jīng)母細(xì)胞瘤細(xì)胞模型，觀察細(xì)胞中HIF-1α及其下游信號(hào)分子轉(zhuǎn)錄水平的變化，探索RIPK3的潛在作用及其信號(hào)通路網(wǎng)絡(luò)。

1材料與方法

1.1實(shí)驗(yàn)細(xì)胞與試劑人神經(jīng)母細(xì)胞瘤細(xì)胞系SH-SY5Y購(gòu)自美國(guó)標(biāo)準(zhǔn)生物品收藏中心(ATCC)，細(xì)胞接種于含有10 %胎牛血清(Gibicol公司)、90% F12與DMEN配比為1∶1的完全培養(yǎng)基(Gibicol公司)中，37 ℃、5% CO2環(huán)境下培養(yǎng)，取對(duì)數(shù)生長(zhǎng)期細(xì)胞用于實(shí)驗(yàn)。質(zhì)粒小提中量試劑盒(天根公司)，Lipofectamine3000(Life公司)，TRIzol(Invitrogen公司)，SYBR Green試劑盒(Roche公司)，β-actin抗體(Sigma公司)，RIPK3抗體(Abcam公司)，辣根過(guò)氧化物酶標(biāo)記的羊抗兔、羊抗小鼠二抗(KPL公司)，QX200 ddPCR EvaGreen Supermix(Bio-Rad公司)。

1.2表達(dá)RIPK3的pCMV6-AC-GFP質(zhì)粒的獲取將RIPK3(NM_006871) Human cDNA ORF Clone質(zhì)粒及對(duì)應(yīng)空載質(zhì)粒分別轉(zhuǎn)化DH5-α感受態(tài)大腸桿菌，菌液接種于含氨芐青霉素(50 μg/mL)的LB固體培養(yǎng)基中，37 ℃培養(yǎng)過(guò)夜。挑取單個(gè)菌落接種于含氨芐青霉素(50 μg/mL)的液體LB培養(yǎng)基中，37 ℃恒溫?fù)u床震蕩16 h，提取質(zhì)粒DNA，測(cè)定濃度后分裝，-80 ℃保存。

1.3質(zhì)粒轉(zhuǎn)染及RIPK3蛋白檢測(cè)SH-SY5Y細(xì)胞接種于6孔板中，分為對(duì)照組與實(shí)驗(yàn)組。將Lipofectamine3000試劑分別與空載質(zhì)粒及RIPK3過(guò)表達(dá)質(zhì)粒DNA混合構(gòu)成質(zhì)粒-脂質(zhì)體復(fù)合體，孵育后的兩種質(zhì)粒-脂質(zhì)體復(fù)合體分別加入對(duì)照組與實(shí)驗(yàn)組培養(yǎng)孔中孵育2~4 d。以含G418(1 000 μg/mL)的完全培養(yǎng)基篩選轉(zhuǎn)染細(xì)胞以獲取穩(wěn)定轉(zhuǎn)染的SH-SY5Y細(xì)胞，傳代2次后G418濃度減為800 μg/mL維持培養(yǎng)，并凍存?zhèn)溆?。取兩組對(duì)數(shù)生長(zhǎng)期細(xì)胞分別提取蛋白，BCA定量法測(cè)定蛋白濃度后分裝置于-80 ℃保存。采用Western blotting法檢測(cè)RIPK3蛋白。

1.5SH-SY5Y細(xì)胞中HIF-1α mRNA檢測(cè)采用微滴式數(shù)字PCR(ddPCR)檢測(cè)兩組細(xì)胞中的HIF-1α mRNA。HIF-1α、GAPDH基因引物序列見(jiàn)表1。采用美國(guó)Bio-Rad公司生產(chǎn)的QX200 ddPCR系統(tǒng)，將由MILIQ水6 μL、cDNA 2 μL、上游引物1 μL、下游引物1 μL及ddPCR EvaGreen Supermix構(gòu)成的反應(yīng)體系進(jìn)行微滴化處理，形成約20 000個(gè)微滴，將微滴轉(zhuǎn)移到PCR管中進(jìn)行擴(kuò)增，完成PCR反應(yīng)后90 ℃維持5 min以穩(wěn)定微滴，對(duì)擴(kuò)增產(chǎn)物進(jìn)行信號(hào)采集及數(shù)據(jù)分析,獲得基因絕對(duì)濃度，以目的基因濃度/GAPDH基因濃度計(jì)算目的基因相對(duì)表達(dá)量。

表1　HIF-1α、GAPDH基因引物序列

1.6HIF-1α信號(hào)通路相關(guān)基因的篩選與檢測(cè)提取兩組細(xì)胞總RNA，用帶有多加多聚T尾標(biāo)記的磁珠及試劑盒處理，以獲取高純度mRNA。將純化的mRNA打斷為短片段，以此為模板合成雙鏈cDNA。純化cDNA并進(jìn)行末端修復(fù)、鏈接多聚A尾及測(cè)序接頭，選擇合適長(zhǎng)度的片段進(jìn)行PCR反應(yīng)，獲得最終的cDNA文庫(kù)。對(duì)cDNA進(jìn)行檢測(cè)，確認(rèn)符合實(shí)驗(yàn)要求后進(jìn)行RNAseq。測(cè)序結(jié)果經(jīng)Ingenuity Pathway Analysis(IPA)軟件進(jìn)行數(shù)據(jù)分析。篩選RIPK3-HIF-1α信號(hào)通路相關(guān)信號(hào)分子，將這些基因輸入到STRING10數(shù)據(jù)庫(kù)網(wǎng)站(http://string-db.org)中獲得其相互作用網(wǎng)絡(luò)。相關(guān)基因表達(dá)檢測(cè)通過(guò)RNAseq技術(shù)完成。

2結(jié)果

2.1兩組細(xì)胞中RIPK3蛋白表達(dá)變化實(shí)驗(yàn)組與對(duì)照組細(xì)胞中RIPK3蛋白相對(duì)表達(dá)量分別為0.45±0.11、0.81±0.12，兩組相比，P<0.05。

2.2兩組細(xì)胞增殖情況比較隨培養(yǎng)時(shí)間延長(zhǎng)，實(shí)驗(yàn)組細(xì)胞增殖受到抑制，培養(yǎng)20、26、32、38 h時(shí)兩組OD值差異有統(tǒng)計(jì)學(xué)意義。見(jiàn)表2。

表2　兩組細(xì)胞增殖情況比較±s)

注：與實(shí)驗(yàn)組相比，*P<0.05，**P<0.01。2.3兩組細(xì)胞中HIF-1α mRNA表達(dá)比較RNAseq結(jié)果顯示實(shí)驗(yàn)組、對(duì)照組細(xì)胞中HIF-1α mRNA相對(duì)表達(dá)量分別為0.046 282 1、0.015 431 0。ddPCR結(jié)果顯示，實(shí)驗(yàn)組、對(duì)照組細(xì)胞中HIF-1α mRNA相對(duì)表達(dá)量分別為0.004 00±0.000 347、0.001 11±0.000 163；實(shí)驗(yàn)組細(xì)胞中HIF-1α mRNA相對(duì)表達(dá)量低于對(duì)照組(P均<0.05)，與RNAseq結(jié)果一致。

2.4HIF-1α信號(hào)通路相關(guān)基因篩選及其表達(dá)在HIF-1α為核心的相互作用關(guān)系網(wǎng)絡(luò)中，共篩選出四種相關(guān)分子：泛素綴合酶樣蛋白(UBC)、希佩爾-林道蛋白(VHL)、轉(zhuǎn)錄延伸因子B多肽1(TCEB1)、血管內(nèi)皮生長(zhǎng)因子A(VEGFA)。

3討論

RIPK3作為細(xì)胞程序性壞死信號(hào)通路中的重要一環(huán)越來(lái)越引起學(xué)者的關(guān)注。作為T(mén)NF-α下游重要的信號(hào)分子，RIPK3參與天冬氨酸特異酶切的半胱氨酸蛋白酶88(Caspase-8)及核轉(zhuǎn)錄因子κB(NF-κB)活化等過(guò)程[9~12]。然而，RIPK3在神經(jīng)系統(tǒng)中的作用機(jī)制尚待進(jìn)一步研究。本實(shí)驗(yàn)以SH-SY5Y細(xì)胞為基礎(chǔ)建立了穩(wěn)定過(guò)表達(dá)RIPK3基因的神經(jīng)元細(xì)胞模型[13]，并通過(guò)RNAseq及IPA數(shù)據(jù)分析尋找潛在的RIPK3下游效應(yīng)分子。本實(shí)驗(yàn)發(fā)現(xiàn)，SH-SY5Y細(xì)胞轉(zhuǎn)染RIPK3基因后，細(xì)胞中HIF-1α的表達(dá)水平明顯下調(diào)。

HIF-1α是一種重要的氧敏感轉(zhuǎn)錄因子。HIF的一個(gè)重要作用是維持氧供需平衡，以減少活性氧簇(ROS)的生成[14]。在氧充足情況下，HIF-1α與VHL結(jié)合，后者能聚集泛素連接酶標(biāo)記HIF-1α而使HIF-1α降解；UBC在此泛素化的過(guò)程中發(fā)揮重要作用，將泛素從泛素相關(guān)蛋白(UBA)的巰基位點(diǎn)轉(zhuǎn)移至蛋白底物上，進(jìn)而進(jìn)入泛素依賴性蛋白水解途徑[15]。有報(bào)道[16, 17]指出，HIF-1α可直接或間接調(diào)節(jié)血管內(nèi)皮細(xì)胞中2%以上的基因。最近研究[18]顯示，HIF-1α在成人體內(nèi)能通過(guò)諸多信號(hào)通路促進(jìn)組織再生，其機(jī)制包括從轉(zhuǎn)錄水平激活修復(fù)基因及其受體，如VEGF、胎盤(pán)生長(zhǎng)因子(PLGF)、血小板衍生因子(PDGF)、血管生成素1(ANGPT1)[19]；通過(guò)調(diào)節(jié)促血管生成因子及其受體如1-磷酸神經(jīng)鞘氨醇(S1P)[20]、趨化因子受體4(CXCR4)，從而促進(jìn)內(nèi)皮祖細(xì)胞在缺氧部位聚集[21]；通過(guò)調(diào)控參與細(xì)胞周期和DNA復(fù)制過(guò)程的基因從而促進(jìn)內(nèi)皮細(xì)胞增殖、分化[22]。本研究中，HIF-1α及其信號(hào)通路相關(guān)分子構(gòu)成一個(gè)復(fù)雜的信號(hào)網(wǎng)絡(luò)。有學(xué)者[23]發(fā)現(xiàn)，海馬神經(jīng)元損傷后，RIPK3表達(dá)明顯上調(diào)且集中分布于細(xì)胞膜，這種效應(yīng)獨(dú)立于RIPK1的調(diào)控。本研究結(jié)果顯示，轉(zhuǎn)染RIPK3基因可對(duì)SH-SY5Y細(xì)胞中HIF-1α及其信號(hào)通路相關(guān)分子表達(dá)水平造成影響，推測(cè)RIPK3在神經(jīng)系統(tǒng)損傷修復(fù)過(guò)程中發(fā)揮十分重要的作用，通過(guò)控制RIPK3的活性有望促進(jìn)神經(jīng)組織再生與修復(fù)。

由于客觀因素限制，我們僅從表觀對(duì)RIPK3過(guò)表達(dá)引起的HIF-1α及其信號(hào)通路相關(guān)分子表達(dá)變化進(jìn)行觀察，且僅對(duì)HIF-1α進(jìn)行驗(yàn)證，未能進(jìn)一步研究RIPK3與HIF-1α具體的相互作用關(guān)系，這仍需通過(guò)后續(xù)實(shí)驗(yàn)進(jìn)一步驗(yàn)證。

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Expression changes of HIF-1α and its signaling pathway related gene in SH-SY5Y cells transfected by RIPK3 gene

ZHANGGuolu1,CHENGShixiang,XUZhongwei,YITailong,LIAOJilian,TUYue,ZHANGSai

(1Neurology&NeurosurgeryHospitalAffiliatedtoLogisticalCollegeofChineseArmedPoliceForces,Tianjin300162,China)

Abstract:ObjectiveTo observe the expression changes of hypoxia-inducible factor 1α (HIF-1α) and its signaling pathway related gene in the neuroblastoma cell line SH-SY5Y transfected by receptor interacting serine/threonine kinase 3 (RIPK3) gene. MethodsThe pCMV6-AC-GFP plasmid expressing RIPK3 gene (recombinant plasmid) was constructed. SH-SY5Y cells were cultured and then were transfected with the recombinant plasmid and empty vector plasmid as the experimental group and control group respectively. Expression of RIPK3 protein was detected by Western blotting, and the OD value was detected by MTT assay at 8, 14, 20, 26, 32 and 38 h. RNA transcriptome sequencing (RNAseq) and Ingenuity Pathway Analysis (IPA) was used to detect and screen the key genes in the downstream signaling pathway of RIPK3-HIF-1α. Droplet Digital PCR (ddPCR) was used to detect the HIF-1α mRNA in the two groups. ResultsThe RIPK3 expression in the experimental group (0.806±0.097 5) was higher than that of the control group (0.455±0.088 6), P<0.05. The proliferation of SH-SY5Y was inhibited as the cell incubation time was prolonged. The expression of HIF-1α mRNA in the experimental group (0.01543±0.00347) was strongly down-regulated as compared with that of the control group (0.04628±0.01026) (P<0.05). In the interaction network of HIF-1α which was taken as the core, we screened the key molecules such as ubiquitin-conjugating enzyme (UBC), von Hippel-Lindau (VHL), transcription elongation factor B polypeptide 1 (TCEB1) and vascular endothelial growth factor A (VEGFA). ConclusionAfter the SY5Y cells were trasfected by RIPK3, the HIF-1α mRNA expression was down-regulated in SH, and the expression levels of HIF-1α signaling pathway-related genes (UBC, VHL, TCEB1 and VEGFA) were affected.

Key words:neuroblastoma; receptor interacting serine/threonine kinase 3; hypoxia-inducible factor 1α; ubiquitin-conjugating enzyme-like protein; Von Hippel-Lindau protein; transcription elongation factor B polypeptide 1; vascular endothelial growth factor A

(收稿日期：2015-09-28)

中圖分類號(hào)：R34

文獻(xiàn)標(biāo)志碼：A

文章編號(hào)：1002-266X(2016)07-0013-04

doi：10.3969/j.issn.1002-266X.2016.07.005

通信作者簡(jiǎn)介：張賽(1956-)，男，醫(yī)學(xué)博士、教授、主任醫(yī)師，主要研究方向?yàn)樯窠?jīng)創(chuàng)傷及危重癥。E-mail： zhangsai718@vip.126.com

作者簡(jiǎn)介：第一張國(guó)祿(1990-)，男，碩士研究生在讀，從事神經(jīng)外科專業(yè)的研究。E-mail： zhangguolu.wjyxy@163.com

基金項(xiàng)目：國(guó)家自然科學(xué)基金項(xiàng)目(31200809)；武警部隊(duì)后勤科研項(xiàng)目(WJHQ2012-20)。

*暨天津市神經(jīng)創(chuàng)傷修復(fù)重點(diǎn)實(shí)驗(yàn)室、武警部隊(duì)腦創(chuàng)傷與神經(jīng)疾病研究所。