ARHI基因轉(zhuǎn)染胰腺癌PANC1細(xì)胞后對(duì)趨化因子及其受體相關(guān)基因表達(dá)的影響

胡珊珊 楊紅 王健 姚堯 朱永健 錢家鳴

·論著·

ARHI基因轉(zhuǎn)染胰腺癌PANC1細(xì)胞后對(duì)趨化因子及其受體相關(guān)基因表達(dá)的影響

胡珊珊 楊紅 王健 姚堯 朱永健 錢家鳴

目的 觀察ARHI基因轉(zhuǎn)染胰腺癌PANC1細(xì)胞后對(duì)趨化因子及其受體相關(guān)基因mRNA表達(dá)的影響。方法 采用脂質(zhì)體法將表達(dá)ARHI基因的質(zhì)粒pLNCX2-ARHI-EGFP、空質(zhì)粒pLNCX2-EGFP轉(zhuǎn)染胰腺癌PANC1細(xì)胞，應(yīng)用G418篩選穩(wěn)定轉(zhuǎn)染細(xì)胞株。采用基因芯片RT2ProfilerTMPCR Array行實(shí)時(shí)定量PCR，分析轉(zhuǎn)染細(xì)胞的基因表達(dá)，包括84個(gè)趨化因子及受體相關(guān)基因，采用Real-time PCR法驗(yàn)證與血管生長(zhǎng)相關(guān)基因mRNA的表達(dá)。結(jié)果 ARHI轉(zhuǎn)染組細(xì)胞有36個(gè)基因mRNA表達(dá)下調(diào)，9個(gè)基因mRNA表達(dá)上調(diào)，39個(gè)基因mRNA表達(dá)變化無(wú)意義。其中腫瘤轉(zhuǎn)移和侵襲相關(guān)基因CXCL12、CXCR4表達(dá)顯著下調(diào)(<-6倍)，MMP-2表達(dá)輕度下調(diào)(<-2倍)；促腫瘤血管生成相關(guān)基因CCL2、CXCL1、CXCL2、CXCL8、CXCL12、CXCR4表達(dá)顯著下調(diào)，CXCL3、CCR1、CCR2表達(dá)輕度下調(diào)；抗腫瘤血管生成相關(guān)基因CXCL9、CXCL10、CXCL11、CXCR3表達(dá)顯著上調(diào)(>6倍)；遠(yuǎn)端器官定位浸潤(rùn)和潛伏相關(guān)基因CXCL12、CXCR4、CCR7表達(dá)顯著下調(diào)，CXCR5表達(dá)輕度下調(diào)；腫瘤免疫調(diào)節(jié)相關(guān)基因CXCL8、CXCR1、CCR7表達(dá)顯著下調(diào)。Real-time PCR驗(yàn)證CXCL1、CXCL8、CXCR4、CXCR3結(jié)果與PCR Array檢測(cè)結(jié)果一致。結(jié)論 ARHI基因可抑制胰腺癌細(xì)胞增殖、轉(zhuǎn)移、血管生成、免疫調(diào)節(jié)相關(guān)趨化因子及其受體的基因表達(dá)。

胰腺腫瘤； ARHI基因； 趨化因子類； 受體，趨化因子

Fund program: National Natural Science Foundation of China(81072055)

ARHI基因是Yu等[1]發(fā)現(xiàn)的一個(gè)母源性印跡基因，為Ras超家族成員之一。既往研究發(fā)現(xiàn)，ARHI基因可抑制胰腺癌細(xì)胞增殖和生長(zhǎng)，誘導(dǎo)細(xì)胞凋亡和自噬[2]。趨化因子是細(xì)胞因子家族成員之一，根據(jù)其氨基酸N端半胱氨酸殘基的不同，可分為CXC、CC、C和CX3C 4個(gè)亞家族；趨化因子通過(guò)與受體結(jié)合參與細(xì)胞的生長(zhǎng)、發(fā)育、分化、凋亡及組織損傷等多種生理和病理過(guò)程。有研究顯示趨化因子及其受體的定向結(jié)合可影響腫瘤發(fā)展、轉(zhuǎn)移和血管生成[3-4]，但ARHI基因?qū)τ谮吇蜃雍褪荏w的影響仍未知。本研究通過(guò)檢測(cè)并分析ARHI基因?qū)σ认侔┘?xì)胞增殖、轉(zhuǎn)移和血管生成相關(guān)的84個(gè)細(xì)胞趨化因子及其受體基因表達(dá)的影響，探討ARHI基因抑制胰腺癌細(xì)胞的相關(guān)分子機(jī)制。

材料與方法

一、穩(wěn)定轉(zhuǎn)染ARHI基因的PANC1胰腺癌細(xì)胞株的建立

胰腺癌細(xì)胞株P(guān)ANC1來(lái)源于ATCC庫(kù)(American type culture collection)，常規(guī)培養(yǎng)及傳代。取對(duì)數(shù)生長(zhǎng)期細(xì)胞接種于6孔板，培養(yǎng)至細(xì)胞融合度達(dá)到70%～80%時(shí)用轉(zhuǎn)染試劑Lipofectamine 2000(Invitrogen公司)按質(zhì)?！胠ipo2000=4.0 μg∶10 μl將插入ARHI質(zhì)粒pLNCX2-ARHI-EGFP(質(zhì)粒為本實(shí)驗(yàn)室構(gòu)建[5])、空載體質(zhì)粒pLNCX2-EGFP轉(zhuǎn)染細(xì)胞，同時(shí)設(shè)單加脂質(zhì)體及僅加培養(yǎng)液的對(duì)照組。每組3個(gè)復(fù)孔。轉(zhuǎn)染48 h后，加入終濃度為9 00 μg/ml的G418篩選14 d，建立穩(wěn)定轉(zhuǎn)染pLNCX2-ARHI-EGFP和pLNCX2-EGFP的細(xì)胞株。

二、實(shí)時(shí)定量PCR芯片檢測(cè)腫瘤血管生成相關(guān)趨化因子及其受體基因mRNA的表達(dá)

收集各組細(xì)胞，采用Trizol(Gibco公司)提取細(xì)胞總RNA，應(yīng)用逆轉(zhuǎn)錄試劑盒(ABI公司)逆轉(zhuǎn)錄成cDNA。采用高通量基因芯片RT2ProfilerTMPCR Array(Qiagen公司)檢測(cè)84個(gè)基因的mRNA表達(dá)，包含CXCL、CXCR、CCL、CCR及相關(guān)家族。以GAPDH為內(nèi)參。PCR反應(yīng)體系為25 μl， 其中2×PCR SYBGREEN mix 12.5 μl，模板量相當(dāng)于100 ng的總RNA。PCR反應(yīng)條件：95℃ 30 s、95℃ 15 s、60℃ 1 min，循環(huán)40次。根據(jù)2-△△Ct公式，以pLNCX2-EGFP組為1，計(jì)算mRNA相對(duì)表達(dá)倍數(shù)。實(shí)驗(yàn)重復(fù)3次，取均值。表達(dá)倍數(shù)>2倍為輕度上調(diào)，<-2倍為輕度下調(diào)，>6倍為顯著上調(diào)，<-6倍為顯著下調(diào)。

三、腫瘤血管生成相關(guān)趨化因子和受體基因mRNA表達(dá)的驗(yàn)證

挑選表達(dá)水平有顯著差異的腫瘤血管生成相關(guān)趨化因子和受體及CXCR2(芯片中無(wú)該基因)。應(yīng)用Beacon Designer 7.0軟件設(shè)計(jì)引物，各引物序列見(jiàn)表1。采用Real-time PCR反應(yīng)進(jìn)行驗(yàn)證，以GAPDH為內(nèi)參。所有條件均同前，根據(jù)公式2-△△Ct計(jì)算mRNA相對(duì)表達(dá)量。

表1 腫瘤血管生成相關(guān)趨化因子和受體的引物序列

四、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、ARHI基因?qū)吇蜃蛹捌涫荏w家族相關(guān)基因表達(dá)的影響

pLNCX2-ARHI-EGFP組與pLNCX2-EGFP組基因表達(dá)見(jiàn)表2。pLNCX2-ARHI-EGFP組中36個(gè)基因mRNA表達(dá)下調(diào)，其中12個(gè)顯著下調(diào)(<-6倍)；9個(gè)基因mRNA表達(dá)上調(diào)，其中5個(gè)顯著上調(diào)(>6倍)；39個(gè)基因mRNA表達(dá)無(wú)變化。其中與腫瘤轉(zhuǎn)移和侵襲相關(guān)基因CXCL12和CXCR4表達(dá)顯著下調(diào)(<-6倍)，MMP-2表達(dá)輕度下調(diào)(<-2倍)；促腫瘤血管生成基因CCL2、CXCL1、CXCL2、CXCL8、CXCL12、CXCR4表達(dá)顯著下調(diào)(<-6倍)，CXCL3、CCR1、CCR2表達(dá)輕度下調(diào)(<-2倍)；抗腫瘤血管生成基因CXCL9、CXCL10、CXCL11、CXCR3表達(dá)顯著上調(diào)(>6倍)，以CXCL9最明顯，達(dá)27.8倍；與對(duì)遠(yuǎn)端器官定位浸潤(rùn)和潛伏相關(guān)基因CXCL12、CXCR4、CCR7表達(dá)顯著下調(diào)(<-6倍)，CXCR5表達(dá)輕度下調(diào)(<-2倍)；與腫瘤免疫調(diào)節(jié)相關(guān)基因CXCL8、CXCR1、CCR7表達(dá)顯著下調(diào)(<-6倍)，其中以CXCR1尤為明顯，達(dá)-41.3倍。

表2 pLNCX2-ARHI-EGFP組和pLNCX2-EGFP組基因表達(dá)的差異

注：a：顯著下調(diào)；b：輕度下調(diào)；c：顯著上調(diào)；d：輕度上調(diào)

二、ARHI基因?qū)δ[瘤血管生成相關(guān)趨化因子和受體mRNA表達(dá)的影響

Real-time PCR驗(yàn)證結(jié)果與PCR Array結(jié)果一致，pLNCX2-ARHI-EGFP組CXCL8、CXCL1、CXCR4 mRNA表達(dá)較pLNCX2-EGFP組顯著下調(diào)(P值分別為0.0243、0.0388、0.0142)，CXCR3表達(dá)顯著上調(diào)(P=0.0481)，CXCR2表達(dá)亦顯著下調(diào)(P=0.0002)，差異均有統(tǒng)計(jì)學(xué)意義(表3)。

表3 穩(wěn)定轉(zhuǎn)染pLNCX2-ARHI-EGFP組與空載體組基因mRNA相對(duì)表達(dá)量的比較

基因pLNCX2-EGFP組pLNCX2-ARHI-EGFP組P值CXCL80.00066±0.000130.00029±0.000130.0243CXCR20.01559±0.000360.00221±0.001650.0002CXCL10.00065±0.000230.00024±5.164e-0050.0388CXCR40.00127±0.000220.00074±3.609e-0050.0142CXCR30.00091±0.000920.00260±0.000490.0481

討 論

ARHI基因是母源性印跡基因，為Ras超家族成員之一。ARHI基因編碼的蛋白在人類多種組織如卵巢、乳腺中表達(dá)，但在乳腺癌、卵巢癌中表達(dá)下調(diào)。本課題組前期研究發(fā)現(xiàn)[6-8]，ARHI基因可以抑制胰腺癌細(xì)胞的增殖，促使細(xì)胞周期停滯，誘導(dǎo)細(xì)胞凋亡及自噬發(fā)生，抑制細(xì)胞遷移，是胰腺癌發(fā)生和發(fā)展過(guò)程中的重要抑癌基因。

研究顯示腫瘤細(xì)胞的生長(zhǎng)和轉(zhuǎn)移受到微環(huán)境的調(diào)控，而趨化因子在微環(huán)境調(diào)節(jié)中起重要作用。趨化因子與受體結(jié)合后促使腫瘤細(xì)胞增殖和腫瘤血管生成從而促進(jìn)腫瘤生長(zhǎng)、轉(zhuǎn)移[8-9]。谷氨酸-亮氨酸-精氨酸陽(yáng)性(glntamic acid-leu-cine-arginine positive, ELR+)的CXC類趨化因子(如CXCL8)具有促腫瘤血管生成的作用，高轉(zhuǎn)移性胰腺癌移植瘤CXCL8高表達(dá)與腫瘤生成和轉(zhuǎn)移高度相關(guān)，給予外源性CXCL8可促進(jìn)血管生成[10]。胰腺癌組織中CXCR4表達(dá)和微血管密度(MVD)均較正常胰腺組織顯著提高，且CXCR4的表達(dá)與MVD有顯著相關(guān)性[11]。ELR陰性以及能被干擾素誘導(dǎo)產(chǎn)生的CXC類趨化因子(如CXCL10)有抗腫瘤血管生成作用[12]。Zhao等[13]研究發(fā)現(xiàn)，其他家族中相關(guān)基因CCL21和CCR7在胰腺癌組織中高表達(dá)，且與MVD呈正相關(guān)，提示CCR7與腫瘤血管生成相關(guān)。

本研究結(jié)果顯示，ARHI基因轉(zhuǎn)染后PANC1細(xì)胞CXCL8/CXCR2、CXCR4和CXCL1的表達(dá)顯著下調(diào)，而CXCR3的表達(dá)顯著上調(diào)，提示ARHI可能通過(guò)抑制CXCL1和CXCR8/CXCR2、CXCL12/CXCR4及CCL21/CCR7通路，上調(diào)CXCL9、CXCL10和CXCL11/CXCR3通路抑制胰腺癌組織血管生成。為了進(jìn)一步驗(yàn)證ARHI基因?qū)@些細(xì)胞因子表達(dá)的影響，本研究對(duì)報(bào)道較多的CXCL1、CXCL8、CXCR3和CXCR4、CXCR2進(jìn)行驗(yàn)證，結(jié)果發(fā)現(xiàn)，這些細(xì)胞因子的表達(dá)差異均有統(tǒng)計(jì)學(xué)意義，證實(shí)ARHI基因能抑制CXCL1、CXCL8、CXCR4和CXCR2 mRNA的表達(dá)，增強(qiáng)CXCR3 mRNA的表達(dá)。

本研究結(jié)果還顯示，pLNCX2-ARHI-EGFP組細(xì)胞CXCL12、CXCR4和CCR7表達(dá)顯著下調(diào)，CXCR5表達(dá)輕度下調(diào)，提示ARHI可能通過(guò)抑制CXCL12/CXCR4、CCL21/CCR7及CXCL13/CXCR5通路進(jìn)一步抑制腫瘤向遠(yuǎn)端器官的定位轉(zhuǎn)移；CXCL8、CXCR1和CCR7表達(dá)顯著下調(diào)，提示ARHI可能通過(guò)CXCR8/CXCR1和CCR7抑制腫瘤的免疫逃避。

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(本文編輯：冀凱宏)

Effects of ARHI gene transfection on chemokines and receptors related gene expression profile of PANC1 cells

HuShanshan,YangHong,WangJian,YaoYao,ZhuYongjian,QianJiaming.

DepartmentofGastroenterology,PekingUnionMedicalHospital,ChineseAcademyofMedicalSciences,Beijing100730,China

QianJiaming,Email:qianjiaming@126.com

Objective To investigate the effects of ARHI transfection on the chemokines and receptors related gene expression profile of PANC1 cells. Methods Plasmids expressing ARHI and empty plasmid were transfected into PANC1 cells, and the stably expressed cell lines were established by using G418. mRNA expression of chemokines and receptors related genes was detected by PCR Array. Real-time PCR was used to detect mRNA expression of the genes related vascular growth. Results In cells transfected with ARHI gene, the expression levels of mRNA of 36 genes were down-regulated, and 9 were up-regulated. Among the genes related to tumor metastasis and invasion CXCL12 and CXCR4 were significantly down-regulated (<-6 folds), and MMP-2 was slightly down-regulated (<-2 folds). Among the genes related to tumor angiogenesis, pro-angiogenesis genes including CCL2, CXCL1, CXCL2, CXCL8, CXCL12 and CXCR4 were significantly down-regulated, and pro-angiogenesis genes including CXCL3, CCR1 and CCR2 was slightly down-regulated. Anti-angiogenesis genes including CXCL9, CXCL10, CXCL11 and CXCR3 were significantly up-regulated (>6 folds). Among the genes related to the localization of distant organ infiltration and latency, CXCL12, CXCR4 and CCR7 were significantly down-regulated,and CXCR5 was slightly down-regulated.Among the gene with tumor immunity,CXCL8,CXCR1 and CCR7 were significantly down-regulated. Gene expression of CXCL1,CXCL8,CXCR4 and CXCR3 detected by Real-time PCR were consistent with PCR array. Conclusions ARHI gene inhibits the expression of chemokines and receptors related to tumor metastasis,angiogenesis and tumor immunity.

Pancreatic neoplasms; ARHI gene; Chemotactic factors; Receptors, chemokine

10.3760/cma.j.issn.1674-1935.2017.03.007

100730 北京，中國(guó)醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院，北京協(xié)和醫(yī)院消化科

錢家鳴，Email：qianjiaming@126.com

國(guó)家自然科學(xué)基金(81072055)

2016-02-04)