蔡松旺, 謝迭來, 翁毅敏, 溫星橋, 張軍航
(中山大學附屬第三醫(yī)院, 1胸外科, 2放射科, 3泌尿外科, 廣東 廣州 510630)
p21活化激酶6對人非小細胞肺癌A549細胞侵襲及遷移能力的影響*
蔡松旺1△, 謝迭來2, 翁毅敏1, 溫星橋3, 張軍航1
(中山大學附屬第三醫(yī)院,1胸外科,2放射科,3泌尿外科, 廣東 廣州 510630)
目的探討p21活化激酶6(PAK6)對非小細胞肺癌侵襲及遷移能力的影響及機制研究。方法實時熒光定量PCR檢測人非小細胞肺癌A548細胞、人支氣管上皮細胞、非小細胞肺癌組織及癌旁組織中PAK6 mRNA的表達。A549細胞分別轉(zhuǎn)染siRNA-PAK6(siPAK6組)和陰性對照(對照組),實時熒光定量PCR技術(shù)檢測PAK6 mRNA,Western blotting檢測PAK6表達量;并行體外遷移及侵襲實驗,檢測轉(zhuǎn)染siRNA-PAK6對A549細胞侵襲及遷移能力的影響;共聚焦顯微鏡觀察細胞骨架的改變。結(jié)果A549細胞中PAK6 mRNA的表達量明顯高于人支氣管上皮細胞中PAK6 mRNA的表達量(3.50±1.16vs1.12±0.42,P<0.05),非小細胞肺癌組織中PAK6 mRNA的表達量明顯高于癌旁組織中PAK6 mRNA的表達量(5.13±1.33vs1.08±0.37,P<0.05)。A549細胞轉(zhuǎn)染siPAK6后,PAK6蛋白表達量下降72%(P<0.05),PAK6 mRNA水平明顯下降(3.72±0.75vs0.69±0.21,P<0.05)。A549細胞轉(zhuǎn)染siPAK6組侵襲及遷移出的細胞數(shù)量明顯少于對照組(P<0.05)。siPAK6組A549細胞骨架應(yīng)力纖維減少明顯,肌動蛋白皺縮。結(jié)論PAK6通過細胞骨架的重構(gòu)影響非小細胞肺癌的侵襲及遷移能力。
p21活化激酶6; 非小細胞肺癌; 腫瘤侵襲; 細胞遷移; 細胞骨架
p21活化激酶-6(p21-activated kinase 6,PAK6)是以雄激素受體的配體結(jié)合上的氨基酸序列629-919, 通過酵母雙雜交識別出來的新的雄激素受體關(guān)聯(lián)蛋白,主要表達于前列腺、睪丸和大腦等器官[1]。我們在前期研究中發(fā)現(xiàn)PAK6可以減弱前列腺癌侵襲、遷移能力[2-3],目前關(guān)于PAK6在肺癌細胞中的作用報道較少,因此,本文擬通過RNA干擾技術(shù),研究PAK6在肺癌A549細胞侵襲及遷移中的作用。
1主要試劑
人支氣管上皮細胞(human bronchial epithelial cells,HBE細胞)和非小細胞肺癌A549細胞購自中國醫(yī)學科學院腫瘤醫(yī)院腫瘤研究所;細胞培養(yǎng)液RPMI-1640和胎牛血清(Gibco),轉(zhuǎn)染試劑Siport(Ambion),Trizol試劑(Invitrogen),siRNA(Santa Cruz Biotechnology),兔抗人PAK6多克隆抗體(Merck),Ⅱ抗(Cell Signal Technology),Transwell小室(Corning,24孔,孔徑8 μm),基質(zhì)膠(BD),F(xiàn)ITC-phalloidin( Sigma)。
2主要方法
2.1細胞培養(yǎng)與轉(zhuǎn)染 HBE及A549細胞用含10%胎牛血清的RPMI-1640培養(yǎng)液于37 ℃、5% CO2的濕化環(huán)境中培養(yǎng)。轉(zhuǎn)染前將細胞接種于6孔板中,待細胞匯合80%左右時,按照siPORT脂質(zhì)體轉(zhuǎn)染法說明書操作,進行siRNA-PAK6 (siPAK6)或陰性對照轉(zhuǎn)染細胞。
2.2實時熒光定量PCR技術(shù)檢測PAK6 mRNA的表達 使用Invitrogen公司的Trizol試劑,按照一步法進行總RNA提取。 以PrimerScriptTMFirst Strand cDNA Synthesis Kit試劑盒逆轉(zhuǎn)錄成單鏈cDNA,GoTaq? Green Master Mix擴增。PAK6上游引物5’- GACTCCATCCTGCTGACCCTC-3’,下游引物5’-CACCTCAGTGGCATACAAAGACC-3’; 擴增片段長159 bp。內(nèi)參照β-actin A0001引物序列TGACGTGGACATCCGCAAAG,β-actin A0002引物序列CTGGAAGGTGGACAGCGAGG。實時熒光定量PCR反應(yīng)(用ΔCt法定量):陰性質(zhì)控標準品采用滅菌雙蒸水,待測樣本按50 μL反應(yīng)體系進行,反應(yīng)條件為:93 ℃ 3 min預變性,然后93 ℃ 30 s、55 ℃ 45 s、72 ℃ 45 s進行40個循環(huán)。
2.3Western blotting檢測 轉(zhuǎn)染48 h后,將6孔板
培養(yǎng)細胞消化離心后,加入蛋白裂解液冰上裂解5 min,冷凍離心提取蛋白,經(jīng)BCA法檢測蛋白濃度。將蛋白樣品25 μg加樣后SDS-PAGE電泳,轉(zhuǎn)膜,封閉,Ⅰ抗(1∶1 000)、Ⅱ抗(1∶2 000)孵育,發(fā)光檢測,GAPDH作為內(nèi)參照。Quantity One軟件定量分析。
2.4CCK-8法檢測細胞活性 按照試劑盒說明書處理細胞,每組設(shè)4個復孔,450 nm作為檢測波長。細胞活性=(處理組A值-空白孔A值)/(對照組A值-空白孔A值)。
2.5體外侵襲及遷移實驗 細胞在無血清RPMI-1640培養(yǎng)液培養(yǎng)24 h后,消化細胞,每個Transwell小室鋪1∶8 基質(zhì)膠80 μL,接種5×104個細胞,加含0.2% BSA無血清RPMI-1640培養(yǎng)液至200 μL,下室加500 μL含10%胎牛血清的RPMI-1640培養(yǎng)液,孵育24 h后棉簽擦去上室未穿過的細胞,4%甲醛固定10 min,0.1%結(jié)晶紫染色。按順序拍照16個視野并計數(shù)取平均值。遷移實驗不需鋪膠,孵育時間為16 h。
2.6共聚焦顯微鏡觀察細胞骨架 A549細胞接種于6孔板中,轉(zhuǎn)染48 h后,預溫PBS液清洗細胞2次,4%甲醛室溫固定10 min,PBS液清洗3次。含1% BSA的PBS液室溫封閉1 h,F(xiàn)ITC-phalloidin(5 mg/L)室溫避光染色1 h,PBS液清洗3次。
3統(tǒng)計學處理
數(shù)據(jù)以均數(shù)±標準誤(mean±SEM)表示,應(yīng)用SPSS統(tǒng)計軟件13.0分析,采用Student’st檢驗進行組間均數(shù)比較,以P<0.05為差異有統(tǒng)計學意義。
1PAK6mRNA在A549細胞、HBE細胞、非小細胞肺癌及癌旁組織中的表達
A549細胞中PAK6 mRNA的表達量明顯高于HBE細胞中PAK6 mRNA的表達量(3.50±1.16vs1.12±0.42,P<0.05),差異有統(tǒng)計學意義,見圖1A;非小細胞肺癌組織中PAK6 mRNA的表達量明顯高于癌旁組織中PAK6 mRNA的表達量(5.13±1.33vs1.08±0.37,P<0.05),差異有統(tǒng)計學意義,見圖1B。
2CCK-8法檢測轉(zhuǎn)染siPAK6后A549細胞活性
A549細胞轉(zhuǎn)染siPAK6后,24 h細胞活性為(97.39±12.72)%,48 h細胞活性為(102.04±12.21)%,72 h細胞活性為(99.90±8.49)%,見圖2。
Figure 1. Expression of PAK6 mRNA in A549 cells, HBE cells(A),non-small-cell lung cancer tissues and adjacent non-tumor tissues (B).Mean±SEM.n=5.*P<0.05vsHBE;#P<0.05vsadjacent non-tumor tissues.
圖1PAK6在A549細胞、HBE細胞中的表達
Figure 2. Viability of A549 cells after siPAK6 transfection.Mean±SEM.n=5.
圖2轉(zhuǎn)染siPAK6對A549細胞活性的影響
3轉(zhuǎn)染siPAK6后A549細胞中PAK6變化
A549細胞轉(zhuǎn)染siPAK6 48 h后,PAK6 mRNA水平下降 (3.72±0.75vs0.69±0.21),PAK6蛋白表達量下降72%,差異均有統(tǒng)計學意義(P<0.05),見圖3。
Figure 3. Knockdown ofPAK6 by siRNA for 48 h efficiently repressed either mRNA (A) or protein (B) levels of endogenous PAK6 in A549 cells.Mean±SEM.n=5.*P<0.05vsNC.
圖3轉(zhuǎn)染siPAK6對A549細胞PAK6mRNA及蛋白水平的影響
4siPAK6對A549細胞侵襲及遷移能力的影響
用30 nmol/L siPAK6轉(zhuǎn)染48 h后,轉(zhuǎn)染siPAK6組A549細胞侵襲及遷移出的細胞數(shù)量明顯少于對照組(P<0.05),說明轉(zhuǎn)染siPAK6后,A549細胞侵襲及遷移能力下降,見圖4。
Figure 4. Knockdown ofPAK6 by siRNA decreased A549 cell migration (A) and invasion (B)invitro(crystal violet staining,×40).Mean±SEM.n=5.*P<0.05vsNC.
圖4轉(zhuǎn)染siPAK6對A549細胞侵襲及遷移能力的影響
5siPAK6對A549細胞骨架的影響
siPAK6組A549細胞骨架應(yīng)力纖維明顯減少,肌動蛋白皺縮,表明轉(zhuǎn)染siPAK6后A549細胞骨架發(fā)生重構(gòu);在×10視野下,放大倍率、實驗數(shù)據(jù)采集像素點、光切聚焦平面和厚度、激發(fā)和檢測波長、檢測電壓等參數(shù)均一致情況下,連續(xù)拍攝10個視野,siPAK6組熒光強度(86.75±5.81)明顯弱于對照組(152.78±9.09),差異有統(tǒng)計學意義(P<0.01),見圖5。
Figure 5. Knockdown ofPAK6 by siRNA impaired cytoskeleton of A549 cells. Stress fibers (polymerised actin) and actin filaments were demonstrated by FITC-phalloidin staining.
圖5轉(zhuǎn)染siPAK6對A549細胞骨架的影響
鑒于PAK6在惡性腫瘤中的作用,我們檢測了非小細胞肺癌A549細胞中PAK6的表達量,結(jié)果表明PAK6在A549細胞中表達量顯著高于正常支氣管上皮細胞。我們進一步檢測了非小細胞肺癌組織及正常肺組織PAK6表達量,結(jié)果顯示PAK6在非小細胞肺癌組織中表達量顯著高于正常肺組織。以上結(jié)果表明PAK6與非小細胞肺癌相關(guān),但目前尚未見到相關(guān)報道。
遠處遷移是癌癥患者最主要的死亡原因[4],為了進一步了解PAK6在非小細胞肺癌中的作用,我們通過轉(zhuǎn)染siPAK6觀察對A549細胞侵襲及遷移能力的影響。結(jié)果表明下調(diào)PAK6表達后A549細胞侵襲及遷移能力明顯減弱。進一步表明PAK6在非小細胞肺癌遷移機制中起重要作用。
細胞骨架是一種存在于細胞漿中的纖維網(wǎng)架結(jié)構(gòu)體系,具有維持細胞形態(tài)及細胞運動的功能,在細胞遷移及侵襲功能中起重要作用[5-6]。我們通過共聚焦顯微鏡觀察,可明顯看到轉(zhuǎn)染siPAK6組A549細胞骨架應(yīng)力纖維均明顯減少,肌動蛋白皺縮,表明PAK6可能通過細胞骨架的重構(gòu)影響其侵襲和遷移能力。
本研究通過體外實驗發(fā)現(xiàn)PAK6在非小細胞肺癌組織及A549細胞中表達量顯著升高,并發(fā)現(xiàn)PAK6通過細胞骨架的重構(gòu)影響非小細胞肺癌的侵襲、遷移能力。我們將在下一步研究中明確PAK6影響非小細胞肺癌侵襲及遷移能力的分子機制。
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Effectsofp21-activatedkinase6oninvasionandmigrationofhumannon-small-celllungcancerA549cells
CAI Song-wang1, XIE Die-lai2, WENG Yi-min1, WEN Xing-qiao3, ZHANG Jun-hang1
(1DepartmentofCardiothoracicSurgery,2DepartmentofRadiology,3DepartmentofUrinarySurgery,theThirdAffiliatedHospital,SunYat-senUniversity,Guangzhou510630,China.E-mail:songwangcai@yahoo.com)
AIM: To investigate the effects of p21-activated kinase 6 (PAK6) on the invasive and migratory abilities of human non-small-cell lung cancer A549 cells.METHODSThe expression of PAK6 mRNA in A549 cells, human bronchial epithelial (HBE) cells, non-small-cell lung cancer tissues and paired adjacent non-tumor tissues was measured by real-time PCR. After A549 cells were transfected with siRNA-PAK6 (siPAK6) or negative control (NC) for 48 h, the expression of PAK6 at mRNA and protein levels was measured by real-time PCR and Western blotting, respectively. The invasion and migration of A549 cells were detected by Matrigel invasion assay and Transwell migration assay. The cytoskeletal changes were observed with FITC-phalloidin staining under confocal microscope.RESULTSThe level of PAK6 mRNA in A549 cells was higher than that in HBE cells (3.50±1.16vs1.12±0.42,P<0.05). The level of PAK6 mRNA in non-small-cell lung cancer tissues was higher than that in paired adjacent non-tumor tissues (5.13±1.33vs1.08±0.37,P<0.05). The expression of PAK6 protein decreased by 72% in A549 cells transfected with siPAK6 (P<0.05), and the level of PAK6 mRNA significantly decreased in A549 cells transfected with siPAK6 (3.72±0.75vs0.69±0.21,P<0.05). Matrigel invasion assay and Transwell migration assay demonstrated that knockdown ofPAK6 markedly attenuated the invasion and migration of A549 cells (P<0.05). The cytoskeletal actin remodeling and reduction of stress fibers in A549 cells transfected with siPAK6 were observed under confocal microscope.CONCLUSIONPAK6 may affect the invasive and migratory abilities of non-small-cell lung cancer cells by cytoskeletal actin remodeling.
p21-activated kinase 6; Non-small-cell lung cancer; Neoplasm invasiveness; Cell migration; Cytoskeleton
R363
A
1000- 4718(2013)09- 1637- 04
2013- 01- 09
2013- 07- 10
廣東省科技計劃(No.2012B031800063);廣東省醫(yī)學科研基金資助項目(No.B2012111)
△通迅作者 Tel: 020-85252230; E-mail: songwangcai@yahoo.com
10.3969/j.issn.1000- 4718.2013.09.017