李祥蘇 熊光蘇 吳叔明

·論著·

Jagged1基因沉默對(duì)人胰腺癌細(xì)胞SW1990和PANC1血管相關(guān)因子分泌及遷移能力的影響

李祥蘇 熊光蘇 吳叔明

目的以RNA干擾技術(shù)靶向沉默人胰腺癌細(xì)胞株Jagged1基因，觀察細(xì)胞VEGF、Angiopoietin2、bFGF、MMP9的分泌及細(xì)胞遷移能力的變化。方法應(yīng)用靶向Jagged1的siRNA、對(duì)照siRNA(c-siRNA)轉(zhuǎn)染人胰腺癌細(xì)胞株SW1990和PANC1，實(shí)時(shí)PCR法和蛋白質(zhì)印跡法檢測(cè)細(xì)胞Jagged1 mRNA 和蛋白的表達(dá)， ELISA法檢測(cè)細(xì)胞培養(yǎng)上清中VEGF、angiopoietin2、bFGF、MMP9的分泌量，Transwell小室觀察細(xì)胞的遷移能力。結(jié)果SW1990和PANC1細(xì)胞均高表達(dá)Jagged1基因。15 nmol/L Jagged1 siRNA轉(zhuǎn)染胰腺癌細(xì)胞72 h后，SW1990、PANC1細(xì)胞的Jagged1 mRNA表達(dá)被抑制，分別為c-siRNA組的(59.62±2.75)%和(76.96±6.16)%，Jagged1蛋白表達(dá)幾乎完全被抑制。SW1990、PANC1細(xì)胞的VEGF分泌量均較c-siRNA組顯著減少[(867.93±58.69)pg/ml比(1516.24±37.58)pg/ml，(951.13±120.49)pg/ml比(1413.68±33.56)pg/ml，P<0.05]，但angiopoietin2、bFGF、MMP9蛋白分泌無明顯變化。另外，Jagged1 siRNA轉(zhuǎn)染后，SW1990細(xì)胞VEGF mRNA表達(dá)水平為c-siRNA組的(52.26±4.85)%(P<0.05)；PANC1細(xì)胞為c-siRNA組的(59.75±4.91)%(P<0.05)。轉(zhuǎn)染后的SW1990和PANC1細(xì)胞的穿膜細(xì)胞數(shù)分別為(65.25±5.56)、(57.50±8.58)個(gè)，較c-siRNA組的(122.25±11.09)、(112.00±12.52)個(gè)顯著減少(P<0.05)。結(jié)論人胰腺癌細(xì)胞高表達(dá)Jagged1，沉默Jagged1基因可明顯抑制人胰腺癌細(xì)胞VEGF的產(chǎn)生和分泌，并且可降低癌細(xì)胞的體外遷移能力。

胰腺腫瘤； Jagged1； 血管生成誘導(dǎo)劑； 血管內(nèi)皮生長(zhǎng)因子； RNA干擾

Jagged1是哺乳動(dòng)物細(xì)胞膜上Notch受體的主要配體之一，除了能與Notch1結(jié)合，還可與Notch2及Notch3結(jié)合。Jagged1與相鄰細(xì)胞表面的Notch胞外域結(jié)合，引起Notch胞內(nèi)域的釋放，然后轉(zhuǎn)移到胞核與轉(zhuǎn)錄因子結(jié)合從而調(diào)控靶基因的表達(dá)。研究發(fā)現(xiàn)，Jagged1在乳腺癌、前列腺癌、肝癌、骨肉瘤等腫瘤中表達(dá)增加，并與腫瘤的浸潤(rùn)轉(zhuǎn)移、復(fù)發(fā)及腫瘤分化程度相關(guān)[1-4]。另外，Jagged1還是血管生長(zhǎng)的調(diào)控因子，與血管的形成有密切關(guān)系。本實(shí)驗(yàn)觀察Jagged1在人胰腺癌細(xì)胞株SW1990和PANC1中的表達(dá)及沉默Jagged1基因?qū)Π┘?xì)胞分泌血管相關(guān)因子及細(xì)胞遷移能力的影響。

材料和方法

一、細(xì)胞培養(yǎng)及分組

人胰腺癌細(xì)胞株SW1990和PANC1由上海市消化疾病研究所實(shí)驗(yàn)室保存，常規(guī)培養(yǎng)、傳代。細(xì)胞以1×106個(gè)/孔密度接種于6孔板，加入不含抗生素的DMEM高糖培養(yǎng)基，待細(xì)胞密度達(dá)30%～50%時(shí)，分為對(duì)照siRNA(c-siRNA)組和Jagged1 siRNA(si-Jagged1)組。根據(jù)QIAGEN公司的siRNA設(shè)計(jì)原則設(shè)計(jì)靶向Jagged1基因的siRNA序列：靶序列為CAGAGTAATCTTGTTGGTTCA；正義鏈為5′-GAG-UAAUCUUGUUGGUUCATT-3′；反義鏈為5′-UGAACC-AACAAGAUUACUCTG-3′，由QIAGEN公司合成。c-siRNA為亂碼siRNA。參照HiPerFect Transfection Reagent(QIAGEN公司)說明書將15 nmol/L的c-siRNA、si-Jagged1分別轉(zhuǎn)染兩組細(xì)胞繼續(xù)培養(yǎng)72 h。

二、方法

1．實(shí)時(shí)PCR法：收集相應(yīng)細(xì)胞，Trizol試劑抽提總RNA，紫外分光光度計(jì)檢測(cè)RNA純度及含量。參照實(shí)時(shí)PCR試劑盒(TaKaRa公司)說明書進(jìn)行操作，引物序列[5-7]見表1，由上海生工生物工程有限公司合成。實(shí)時(shí)PCR反應(yīng)條件：95℃ 30 s，95℃ 5 s、60℃ 31 s， 40次循環(huán)。以SDS2.3軟件分析Ct值，以2-△△ct公式計(jì)算mRNA表達(dá)量。

表1 PCR引物序列表

2．蛋白質(zhì)印跡法：收集細(xì)胞，RIPA裂解液冰上裂解30 min，提取細(xì)胞總蛋白，BCA法測(cè)蛋白質(zhì)濃度。常規(guī)行蛋白質(zhì)印跡法檢測(cè)Jagged1蛋白表達(dá)，以β-actin作為內(nèi)參。兔抗人Jagged1單抗(Cell Signaling Technology公司)1∶1000稀釋，HRP標(biāo)記的羊抗兔二抗1∶5000稀釋，化學(xué)發(fā)光法自顯影。

3．酶聯(lián)免疫吸附法(ELISA)：收集細(xì)胞培養(yǎng)上清，4℃ 800 g離心5 min，上清液-80℃保存。應(yīng)用ELISA法檢測(cè)培養(yǎng)上清中血管內(nèi)皮生長(zhǎng)因子(VEGF)、血管生成素-2(angiopoietin 2)、堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF)、基質(zhì)金屬蛋白酶9(MMP9)含量，按ELISA試劑盒(R&D公司)說明書操作。

4．細(xì)胞遷移實(shí)驗(yàn)：收集細(xì)胞，用無血清DMEM培養(yǎng)基重懸，加入Transwell小室(Millipore公司)的上室，每室200 μl(1×105個(gè)細(xì)胞)，下室內(nèi)加入600 μl含20%FBS的培養(yǎng)基，常規(guī)培養(yǎng)12 h后，去除濾膜未穿膜細(xì)胞，用4%多聚甲醛固定，0.1%結(jié)晶紫室溫染色30 min，高倍鏡下計(jì)數(shù)穿膜細(xì)胞數(shù)。

三、統(tǒng)計(jì)學(xué)分析

結(jié) 果

一、細(xì)胞Notch受體、配體及靶基因mRNA表達(dá)

SW1990和PANC1細(xì)胞Notch受體(Notch1、Notch2、Notch3、Notch4)、配體(Jagged1、Jagged2)及靶基因Hes1 mRNA表達(dá)量見圖1，均以Jagged1及Notch2的表達(dá)最高。

SW1990細(xì)胞的c-siRNA組、si-Jagged1組細(xì)胞Jagged1 mRNA相對(duì)表達(dá)量分別為1.13±0.06、0.40±0.03，Jagged1 mRNA表達(dá)抑制率為(59.62±2.75)%；PANC1細(xì)胞的c-siRNA組、si-Jagged1組的Jagged1 mRNA相對(duì)表達(dá)量分別為1.14±0.09、0.23±0.06，抑制率達(dá)(76.96±6.16)%。兩株細(xì)胞的Jagged1蛋白表達(dá)幾乎完全被抑制(圖2)。

圖1SW1990及PANC1細(xì)胞Notch信號(hào)通路相關(guān)基因mRNA的表達(dá)

圖2SW1990及PANC1細(xì)胞的Jagged1蛋白表達(dá)

二、細(xì)胞血管相關(guān)因子的分泌量

Jagged1 siRNA轉(zhuǎn)染后72 h，SW1990和PANC1細(xì)胞VEGF mRNA表達(dá)量分別為c-siRNA組的(52.26±4.85)%和(59.75±4.91)%(P<0.05)；VEGF分泌量也均顯著減少(P<0.05)，而angiopoietin2、bFGF、MMP9的分泌量無明顯變化(表2)。

三、細(xì)胞遷移能力的變化

SW1990和PANC1細(xì)胞的si-Jagged1組穿膜細(xì)胞數(shù)分別為(65.25±5.56)、(57.50±8.58)個(gè)，較c-siRNA組的(122.25±11.09)、(112.00±12.52)個(gè)明顯減少(P<0.05，圖3)。

表2 兩株胰腺癌細(xì)胞血管相關(guān)因子的分泌量

注：與c-siRNA組比較，aP<0.05

圖3SW1990細(xì)胞的c-siRNA組(a)、si-Jagged1組(b)和PANC1細(xì)胞的c-siRNA組(c)、si-Jagged1組(d)的穿膜細(xì)胞( ×400)

討 論

人Jagged1基因定位于染色體20p12，全長(zhǎng)36 000 bp，開放閱讀框編碼3657個(gè)氨基酸。Jagged1屬于DSL(Delta、Serrate、Lag-2)蛋白家族，在包括胰腺癌的多種腫瘤組織中高表達(dá)。本結(jié)果也顯示胰腺癌PANC1和SW1990細(xì)胞均高表達(dá)Jagged1基因。

Jagged1是近年來新發(fā)現(xiàn)的血管調(diào)控因子，敲除Jagged1基因的小鼠于胚胎10.5 d死亡，表現(xiàn)為血管發(fā)育異常和廣泛出血，血管重塑嚴(yán)重缺陷[8]。Zeng等[9]報(bào)道，Notch1、Jagged1及VEGF蛋白兩兩間呈正相關(guān)，Notch1和Jagged1表達(dá)增強(qiáng)可促進(jìn)癌細(xì)胞VEGF的表達(dá)。有研究顯示[10]，Jagged1可通過影響NF-κB p65 DNA結(jié)合活性來影響其靶基因，如VEGF(VEGF121、VEGF165及VEGF189)、MMP9及uPA(尿激酶型纖溶酶原激活物)等的表達(dá)。本結(jié)果顯示， Jagged1 siRNA轉(zhuǎn)染胰腺癌細(xì)胞后可明顯抑制VEGF的產(chǎn)生，但對(duì)angiopoietin2、bFGF、MMP9的表達(dá)無影響。提示Jagged1通過影響VEGF的產(chǎn)生和分泌進(jìn)而參與血管形成的過程。但本結(jié)果與上述結(jié)果不同，對(duì)其他基因表達(dá)無影響，可能與Jagged1在不同腫瘤中的作用機(jī)制不完全相同有關(guān)。

另外，本結(jié)果還顯示，Jagged1表達(dá)被抑制后人胰腺癌細(xì)胞的體外遷移能力明顯減弱，與Büchler等[11]報(bào)道的Jagged1蛋白可增強(qiáng)胰腺癌細(xì)胞的體外侵襲能力的結(jié)果一致。臨床試驗(yàn)也發(fā)現(xiàn)，Jagged1 mRNA和蛋白水平均高表達(dá)的乳腺癌患者預(yù)后差，與其侵襲性增加有關(guān)[1]。

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[5] Sj?lund J, Johansson M, Manna S, et al. Suppression of renal cell carcinoma growth by inhibition of Notch signaling in vitro and in vivo. J Clin Invest, 2008,118: 217-228.

[6] Vercauteren SM, Sutherland HJ. Constitutively active Notch4 promotes early human hematopoietic progenitor cell maintenance while inhibiting differentiation and causes lymphoid abnormalities in vivo. Blood,2004,104:2315-2322.

[7] Wang Z, Banerjee S, Li Y,et al. Down-regulation of notch-1 inhibits invasion by inactivation of nuclear factor-kappaB, vascular endothelial growth factor, and matrix metalloproteinase-9 in pancreatic cancer cells. Cancer Res, 2006,66:2778-2784.

[8] Roca C, Adams RH. Regulation of vascular morphogenesis by Notch signaling.Genes Dev,2007,21:2511-2524.

[9] Zeng Q, Li S, Chepeha DB, et al. Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling. Cancer Cell,2005,8:13-23.

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2010-11-09)

(本文編輯：屠振興)

EffectsofJagged1genesilencingonsecretionofvascular-relatedfactorsandcellmigrationinhumanpancreaticcancercellsSW1990andPANC1

LIXiang-su,XIONGGuang-su,WUShu-ming．

DepartmentofGastroenterology,RenjiHospital,SchoolofMedicine,ShanghaiJiaotongUniversity,ShanghaiInstituteofDigestiveDisease,Shanghai200001,China

WUShu-ming,Email：wushuming@vip.sina.com

ObjectiveTo investigate the effects of Jagged1 gene silencing on the expression of VEGF, angiopoietin 2, bFGF, MMP 9, and the migration of human pancreatic cancer cells SW1990 and PANC1.MethodsJagged1 siRNA and control siRNA were transfected into human pancreatic cancer cells SW1990 and PANC1 respectively. The expressions of Jagged1 mRNA and protein were detected by real-time PCR and Western blotting. ELISA was used to detect the concentration of VEGF, angiopoietin 2, bFGF, MMP9 in cell supernatants. Transwell was used to detect the migration of SW1990 and PANC1 cells.ResultsJagged1 mRNA and protein were highly expressed in human pancreatic cancer cells SW1990 and PANC1. After transfected with 15 nmol/L Jagged1 siRNA for 72 h, the expression of Jagged1 mRNA in SWl990 was inhibited and reduced by (59.62±2.75)% and in PANC1 reduced by (76.96±6.16)% when compared with that in control siRNA group. The Jagged1 protein expression was almost inhibited. The concentration of VEGF in the culture supernatants of SWl990 and PANC1 cells was significantly decreased [(867.93±58.69)pg/mlvs. (1516.24±37.58)pg/ml, 951.13±120.49)pg/mlvs. (1413.68±33.56)pg/ml,P<0.05], but the protein concentration of angiopoietin 2, bFGF, MMP9 were not significantly changed. In addition, after transfected with Jagged1 siRNA, tne expression of VEGF mRNA was (52.26±4.85)% of the control levels in SW1990 (P<0.05), and (59.75±4.91)% of the control levels(P<0.05) in PANC1. The number of cell migration of transfected SW1990 and PANC1 was 65.25±5.56 and 57.50±8.58, which were significantly lower than those in the control siRNA group (122.25±11.09, 112.00±12.52,P<0.05).ConclusionsJagged1 is highly expressed in human pancreatic cancer cells. Jagged1 gene silencing significantly inhibits the production and secretion of VEGF in SW1990 and PANC1 cells, and reduces the migration of cancer cells in vitro.

Pancreatic neoplasms; Jagged1; Angiogenesis inducing agents; Vascular endothelial growth factor; RNA interference

10.3760/cma.j.issn.1674-1935.2011.05.005

200001 上海，上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院消化科，上海市消化疾病研究所

吳叔明，Email：wushuming@vip.sina.com