Snail基因沉默對(duì)胰腺癌PANC1細(xì)胞侵襲和增殖的影響

楊靜 吳洪玉 李理想 任洪波

·論著·

Snail基因沉默對(duì)胰腺癌PANC1細(xì)胞侵襲和增殖的影響

楊靜 吳洪玉 李理想 任洪波

目的 觀察Snail基因沉默后對(duì)胰腺癌PANC1細(xì)胞侵襲和增殖能力的影響。方法 構(gòu)建針對(duì)Snail的小發(fā)卡RNA(shRNA-Snail)的慢病毒載體和不針對(duì)任何已知mRNA序列的shRNA(shRNA-NC)慢病毒載體，分別感染PANC1細(xì)胞，以未感染細(xì)胞作為對(duì)照組。應(yīng)用實(shí)時(shí)熒光定量PCR法和蛋白質(zhì)印跡法檢測(cè)Snail、α-平滑肌肌動(dòng)蛋白(α-SMA)及E-鈣黏素(E-cadherin)mRNA和蛋白的表達(dá)；Transwell小室檢測(cè)細(xì)胞體外侵襲能力，CCK-8法檢測(cè)細(xì)胞增殖能力。結(jié)果 與shRNA-NC組比較，shRNA-Snail組細(xì)胞Snail mRNA和蛋白表達(dá)水平顯著下降[(0.27±0.02)比(0.92±0.03)，(0.26±0.02)比(0.80±0.02)]，α-SMA mRNA和蛋白表達(dá)水平亦顯著下降[(0.33±0.04)比(0.97±0.07)，(0.31±0.04)比(0.74±0.06)]，E-cadherin mRNA和蛋白表達(dá)水平則顯著升高[(1.57±0.45)比(0.95±0.08)，(0.86±0.03)比(0.20±0.03)]，穿膜細(xì)胞數(shù)顯著減少[(6.80±0.73)比(26.80±2.52)個(gè)/400倍視野]，細(xì)胞增殖明顯被抑制[(0.74±0.05)比(1.47±0.04)]，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。shRNA-NC組與對(duì)照組細(xì)胞各指標(biāo)的差異均無(wú)統(tǒng)計(jì)學(xué)意義。結(jié)論 沉默Snail基因表達(dá)可在一定程度上抑制胰腺癌PANC1細(xì)胞的侵襲和增殖能力。

胰腺腫瘤； Snail因子； RNA，小分子干擾； 腫瘤轉(zhuǎn)移； 細(xì)胞增殖

Fund program：Outstanding Yong and Middle-aged Scientific Research Fund of Shandong Province(BS2011YY023)

轉(zhuǎn)錄因子Snail可通過(guò)抑制E-鈣黏素(E-cadherin)的表達(dá)促進(jìn)上皮-間質(zhì)轉(zhuǎn)化(epithelial-to-mesenchymal transition，EMT)及細(xì)胞存活，促進(jìn)腫瘤的增殖、侵襲和擴(kuò)散[1]。已有研究證明，伴隨淋巴結(jié)和遠(yuǎn)處轉(zhuǎn)移的胰腺癌組織高表達(dá)Snail[2]。將Snail基因轉(zhuǎn)染不表達(dá)Snail的高分化胰腺癌細(xì)胞BxPC3，結(jié)果該株細(xì)胞發(fā)生EMT，且在體內(nèi)的生長(zhǎng)能力和侵襲性明顯增強(qiáng)[3]。本研究通過(guò)shRNA-慢病毒載體干擾技術(shù)沉默胰腺癌PANC1細(xì)胞Snail基因表達(dá)，觀察其對(duì)細(xì)胞侵襲和增殖的影響。

材料與方法

一、材料與試劑

人胰腺癌細(xì)胞株P(guān)ANC1由第二軍醫(yī)大學(xué)長(zhǎng)海醫(yī)院消化內(nèi)科實(shí)驗(yàn)室惠贈(zèng)；胰酶、胎牛血清及DMEM培養(yǎng)液購(gòu)自美國(guó)Gibco公司；CCK-8試劑盒購(gòu)自日本同仁公司；Transwell小室購(gòu)自美國(guó)Corning公司；實(shí)時(shí)熒光定量PCR相關(guān)試劑均購(gòu)自日本Takara公司；相關(guān)引物由Invitrogen公司設(shè)計(jì)合成；羊抗人Snail多克隆抗體(sc-10432)、兔抗人E-cadherin多克隆抗體(sc-7870)、羊抗人GAPDH單克隆抗體以及辣根過(guò)氧化物酶標(biāo)記相關(guān)二抗均購(gòu)自Santa Cruz 公司。

二、shRNA合成與慢病毒載體構(gòu)建

體外合成針對(duì)Snail的shRNA(shRNA-Snail)，正義序列為5′-GATCCGGCCACTCAGATGTCAA-GAAGTAGGTACCTACTTCTTGACATCTGAGTGGTTT-TTG-3′，反義序列為5′-AATTCAAAAACCACTCAGATGTCAAGAAGTA-GGTACCTACTTCTTGACATCTGAGTGGCCG-3′，兩端分別帶EcoRⅠ和BamHⅠ酶切位點(diǎn)；另設(shè)不針對(duì)任何已知mRNA序列的陰性對(duì)照shRNA(shRNA-NC)。采用雙酶切方法將shRNA插入質(zhì)粒載體，重組質(zhì)粒轉(zhuǎn)化GeneHogs化學(xué)感受態(tài)細(xì)菌，常規(guī)擴(kuò)增、收集細(xì)菌，提取質(zhì)粒mRNA，應(yīng)用RT-PCR鑒定插入片段正確后應(yīng)用慢病毒載體包裝重組質(zhì)粒，按試劑盒說(shuō)明書(shū)操作，然后將慢病毒表達(dá)載體感染293T細(xì)胞，收集上清病毒懸液，分裝保存。

三、細(xì)胞感染與分組

PANC1細(xì)胞常規(guī)培養(yǎng)傳代。取104個(gè)細(xì)胞接種96孔板，培養(yǎng)24 h待細(xì)胞融合達(dá)80%時(shí)換含有5 μg/ml Polybrene的新鮮培養(yǎng)液，分別加入插有Snail-shRNA、shRNA-NC的慢病毒感染24 h，更換新鮮培養(yǎng)液繼續(xù)培養(yǎng)72 h，以未感染慢病毒的細(xì)胞作為對(duì)照組。熒光顯微鏡下觀察熒光信號(hào)，熒光細(xì)胞超過(guò)90%視為感染成功。

四、實(shí)時(shí)熒光定量PCR檢測(cè)

提取各組感染細(xì)胞總RNA，先反轉(zhuǎn)錄為cDNA，再行實(shí)時(shí)熒光定量PCR。Snail、α-SMA、E-cadherin及內(nèi)參GAPDH引物序列如下：Snail正義序列5′-TCGGAAGCCTAACTACAGCGA-3′，反義序列5′-AGATGAGCATTGGCAGCGAG-3′；α-SMA正義序列5′-CCAGCTATGTGAAGAAGAAGAGG- 3′，反義序列5′-GTGATCTCCTTCTGCATTCGGT-3′；E-cadherin正義序列5′ -ATCCAAAGCCTCAGGTCATAAACA- 3′，反義序列5′-AAGAAACAGCAAGAGCAGCAGAAT-3′；GAPDH正義序列5′-CGGGAAACTGTGGCGTGAT-3′，反義序列5′-CAAAGGTGGAGGAGTGGGT-3′。PCR反應(yīng)體系：2×熒光定量SYBR Green mix 10 μl，上下游引物各0.5 μl，DyeⅡ0.4 μl，cDNA 5 μl，加雙蒸水至20 μl。PCR反應(yīng)條件：95℃ 10 min；95℃ 15 s、60℃ 30 s、72℃ 30 s，40個(gè)循環(huán)；最后72℃延伸10 min。每個(gè)樣本設(shè)3個(gè)復(fù)孔。采用公式2-ΔΔCt計(jì)算mRNA相對(duì)表達(dá)量。

五、蛋白質(zhì)印跡法檢測(cè)

收集各組感染細(xì)胞，離心并用預(yù)冷PBS沖洗后加入RIPA及蛋白酶抑制劑置冰上裂解細(xì)胞，所得細(xì)胞勻漿以12 000 r/min 4℃離心20 min，取上清定量蛋白。取30 μg蛋白常規(guī)行蛋白質(zhì)印跡法檢測(cè)Snail、α-SMA、E-cadherin蛋白表達(dá)，以GAPDH為內(nèi)參，實(shí)驗(yàn)重復(fù)3次。應(yīng)用ImageJ軟件掃描各條帶灰度值，以目的條帶與內(nèi)參條帶灰度值的比值表示蛋白相對(duì)表達(dá)量。

六、細(xì)胞侵襲實(shí)驗(yàn)

采用聚碳酸酯膜孔徑為8 μm的Transwell小室，小室隔膜表面預(yù)先用Matrigel包被后分別在上室和下室加入無(wú)血清DMEM培養(yǎng)液4℃過(guò)夜，棄培養(yǎng)液。取各組對(duì)數(shù)生長(zhǎng)期細(xì)胞，用PBS和無(wú)血清培養(yǎng)液先后洗滌1次，以無(wú)血清培養(yǎng)液重懸細(xì)胞，調(diào)整細(xì)胞密度為5×104/L。上室加細(xì)胞懸液100 μl，下室加含10%血清的培養(yǎng)液600 μl，培養(yǎng)24 h后取出小室，用棉簽擦掉膜上方的Matrigel及未穿膜細(xì)胞，置95%乙醇溶液中固定10 min，結(jié)晶紫染色8 min，封固于載玻片上。顯微鏡下隨機(jī)取8個(gè)高倍鏡視野(400倍)，計(jì)數(shù)每個(gè)視野的穿膜細(xì)胞。實(shí)驗(yàn)重復(fù)3次，取均值。

七、CCK-8法檢測(cè)

取各組對(duì)數(shù)生長(zhǎng)期細(xì)胞，以5×103個(gè)細(xì)胞/孔接種96孔板(100 μl)，每組設(shè)4個(gè)復(fù)孔，置37℃、5% CO2條件下培養(yǎng)4 h，加入10 μl CCK-8溶液，繼續(xù)培養(yǎng)4 h，上酶標(biāo)儀測(cè)定各孔450 nm處的吸光度值(A450值)。實(shí)驗(yàn)重復(fù)3次，取均值。

八、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、各組PANC1細(xì)胞Snail、α-SMA、E-cadherin mRNA表達(dá)

shRNA-Snail組、shRNA-NC組、對(duì)照組PANC1細(xì)胞的Snail mRNA表達(dá)量分別為0.27±0.02、0.92±0.03、0.93±0.04；α-SMA mRNA表達(dá)量為0.33±0.04、0.97±0.07、0.98±0.06；E-cadherin mRNA為1.57±0.45、0.95±0.08、0.96±0.05。shRNA-Snail組Snail、α-SMA的mRNA表達(dá)量較shRNA-NC組顯著下降(t值分別為19.66、7.97)，而E-cadherin mRNA表達(dá)量顯著增加(t=6.77)，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。shRNA-NC組與對(duì)照組間的差異均無(wú)統(tǒng)計(jì)學(xué)意義。

二、各組PANC1細(xì)胞Snail、α-SMA、E-cadherin蛋白表達(dá)

shRNA-Snail組、shRNA-NC組、對(duì)照組PANC1細(xì)胞的Snail 蛋白表達(dá)量分別為0.26±0.02、0.80±0.02、0.83±0.03；α-SMA蛋白為0.31±0.04、0.74±0.06、0.82±0.02；E-cadherin蛋白為0.86±0.03、0.20±0.03、0.19±0.01。shRNA-Snail組Snail蛋白、α-SMA蛋白表達(dá)量較shRNA-NC組顯著下降(t值分別為19.02、5.85)，而E-cadherin蛋白表達(dá)量顯著增加(t=14.62)，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。shRNA-NC組與對(duì)照組間的差異均無(wú)統(tǒng)計(jì)學(xué)意義。

三、各組PANC1細(xì)胞體外侵襲能力

shRNA-Snail組、shRNA-NC組、對(duì)照組PANC1細(xì)胞的穿膜細(xì)胞數(shù)分別為(6.80±0.73)、(26.80±2.52)、(31.20±2.48)個(gè)/400倍視野(圖1)，shRNA-Snail組較shRNA-NC組顯著減少，差異有統(tǒng)計(jì)學(xué)意義(t=7.63，P<0.01)，而shRNA-NC組與對(duì)照組之間差異無(wú)統(tǒng)計(jì)學(xué)意義。

四、各組PANC1 細(xì)胞的增殖能力

shRNA-Snail組、shRNA-NC組、對(duì)照組PANC1細(xì)胞培養(yǎng)4 h后A450值分別為0.74±0.05、1.47±0.04、1.68±0.01。shRNA-Snail組細(xì)胞增殖較shRNA-NC組顯著被抑制，差異有統(tǒng)計(jì)學(xué)意義(t=11.23，P<0.01)，而shRNA-NC組與對(duì)照組之間差異無(wú)統(tǒng)計(jì)學(xué)意義。

討 論

胰腺癌是消化系統(tǒng)惡性度極高的腫瘤，總體病死率高達(dá)85%[4]，主要原因是其具有高侵襲性，早期即可發(fā)生轉(zhuǎn)移[5-7]。EMT能使細(xì)胞失去極性和細(xì)胞間連接，獲得游走能力，是胰腺癌發(fā)生侵襲轉(zhuǎn)移的主要生物學(xué)機(jī)制[8]。Yamada等[9]報(bào)道，EMT可作為預(yù)測(cè)胰腺癌預(yù)后的獨(dú)立危險(xiǎn)因素。EMT過(guò)程的始動(dòng)因素為E-cadherin表達(dá)缺失[10]。轉(zhuǎn)錄因子Snail作為E-cadherin的直接抑制子促進(jìn)EMT的發(fā)生[11-14]，被視為EMT的關(guān)鍵控制因素。

圖1 shRNA-Snail組(1A)、shRNA-NC組(1B)、對(duì)照組(1C)的PANC1細(xì)胞侵襲能力(×400)

Tao和Tagare[15]報(bào)道，約36%的胰腺癌Snail 陽(yáng)性表達(dá)，同時(shí)伴隨E-cadherin低表達(dá)，而且有淋巴結(jié)轉(zhuǎn)移和遠(yuǎn)處轉(zhuǎn)移的胰腺癌的Snail陽(yáng)性表達(dá)率明顯升高。Hotz等[10]報(bào)道，低分化的胰腺癌細(xì)胞株(如PANC1)Snail的陽(yáng)性表達(dá)率明顯高于高分化的胰腺癌細(xì)胞株(如HPAF-2、AsPC-1)，而E-cadherin表達(dá)則被抑制。 Nishioka等[3]將Snail基因轉(zhuǎn)染不表達(dá)Snail的高分化胰腺癌細(xì)胞株BxPC3，結(jié)果該細(xì)胞株的細(xì)胞形態(tài)發(fā)生了EMT，將轉(zhuǎn)染Snail基因的細(xì)胞種植于裸鼠后，種植瘤的侵襲性明顯增強(qiáng)，早期即發(fā)生轉(zhuǎn)移。此外有報(bào)道，抑制Snail表達(dá)可以提高胰腺癌對(duì)放化療的敏感性[16]。

本研究選擇低分化的PANC1胰腺癌細(xì)胞株進(jìn)行實(shí)驗(yàn)，結(jié)果顯示PANC1細(xì)胞Snail基因高表達(dá)，與Hotz等[10]的研究一致。應(yīng)用插入shRNA-Snail的慢病毒載體沉默PANC1細(xì)胞Snail基因表達(dá)后，該細(xì)胞的α-SMA表達(dá)顯著下降，而E-cadherin表達(dá)顯著升高，說(shuō)明抑制Snail表達(dá)后細(xì)胞恢復(fù)上皮表型。此外，沉默Snail基因表達(dá)后細(xì)胞的增殖及侵襲能力下降，與α-SMA和E-cadherin表達(dá)變化趨勢(shì)相吻合。由于本研究?jī)H應(yīng)用PANC1一種細(xì)胞株，因此尚不能說(shuō)明在所有胰腺癌細(xì)胞中抑制Snail表達(dá)均可以作為有效的干預(yù)措施。

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(本文編輯：冀凱宏)

The effect of Snail gene silencing on cell invasion and proliferation in human pancreatic cancer cell line PANC1

YangJing,Wuhongyu,LiLixiang,RenHongbo.

DepartmentofGastroenterology,QiluHospital,ShandongUniversity,Jinan250012，China

RenHongbo，Email：rhb2229@medmail.com.cn

Objective To observe the effect of silencing Snail gene on the invasion and proliferation ability of human pancreatic cancer cell line PANC1. Methods Lentiviral vectors that can express small hairpin RNA(shRNA) targeting human Snail gene(shRNA-Snail) or shRNA sequence that did not match any known mRNA(shRNA-NC) were constructed，and transfected into PANC1 cells. Untransfected cells served as control. mRNA and protein expression of Snail，α-smooth muscle actin(α-SMA) and E-cadherin was determined by real time quantitative PCR and Western blotting, respectively. In vitro invasion ability was tested by Transwell model. Proliferation ability was measured by CCK-8 assay. Results Compared with those in shRNA-NC group， Snail mRNA (0.27±0.02vs0.92±0.03) and protein level (0.26±0.02vs0.80±0.02)，and α-SMA mRNA (0.33±0.04vs0.97±0.07) and protein level (0.31±0.04vs0.74±0.06) in shRNA-Snail group were obviously decreased, but E-cadherin mRNA (1.57±0.45vs0.95±0.08) and protein level (0.86±0.03vs0.20±0.03) were greatly increased. The number of cells permeating the septum of transwell [(6.80±0.73)/400 magnificationvs(26.80±2.52)/400 magnification,P<0.01] was significantly decreased, and cell proliferation was inhibited(0.74±0.05vs1.47±0.04,P<0.01). All the differences above were statistically significant (allP<0.01). No significant differences were observed between shRNA-NC and normal control group. Conclusions Silencing Snail gene may restrain the invasion and proliferation ability of PANC1 cells to a certain degree.

Pancreatic neoplasms； Transcriptional factor Snail； RNA, Small interfering; Neoplasms metastasis; Cell proliferation

10.3760/cma.j.issn.1674-1935.2017.01.003

250012 濟(jì)南，山東大學(xué)齊魯醫(yī)院消化內(nèi)科(楊靜、李理想、任洪波)；第二軍醫(yī)大學(xué)長(zhǎng)海醫(yī)院消化內(nèi)科(吳洪玉)

任洪波，Email: rhb2229@medmail.com.cn

山東省優(yōu)秀中青年科研獎(jiǎng)勵(lì)基金(BS2011YY023)

2015-10-23)