Snail基因沉默對胰腺癌PANC1細胞侵襲和增殖的影響

楊靜 吳洪玉 李理想 任洪波

·論著·

Snail基因沉默對胰腺癌PANC1細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基因沉默后對胰腺癌PANC1細胞侵襲和增殖能力的影響。方法 構建針對Snail的小發卡RNA(shRNA-Snail)的慢病毒載體和不針對任何已知mRNA序列的shRNA(shRNA-NC)慢病毒載體，分別感染PANC1細胞，以未感染細胞作為對照組。應用實時熒光定量PCR法和蛋白質印跡法檢測Snail、α-平滑肌肌動蛋白(α-SMA)及E-鈣黏素(E-cadherin)mRNA和蛋白的表達；Transwell小室檢測細胞體外侵襲能力，CCK-8法檢測細胞增殖能力。結果 與shRNA-NC組比較，shRNA-Snail組細胞Snail mRNA和蛋白表達水平顯著下降[(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)]，穿膜細胞數顯著減少[(6.80±0.73)比(26.80±2.52)個/400倍視野]，細胞增殖明顯被抑制[(0.74±0.05)比(1.47±0.04)]，差異均有統計學意義(P值均<0.01)。shRNA-NC組與對照組細胞各指標的差異均無統計學意義。結論 沉默Snail基因表達可在一定程度上抑制胰腺癌PANC1細胞的侵襲和增殖能力。

胰腺腫瘤； Snail因子； RNA，小分子干擾； 腫瘤轉移； 細胞增殖

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

轉錄因子Snail可通過抑制E-鈣黏素(E-cadherin)的表達促進上皮-間質轉化(epithelial-to-mesenchymal transition，EMT)及細胞存活，促進腫瘤的增殖、侵襲和擴散[1]。已有研究證明，伴隨淋巴結和遠處轉移的胰腺癌組織高表達Snail[2]。將Snail基因轉染不表達Snail的高分化胰腺癌細胞BxPC3，結果該株細胞發生EMT，且在體內的生長能力和侵襲性明顯增強[3]。本研究通過shRNA-慢病毒載體干擾技術沉默胰腺癌PANC1細胞Snail基因表達，觀察其對細胞侵襲和增殖的影響。

材料與方法

一、材料與試劑

人胰腺癌細胞株PANC1由第二軍醫大學長海醫院消化內科實驗室惠贈；胰酶、胎牛血清及DMEM培養液購自美國Gibco公司；CCK-8試劑盒購自日本同仁公司；Transwell小室購自美國Corning公司；實時熒光定量PCR相關試劑均購自日本Takara公司；相關引物由Invitrogen公司設計合成；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氧化物酶標記相關二抗均購自Santa Cruz 公司。

二、shRNA合成與慢病毒載體構建

體外合成針對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；另設不針對任何已知mRNA序列的陰性對照shRNA(shRNA-NC)。采用雙酶切方法將shRNA插入質粒載體，重組質粒轉化GeneHogs化學感受態細菌，常規擴增、收集細菌，提取質粒mRNA，應用RT-PCR鑒定插入片段正確后應用慢病毒載體包裝重組質粒，按試劑盒說明書操作，然后將慢病毒表達載體感染293T細胞，收集上清病毒懸液，分裝保存。

三、細胞感染與分組

PANC1細胞常規培養傳代。取104個細胞接種96孔板，培養24 h待細胞融合達80%時換含有5 μg/ml Polybrene的新鮮培養液，分別加入插有Snail-shRNA、shRNA-NC的慢病毒感染24 h，更換新鮮培養液繼續培養72 h，以未感染慢病毒的細胞作為對照組。熒光顯微鏡下觀察熒光信號，熒光細胞超過90%視為感染成功。

四、實時熒光定量PCR檢測

提取各組感染細胞總RNA，先反轉錄為cDNA，再行實時熒光定量PCR。Snail、α-SMA、E-cadherin及內參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反應條件：95℃ 10 min；95℃ 15 s、60℃ 30 s、72℃ 30 s，40個循環；最后72℃延伸10 min。每個樣本設3個復孔。采用公式2-ΔΔCt計算mRNA相對表達量。

五、蛋白質印跡法檢測

收集各組感染細胞，離心并用預冷PBS沖洗后加入RIPA及蛋白酶抑制劑置冰上裂解細胞，所得細胞勻漿以12 000 r/min 4℃離心20 min，取上清定量蛋白。取30 μg蛋白常規行蛋白質印跡法檢測Snail、α-SMA、E-cadherin蛋白表達，以GAPDH為內參，實驗重復3次。應用ImageJ軟件掃描各條帶灰度值，以目的條帶與內參條帶灰度值的比值表示蛋白相對表達量。

六、細胞侵襲實驗

采用聚碳酸酯膜孔徑為8 μm的Transwell小室，小室隔膜表面預先用Matrigel包被后分別在上室和下室加入無血清DMEM培養液4℃過夜，棄培養液。取各組對數生長期細胞，用PBS和無血清培養液先后洗滌1次，以無血清培養液重懸細胞，調整細胞密度為5×104/L。上室加細胞懸液100 μl，下室加含10%血清的培養液600 μl，培養24 h后取出小室，用棉簽擦掉膜上方的Matrigel及未穿膜細胞，置95%乙醇溶液中固定10 min，結晶紫染色8 min，封固于載玻片上。顯微鏡下隨機取8個高倍鏡視野(400倍)，計數每個視野的穿膜細胞。實驗重復3次，取均值。

七、CCK-8法檢測

取各組對數生長期細胞，以5×103個細胞/孔接種96孔板(100 μl)，每組設4個復孔，置37℃、5% CO2條件下培養4 h，加入10 μl CCK-8溶液，繼續培養4 h，上酶標儀測定各孔450 nm處的吸光度值(A450值)。實驗重復3次，取均值。

八、統計學處理

結 果

一、各組PANC1細胞Snail、α-SMA、E-cadherin mRNA表達

shRNA-Snail組、shRNA-NC組、對照組PANC1細胞的Snail mRNA表達量分別為0.27±0.02、0.92±0.03、0.93±0.04；α-SMA mRNA表達量為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表達量較shRNA-NC組顯著下降(t值分別為19.66、7.97)，而E-cadherin mRNA表達量顯著增加(t=6.77)，差異均有統計學意義(P值均<0.01)。shRNA-NC組與對照組間的差異均無統計學意義。

二、各組PANC1細胞Snail、α-SMA、E-cadherin蛋白表達

shRNA-Snail組、shRNA-NC組、對照組PANC1細胞的Snail 蛋白表達量分別為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蛋白表達量顯著增加(t=14.62)，差異均有統計學意義(P值均<0.01)。shRNA-NC組與對照組間的差異均無統計學意義。

三、各組PANC1細胞體外侵襲能力

shRNA-Snail組、shRNA-NC組、對照組PANC1細胞的穿膜細胞數分別為(6.80±0.73)、(26.80±2.52)、(31.20±2.48)個/400倍視野(圖1)，shRNA-Snail組較shRNA-NC組顯著減少，差異有統計學意義(t=7.63，P<0.01)，而shRNA-NC組與對照組之間差異無統計學意義。

四、各組PANC1 細胞的增殖能力

shRNA-Snail組、shRNA-NC組、對照組PANC1細胞培養4 h后A450值分別為0.74±0.05、1.47±0.04、1.68±0.01。shRNA-Snail組細胞增殖較shRNA-NC組顯著被抑制，差異有統計學意義(t=11.23，P<0.01)，而shRNA-NC組與對照組之間差異無統計學意義。

討 論

胰腺癌是消化系統惡性度極高的腫瘤，總體病死率高達85%[4]，主要原因是其具有高侵襲性，早期即可發生轉移[5-7]。EMT能使細胞失去極性和細胞間連接，獲得游走能力，是胰腺癌發生侵襲轉移的主要生物學機制[8]。Yamada等[9]報道，EMT可作為預測胰腺癌預后的獨立危險因素。EMT過程的始動因素為E-cadherin表達缺失[10]。轉錄因子Snail作為E-cadherin的直接抑制子促進EMT的發生[11-14]，被視為EMT的關鍵控制因素。

圖1 shRNA-Snail組(1A)、shRNA-NC組(1B)、對照組(1C)的PANC1細胞侵襲能力(×400)

Tao和Tagare[15]報道，約36%的胰腺癌Snail 陽性表達，同時伴隨E-cadherin低表達，而且有淋巴結轉移和遠處轉移的胰腺癌的Snail陽性表達率明顯升高。Hotz等[10]報道，低分化的胰腺癌細胞株(如PANC1)Snail的陽性表達率明顯高于高分化的胰腺癌細胞株(如HPAF-2、AsPC-1)，而E-cadherin表達則被抑制。 Nishioka等[3]將Snail基因轉染不表達Snail的高分化胰腺癌細胞株BxPC3，結果該細胞株的細胞形態發生了EMT，將轉染Snail基因的細胞種植于裸鼠后，種植瘤的侵襲性明顯增強，早期即發生轉移。此外有報道，抑制Snail表達可以提高胰腺癌對放化療的敏感性[16]。

本研究選擇低分化的PANC1胰腺癌細胞株進行實驗，結果顯示PANC1細胞Snail基因高表達，與Hotz等[10]的研究一致。應用插入shRNA-Snail的慢病毒載體沉默PANC1細胞Snail基因表達后，該細胞的α-SMA表達顯著下降，而E-cadherin表達顯著升高，說明抑制Snail表達后細胞恢復上皮表型。此外，沉默Snail基因表達后細胞的增殖及侵襲能力下降，與α-SMA和E-cadherin表達變化趨勢相吻合。由于本研究僅應用PANC1一種細胞株，因此尚不能說明在所有胰腺癌細胞中抑制Snail表達均可以作為有效的干預措施。

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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 濟南，山東大學齊魯醫院消化內科(楊靜、李理想、任洪波)；第二軍醫大學長海醫院消化內科(吳洪玉)

任洪波，Email: rhb2229@medmail.com.cn

山東省優秀中青年科研獎勵基金(BS2011YY023)

2015-10-23)