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細胞后對趨化因子及其受體相關(guān)基因表達的影響

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細胞后對趨化因子及其受體相關(guān)基因mRNA表達的影響。方法 采用脂質(zhì)體法將表達ARHI基因的質(zhì)粒pLNCX2-ARHI-EGFP、空質(zhì)粒pLNCX2-EGFP轉(zhuǎn)染胰腺癌PANC1細胞，應用G418篩選穩(wěn)定轉(zhuǎn)染細胞株。采用基因芯片RT2ProfilerTMPCR Array行實時定量PCR，分析轉(zhuǎn)染細胞的基因表達，包括84個趨化因子及受體相關(guān)基因，采用Real-time PCR法驗證與血管生長相關(guān)基因mRNA的表達。結(jié)果 ARHI轉(zhuǎn)染組細胞有36個基因mRNA表達下調(diào)，9個基因mRNA表達上調(diào)，39個基因mRNA表達變化無意義。其中腫瘤轉(zhuǎn)移和侵襲相關(guān)基因CXCL12、CXCR4表達顯著下調(diào)(<-6倍)，MMP-2表達輕度下調(diào)(<-2倍)；促腫瘤血管生成相關(guān)基因CCL2、CXCL1、CXCL2、CXCL8、CXCL12、CXCR4表達顯著下調(diào)，CXCL3、CCR1、CCR2表達輕度下調(diào)；抗腫瘤血管生成相關(guān)基因CXCL9、CXCL10、CXCL11、CXCR3表達顯著上調(diào)(>6倍)；遠端器官定位浸潤和潛伏相關(guān)基因CXCL12、CXCR4、CCR7表達顯著下調(diào)，CXCR5表達輕度下調(diào)；腫瘤免疫調(diào)節(jié)相關(guān)基因CXCL8、CXCR1、CCR7表達顯著下調(diào)。Real-time PCR驗證CXCL1、CXCL8、CXCR4、CXCR3結(jié)果與PCR Array檢測結(jié)果一致。結(jié)論 ARHI基因可抑制胰腺癌細胞增殖、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。

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胞增殖和生長，誘導細胞凋亡和自噬[2]。趨化因子是細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個亞家族；趨化因子通過與受體結(jié)合參與細胞的生長、發(fā)育、分化、凋亡及組織損傷等多種生理和病理過程。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。本研究通過檢測并分析ARHI基因?qū)σ认侔┘毎鲋?、轉(zhuǎn)移和血管生成相關(guān)的84個細胞趨化因子及其受體基因表達的影響，探討ARHI基因抑制胰腺癌細胞的相關(guān)分子機制。

材料與方法

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

胰腺癌細胞株P(guān)ANC1來源于ATCC庫(American type culture collection)，常規(guī)培養(yǎng)及傳代。取對數(shù)生長期細胞接種于6孔板，培養(yǎng)至細胞融合度達到70%～80%時用轉(zhuǎn)染試劑Lipofectamine 2000(Invitrogen公司)按質(zhì)粒∶lipo2000=4.0 μg∶10 μl將插入ARHI質(zhì)粒pLNCX2-ARHI-EGFP(質(zhì)粒為本實驗室構(gòu)建[5])、空載體質(zhì)粒pLNCX2-EGFP轉(zhuǎn)染細胞，同時設單加脂質(zhì)體及僅加培養(yǎng)液的對照組。每組3個復孔。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的細胞株。

二、實時定量PCR芯片檢測腫瘤血管生成相關(guān)趨化因子及其受體基因mRNA的表達

收集各組細胞，采用Trizol(Gibco公司)提取細胞總RNA，應用逆轉(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84個基因的mRNA表達，包含CXCL、CXCR、CCL、CCR及相關(guān)家族。以GAPDH為內(nèi)參。PCR反應體系為25 μl， 其中2×PCR SYBGREEN mix 12.5 μl，模板量相當于100 ng的總RNA。PCR反應條件：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，計算mRNA相對表達倍數(shù)。實驗重復3次，取均值。表達倍數(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表達的驗證

挑選表達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(芯片中無該基因)。應用Beacon Designer 7.0軟件設計引物，各引物序列見表1。采用Real-time PCR反應進行驗證，以GAPDH為內(nèi)參。所有條件均同前，根據(jù)公式2-△△Ct計算mRNA相對表達量。

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)計學處理

結(jié) 果

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

pLNCX2-ARHI-EGFP組與pLNCX2-EGFP組基因表達見表2。pLNCX2-ARHI-EGFP組中36個基因mRNA表達下調(diào)，其中12個顯著下調(diào)(<-6倍)；9個基因mRNA表達上調(diào)，其中5個顯著上調(diào)(>6倍)；39個基因mRNA表達無變化。其中與腫瘤轉(zhuǎn)移和侵襲相關(guān)基因CXCL12和CXCR4表達顯著下調(diào)(<-6倍)，MMP-2表達輕度下調(diào)(<-2倍)；促腫瘤血管生成基因CCL2、CXCL1、CXCL2、CXCL8、CXCL12、CXCR4表達顯著下調(diào)(<-6倍)，CXCL3、CCR1、CCR2表達輕度下調(diào)(<-2倍)；抗腫瘤血管生成基因CXCL9、CXCL10、CXCL11、CXCR3表達顯著上調(diào)(>6倍)，以CXCL9最明顯，達27.8倍；與對遠端器官定位浸潤和潛伏相關(guān)基因CXCL12、CXCR4、CCR7表達顯著下調(diào)(<-6倍)，CXCR5表達輕度下調(diào)(<-2倍)；與腫瘤免疫調(diào)節(jié)相關(guān)基因CXCL8、CXCR1、CCR7表達顯著下調(diào)(<-6倍)，其中以CXCR1尤為明顯，達-41.3倍。

表2 pLNCX2-ARHI-EGFP組和pLNCX2-EGFP組基因表達的差異

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表達的影響

Real-time PCR驗證結(jié)果與PCR Array結(jié)果一致，pLNCX2-ARHI-EGFP組CXCL8、CXCL1、CXCR4 mRNA表達較pLNCX2-EGFP組顯著下調(diào)(P值分別為0.0243、0.0388、0.0142)，CXCR3表達顯著上調(diào)(P=0.0481)，CXCR2表達亦顯著下調(diào)(P=0.0002)，差異均有統(tǒng)計學意義(表3)。

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

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，但在乳腺癌、卵巢癌中表達下調(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胞的增殖，促使細胞周期停滯，誘導細胞凋亡及自噬發(fā)生，抑制細胞遷移，是胰腺癌發(fā)生和發(fā)展過程中的重要抑癌基因。

研究顯示腫瘤細胞的生長和轉(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)合后促使腫瘤細胞增殖和腫瘤血管生成從而促進腫瘤生長、轉(zhuǎn)移[8-9]。谷氨酸-亮氨酸-精氨酸陽性(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高表達與腫瘤生成和轉(zhuǎn)移高度相關(guān)，給予外源性CXCL8可促進血管生成[10]。胰腺癌組織中CXCR4表達和微血管密度(MVD)均較正常胰腺組織顯著提高，且CXCR4的表達與MVD有顯著相關(guān)性[11]。ELR陰性以及能被干擾素誘導產(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，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細胞CXCL8/CXCR2、CXCR4和CXCL1的表達顯著下調(diào)，而CXCR3的表達顯著上調(diào)，提示ARHI可能通過抑制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。為了進一步驗證ARHI基因?qū)@些細胞因子表達的影響，本研究對報道較多的CXCL1、CXCL8、CXCR3和CXCR4、CXCR2進行驗證，結(jié)果發(fā)現(xiàn)，這些細胞因子的表達差異均有統(tǒng)計學意義，證實ARHI基因能抑制CXCL1、CXCL8、CXCR4和CXCR2 mRNA的表達，增強CXCR3 mRNA的表達。

本研究結(jié)果還顯示，pLNCX2-ARHI-EGFP組細胞CXCL12、CXCR4和CCR7表達顯著下調(diào)，CXCR5表達輕度下調(diào)，提示ARHI可能通過抑制CXCL12/CXCR4、CCL21/CCR7及CXCL13/CXCR5通路進一步抑制腫瘤向遠端器官的定位轉(zhuǎn)移；CXCL8、CXCR1和CCR7表達顯著下調(diào)，提示ARHI可能通過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，中國醫(yī)學科學院北京協(xié)和醫(yī)學院，北京協(xié)和醫(yī)院消化科

錢家鳴，Email：qianjiaming@126.com

國家自然科學基金(81072055)

2016-02-04)