宋博　馬洪運　李森　邵卓　宋彬　郝俊　程鵬　金鋼

200433　上海，第二軍醫(yī)大學附屬長海醫(yī)院胰腺肝膽外科(宋博、馬洪運、李森、邵卓、宋彬、郝俊、程鵬、金鋼)；解放軍第454醫(yī)院普外科(宋博)

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·論著·

沉默糖原合成激酶3β表達對胰腺癌PANC1細胞生長及上皮間質轉化的影響

宋博馬洪運李森邵卓宋彬郝俊程鵬金鋼

200433上海，第二軍醫(yī)大學附屬長海醫(yī)院胰腺肝膽外科(宋博、馬洪運、李森、邵卓、宋彬、郝俊、程鵬、金鋼)；解放軍第454醫(yī)院普外科(宋博)

【摘要】目的觀察沉默糖原合成激酶3β(GSK3β)的表達對胰腺癌PANC1細胞增殖及上皮間質轉化(EMT)的影響，探討其作用機制。方法設計并合成靶向GSK3β的3個siRNA(siRNA-GSK3β)及陰性對照siRNA(siRNA-NC)，分別轉染PANC1細胞。通過RT-PCR法檢測各組細胞GSK3β mRNA表達量，篩選基因沉默效果最佳的siRNA進行后續(xù)實驗。采用MTT法檢測轉染細胞的增殖。采用RT-PCR法檢測轉染細胞Wnt/β-catenin通路成員Wnt、β-catenin、C-myc、CyclinD1mRNA；Hedgehog通路成員Shh、Smo mRNA；PI3K/Akt/mTOR通路成員PI3K、Akt、mTOR、4E-BP1、p70S6K mRNA以及EMT相關分子Slug、Snail、Twist、E-cadherin、N-cadherin、Vimentin mRNA的表達。結果轉染siRNA-NC的PANC1細胞GSK3β mRNA表達量為1.00±0.15，3個轉染siRNA-GSK3β的GSK3β mRNA表達量分別為0.25±0.08、0.62±0.09、0.70±0.11，轉染siRNA-GSK3β細胞均顯著低于轉染siRNA-NC細胞，差異有統(tǒng)計學意義(P值均<0.0001 )。轉染后siRNA-GSK3β組細胞的增殖顯著低于轉染siRNA-NC組細胞，差異有統(tǒng)計學意義(P<0.05)。轉染后siRNA-GSK3β組細胞的Wnt、β-catenin、C-myc、CyclinD1、PI3K、Akt、mTOR、4E-BP1、p70S6K、Slug、Snail、N-cadherin、Vimentin mRNA表達量分別為0.28±0.04、0.47±0.05、0.37±0.05、0.62±0.08、0.22±0.03、0.47±0.06、0.65±0.08、0.39±0.04、0.56±0.07、0.33±0.05、0.46±0.07、0.55±0.06、0.38±0.04，均顯著低于轉染siRNA-NC組細胞，差異有統(tǒng)計學意義(P值均<0.05)；Shh、Smo mRNA表達量分別為1.10±0.13、1.05±0.11，與轉染siRNA-NC組細胞的差異無統(tǒng)計學意義；Twist mRNA表達量為0.62±0.08，低于轉染siRNA-NC組細胞，E-cadherin mRNA表達量為2.12±0.25，高于轉染siRNA-NC組細胞，但差異均無統(tǒng)計學意義。結論沉默PANC1細胞GSK3β基因表達后能夠通過Wnt/β-catenin、PI3K/Akt通路抑制胰腺癌細胞的增殖，并抑制其上皮間質轉化過程。

【關鍵詞】胰腺腫瘤；糖原合成酶激酶3β；細胞增殖；上皮間質轉化

胰腺癌是消化系統(tǒng)惡性程度極高、預后極差的惡性腫瘤，流行病學資料顯示，胰腺癌患者的中位生存時間不足6個月、5年生存率更低于5%[1-2]。胰腺癌的早期診斷較為困難，確診后又缺乏有效的靶向治療手段，因此患者預后不佳[3]。近年來，糖原合成激酶3β(glycogen synthase kinase 3β, GSK3β)的生物學功能受到了越來越多的重視。GSK3β定位于胞質，屬于絲氨酸/蘇氨酸蛋白激酶，對細胞增殖、分化、侵襲等均具有調控作用[4-5]。本研究應用RNA干擾技術沉默胰腺癌細胞株PANC1的GSK3β表達，觀察其對胰腺癌細胞增殖及上皮間質轉化(epithelial-mesenchymal transition，EMT)的影響，探討其作用機制。

材料與方法

一、材料

胰腺癌細胞株PANC1購自中科院細胞庫，DMEM培養(yǎng)基、胎牛血清、胰蛋白酶均購自Gibco公司，轉染試劑盒購自Invitrogen公司，MTT細胞活力試劑盒購自Promega公司，RNAiso提取液、反轉錄試劑盒以及PCR擴增試劑盒均購自Takara公司。

二、方法

1．細胞培養(yǎng)及siRNA轉染：PANC1細胞復蘇后用含有10%胎牛血清的DMEM在培養(yǎng)瓶中培養(yǎng)，待細胞生長至70%～80%融合后用0.125%的胰蛋白酶消化并傳代。傳代細胞接種于96孔培養(yǎng)板，每孔1×103個細胞。培養(yǎng)過夜后棄含血清的DMEM更換為無血清的DMEM繼續(xù)培養(yǎng)24 h。委托吉瑪公司設計及合成3個靶向GSK3β的siRNA，分別命名為siRNA-GSK3β-1、siRNA-GSK3β-2、siRNA-GSK3β-3。siRNA-GSK3β-1正義序列為5′-CAAGUC-GAUUUAGCUAGUC-3′，反義序列5′-UACCAAGUGUCCGUAGCGU-3′；siRNA-GSK3β-2正義序列5′-AUUUACACUUAAUCAUUC-3′，反義序列5′-UUAUUGCCGCUUAAGCUAGC-3′；siRNA-GSK3β-3正義序列5′-CGUAAAGCUAGCUAGCUG-3′，反義序列5′-AAUGGCAGCAAGUGCAU-3′。同時設計及合成陰性對照siRNA(siRNA-NC)。將轉染試劑分別與3個siRNA-GSK3β及siRNA-NC按1∶2的比例混合，采用脂質體法將它們分別轉染PANC1細胞，以不加siRNA的細胞作為陰性對照，每組設3個復孔，按試劑盒說明書操作。

2．細胞增殖檢測：PANC1細胞轉染siRNA-GSK3β或siRNA-NC前及轉染后6、12、18、24 h分別在細胞培養(yǎng)孔內加入MTT 20 μl，孵育4 h，上酶標儀上讀取各孔波長560 nm處的吸光值(A560值)。

3．轉染細胞相關基因的mRNA表達檢測：取PANC1細胞接種于12孔細胞板，每孔5×104個細胞，轉染siRNA-GSK3β或siRNA-NC后24 h棄培養(yǎng)液，加入RNAiso提取液0.5 ml，用細胞刮刀充分裂解細胞，并依次加入三氯甲烷、異丙醇、95%乙醇抽提總RNA，加入DEPC水溶解。采用逆轉錄試劑盒及PCR試劑盒進行RT-PCR擴增。各基因的引物序列及退火溫度見表1。引物由上海吉瑪公司合成。逆轉錄反應條件：25℃ 10 min，42℃ 30 min，85℃ 5 min；PCR擴增條件：95℃ 10 s、退火溫度15 s、72℃ 20 s，40個循環(huán)。由儀器自帶軟件獲得Ct值，以siRNA-NC組細胞GSK3β mRNA表達量為1，計算siRNA-GSK3β組細胞GSK3β mRNA的相對表達量。

三、統(tǒng)計學處理

結果

一、轉染細胞的沉默效果

轉染siRNA-GSK3β-1、siRNA-GSK3β-2、siRNA-GSK3β-3、siRNA-NC的PANC1細胞GSK3β mRNA相對表達量分別為0.62±0.09、0.25±0.08、0.70±0.11、1.00±0.15。轉染siRNA-GSK3β的3組細胞表達量均顯著低于轉染siRNA-NC細胞，差異有統(tǒng)計學意義(P值均<0.0001 )，其中以轉染siRNA-GSK3β-2細胞的GSK3β沉默效率最高，以此轉染細胞用于后續(xù)實驗。

二、細胞增殖變化

轉染前兩組細胞的增殖差異無統(tǒng)計學意義。轉染后siRNA-GSK3β組PANC1細胞的增殖顯著低于siRNA-NC組，差異均有統(tǒng)計學意義(表2)。

三、轉染細胞Wnt/β-catenin、Hedgehog通路成員mRNA表達的變化

轉染24 h后siRNA-GSK3β組細胞Wnt/β-catenin通路成員Wnt、β-catenin、C-myc、CyclinD1 mRNA表達量均顯著低于siRNA-NC組，差異有統(tǒng)計學意義；Hedgehog通路成員Shh、Smo mRNA表達量與siRNA-NC組的差異無統(tǒng)計學意義(表3)。

表1　轉染細胞相關基因mRNA的引物序列

表2　siRNA-GSK3β組和siRNA-NC組細胞的增殖變化(A560值，

表3　轉染細胞Wnt/β-catenin、Hedgehog通路成員mRNA表達量

四、轉染細胞PI3K/Akt/mTOR通路成員 mRNA表達的變化

轉染24 h后siRNA-GSK3β組細胞PI3K/Akt/mTOR通路成員PI3K、Akt、mTOR、4E-BP1、p70S6K mRNA表達量均顯著低于siRNA-NC組，差異有統(tǒng)計學意義(表4)。

五、轉染細胞EMT相關分子mRNA表達的變化

轉染24 h后siRNA-GSK3β組細胞EMT相關分子Slug、Snail、N-cadherin、Vimentin mRNA表達量均顯著低于siRNA-NC組，差異均有統(tǒng)計學意義。而Twist mRNA表達量低于siRNA-NC組，E-cadherin mRNA表達量高于siRNA-NC組，但差異均無統(tǒng)計學意義(表4)。

討論

GSK3β是近年來倍受重視的一類蛋白激酶，它不但能使多種底物磷酸化，而且參與Wnt/β-catenin、PI3K/Akt及Hedgehog等信號通路的調控，對細胞的增殖、分化、侵襲等生物學行為均具有調控作用[6]。Shakoori等[7]報道，結腸癌組織GSK3β及其活性形式的表達水平顯著高于其配對的周圍正常組織，并且多種結腸癌細胞系GSK3β基因均高表達；應用GSK3β特異性抑制劑或RNA干擾技術抑制GSK3β的表達均能顯著抑制多種結腸癌細胞的增殖，促進細胞凋亡，提示GSK3β表達異常與腫瘤的發(fā)生、發(fā)展有關。但GSK3β究竟發(fā)揮促腫瘤生長的作用還是抑腫瘤生長的作用尚無統(tǒng)一認識。Shao等[8]報道，GSK3β通過降解c-Jun以抑制乳腺癌細胞的生長和成瘤過程，但Grassilli等[9]和Liu等[10]的研究則認為GSK3β對結腸癌、肺癌細胞的生長、上皮充質轉化具有促進作用。目前關于GSK3β是否參與胰腺癌的發(fā)生和發(fā)展以及如何調控胰腺癌細胞的生物學行為的研究較少。

本研究將siRNA-GSK3β轉染胰腺癌細胞PANC1，結果顯示轉染細胞的GSK3β mRNA表達量顯著下調，細胞的增殖明顯被抑制。

GSK3β的下游信號通路涉及Wnt/β-catenin、PI3K/Akt、Hedgehog等[4, 11-12]。 Wnt/β-catenin和Hedgehog信號通路調節(jié)的下游靶基因均為C-myc、CyclinD1等促增殖基因。Wnt分子被活化后，β-catenin的降解過程受阻并在胞質中大量蓄積，轉位進入細胞核后能啟動C-myc、CyclinD1的轉錄過程[13-14]。Hedgehog通路中Shh被活化后Ptch對Smo的結合和抑制作用喪失，Smo釋放并通過多種轉錄因子促進C-myc、CyclinD1的表達[15]。PI3K/Akt通路的上游激活信號由EGFR介導，而下游的效應分子為mTOR。mTOR能夠通過4E-BP1和p70S6K抑制細胞凋亡、促進細胞增殖[16]。本研究結果顯示，PANC1細胞轉染siRNA-GSK3β后，細胞Wnt、β-catenin、C-myc、CyclinD1、PI3K、Akt、mTOR、4E-BP1、p70S6K mRNA表達量均顯著低于轉染siRNA-NC的PANC1細胞，差異均有統(tǒng)計學意義，而兩組細胞Shh、Smo mRNA表達量的差異無統(tǒng)計學意義，提示PANC1細胞的Wnt/β-catenin、PI3K/Akt信號通路調控GSK3β基因的表達，而Hedgehog通路不參與GSK3β基因的調控。

EMT是指上皮細胞失去細胞極性并向具有較高侵襲、遷移能力的間質細胞轉化。上皮細胞是人類進化過程中的終端細胞，通過細胞兩面的極性形成緊密黏附，進而阻滯細胞的遷移過程；間質細胞具有較強的遷移和侵襲能力，細胞間結構松散且連接較少，能夠促進細胞發(fā)生原位浸潤以及遠處轉移[17-18]。Slug、Snail、Twist是調節(jié)EMT過程重要的轉錄因子，能夠抑制上皮細胞標志分子E-cadherin的表達、增加間質細胞標志分子N-cadherin、Vimentin的表達[19]。近年來關于胰腺癌生物學特征的研究證實，EMT是造成胰腺癌病情進展的重要環(huán)節(jié)。本研究結果顯示，PANC1細胞轉染siRNA-GSK3β后，細胞Slug、Snail、Twist、N-cadherin、Vimentin mRNA表達量低于轉染siRNA-NC的細胞，E-cadherin mRNA表達量高于轉染siRNA-NC的細胞，提示抑制GSK3β基因表達能夠阻礙胰腺癌細胞的EMT過程，為胰腺癌靶向GSK3β的基因治療提供實驗依據。

表4　轉染細胞PI3K/Akt/mTOR通路成員及EMT相關分子mRNA表達量

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(本文編輯：屠振興)

Effects of glycogen synthase kinase3β silencing on cell growth and epithelial-mesenchymal transition in pancreatic cancer PANC1 cells

SongBo,MaHongyun,LiSeng,ShaoZhuo,SongBin,HaoJun,ChenPeng,JinGang.DepartmentofGeneralSurgery,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China

【Abstract】ObjectiveTo study the effect of inhibiting glycogen synthase kinase3β (GSK3β) on cell proliferation and epithelial mesenchymal transition (EMT) in pancreatic cancer PANC1 cells. MethodsThe siRNA trageting GSK3β(siRNA-GSK3β) and negative control siRNA (siRNA-NC) were synthesized to transfect pancreatic cancer PANC1 cells. MTT assay was used to detect cell proliferation. mRNA expression of Wnt, β-catenin, C-myc, CyclinD1 in Wnt/β-catenin signaling, Shh and Smo in Hedgehog pathway, PI3K, Akt, mTOR, 4E-BP1, p70S6K in PI3K/Akt/mTOR signal pathway and Slug, Snail, Twist, E-cadherin, N-cadherin, Vimentin involved in EMT were detected by RT-PCR. ResultsGSK3β mRNA expression was 1in cells transfected with siRNA-NC, which was 0.25±0.08, 0.62±0.09, 0.70±0.11 in cells with 3 different siRNA-GSK3βs, respectively. GSK3β mRNA was significantly decreased by transfection of siRNA-GSK3β(P<0.0001). The cell proliferation of siRNA-GSK3β group was significantly lower than that of siRNA-NC group(P<0.05). Wnt, β-catenin, C-myc, CyclinD1, PI3K, Akt, mTOR, 4E-BP1, p70S6K, Slug, Snail, N-cadherin, Vimentin mRNA in cells transfected with siRNA-GSK3β was 0.28±0.04, 0.47±0.05, 0.37±0.05, 0.62±0.08, 0.22±0.03, 0.47±0.06, 0.65±0.08, 0.39±0.04, 0.56±0.07, 0.33±0.05, 0.46±0.07, 0.55±0.06, 0.38±0.04, which were greatly lower than those in cells transfected with siRNA-NC, and the difference was statistically significant (all P<0.05). Shh and Smo mRNA was 1.10±0.13 and 1.05±0.11， which was comparable with those in cells with siRNA-NC. Twist mRNA was 0.62±0.08, which was lower than that in cells with siRNA-NC, while E-cadherin mRNA was 2.12±0.25, which was higher than that in cells with siRNA-NC, but the differences were not statistically significant. ConclusionsGSK3β silencing in PANC1 cells can suppress cell proliferation and EMT of pancreatic cancer cells by regulating Wnt/β-catenin and PI3K/Akt pathway.

【Key words】Pancreatic neoplasms;Glycogen synthase kinase 3β;Cell proliferation;Epithelial mesenchymal transition

(收稿日期：2016-01-06)

Corresponding author:Jin Gang, Email：jin_gang@outlook.com

通信作者：金鋼，Email：jin_gang@outlook.com

DOI:10.3760/cma.j.issn.1674-1935.2016.02.002