PAK4-LIMK1-Cofilin信號(hào)通路對(duì)非小細(xì)胞肺癌遷移及侵襲能力的影響*

李冬霞，　王永玲，　蔡松旺

(1新鄉(xiāng)醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)院，河南 新鄉(xiāng) 453003；2中山大學(xué)附屬第三醫(yī)院胸外科，廣東 廣州 510630)

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PAK4-LIMK1-Cofilin信號(hào)通路對(duì)非小細(xì)胞肺癌遷移及侵襲能力的影響*

李冬霞1，王永玲1，蔡松旺2△

(1新鄉(xiāng)醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)院，河南 新鄉(xiāng) 453003；2中山大學(xué)附屬第三醫(yī)院胸外科，廣東 廣州 510630)

[摘要]目的： 探討p21活化激酶4(PAK4)對(duì)非小細(xì)胞肺癌(NSCLC)侵襲和遷移能力的影響及相關(guān)機(jī)制。方法: PAK4-siRNA或陰性對(duì)照轉(zhuǎn)染A549和NCI-p20細(xì)胞株，實(shí)時(shí)熒光定量PCR和Western blot分別檢測(cè)細(xì)胞PAK4 mRNA和蛋白表達(dá)水平，Transwell小室法檢測(cè)其對(duì)細(xì)胞侵襲和遷移能力的影響；Western blot檢測(cè)其LIMK1、cofilin及其磷酸化水平；免疫共沉淀檢測(cè)PAK4和LIMK1蛋白是否直接綁定；體外激酶分析實(shí)驗(yàn)檢測(cè)LIMK1是否是PAK4的激酶底物；Western blot檢測(cè)10例非小細(xì)胞肺癌組織中PAK4和磷酸化LIMK1的相關(guān)性；PAK4-siRNA和LIMK1質(zhì)粒共轉(zhuǎn)染A549和NCI-p20細(xì)胞后觀察細(xì)胞遷移和侵襲能力變化。結(jié)果: 沉默PAK4后A549和NCI-p20細(xì)胞遷移和侵襲能力明顯減弱(P<0.05)；LIMK1和cofilin蛋白水平無顯著變化，而磷酸化LIMK1和磷酸化cofilin水平顯著下調(diào)；免疫共沉淀結(jié)果示PAK4與LIMK1相互綁定；激酶分析實(shí)驗(yàn)結(jié)果顯示，激酶缺陷型PAK4(K350M)使LIMK1磷酸化的作用顯著低于野生型PAK4或活化型PAK4(S445N)(P<0.05)。Western blot檢測(cè)結(jié)果示非小細(xì)胞肺癌組織中PAK4表達(dá)上調(diào)的程度與磷酸化LIMK1含量呈正相關(guān)(P<0.05)；PAK4-siRNA和LIMK1質(zhì)粒共轉(zhuǎn)染A549和NCI-p20細(xì)胞后，遷移和侵襲細(xì)胞數(shù)均高于PAK4-siRNA轉(zhuǎn)染組(P<0.05)。結(jié)論: PAK4通過直接磷酸化LIMK1而促進(jìn)非小細(xì)胞肺癌的遷移及侵襲能力。

[關(guān)鍵詞]p21活化激酶4； LIM激酶1； 非小細(xì)胞肺癌； 細(xì)胞遷移； 細(xì)胞侵襲

p21活化激酶4(p21-activated kinase 4，PAK4)屬于絲氨酸/蘇氨酸蛋白激酶家族中的成員，是Rho家族Rac、Cdc42等下游的靶蛋白，具有調(diào)節(jié)細(xì)胞骨架、細(xì)胞增殖、凋亡和遷移等功能[1-3]。目前已發(fā)現(xiàn)人體多種惡性腫瘤的發(fā)生發(fā)展與PAK4異常有關(guān)[4-5]，而PAK4在人非小細(xì)胞肺癌(non-small-cell lung cancer，NSCLC)中的生物學(xué)作用及其機(jī)制未見相關(guān)報(bào)道。我們前期的研究發(fā)現(xiàn)PAK4與NSCLC的分化程度、淋巴結(jié)轉(zhuǎn)移、遠(yuǎn)處轉(zhuǎn)移和臨床分期相關(guān)，預(yù)后分析提示PAK4高表達(dá)與預(yù)后差相關(guān)。本研究旨在通過RNA干擾技術(shù)，探討PAK4在非小細(xì)胞肺癌侵襲及遷移中的作用及相關(guān)機(jī)制。

材料和方法

1材料

A549和NCI-p20細(xì)胞株購自中國(guó)醫(yī)學(xué)科學(xué)院上海生科院細(xì)胞資源中心；選用中山大學(xué)第三附屬醫(yī)院2012～2013年10例原發(fā)性非小細(xì)胞肺癌手術(shù)新鮮切除標(biāo)本(切除后標(biāo)本迅速放入液氮中，標(biāo)本均經(jīng)病理醫(yī)生檢查驗(yàn)證和歸類)；RPMI-1640培養(yǎng)基和胎牛血清(Gibco)；siPORTTMNeoFXTM轉(zhuǎn)染劑(Am-bion)；PrimeScript RT試劑盒(Promega)；醋酸纖維膜(Bio-Rad)；PAK4-siRNA(si-PAK4; 圣克魯斯生物技術(shù)公司)；LIMK1質(zhì)粒(上海吉瑪生物有限公司)；TRIzol試劑和LIMK2純化蛋白(Life Technologies)；兔抗人PAK4抗體、LIMK1抗體、p-LIMK1、cofilin 抗體、p-cofilin 抗體和GAPDH內(nèi)參照抗體(CST)；ECL顯色試劑盒(Pierce)。

2方法

2.1細(xì)胞培養(yǎng)與質(zhì)粒轉(zhuǎn)染A549和NCI-p20細(xì)胞于含10%胎牛血清的RPMI-1640培養(yǎng)基中培養(yǎng)，置于37 ℃、5% CO2培養(yǎng)箱培養(yǎng)，隔天傳代1次。轉(zhuǎn)染前24 h將A549和NCI-p20 細(xì)胞分別接種于6孔板中，細(xì)胞密度大約為5×108/L，待細(xì)胞匯合80%左右時(shí)，按照siPORTTMNeoFXTM轉(zhuǎn)染試劑操作手冊(cè)將si-PAK4、si-PAK4+LIMK1質(zhì)粒、LIMK1質(zhì)粒或陰性對(duì)照轉(zhuǎn)染細(xì)胞，繼續(xù)在37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)。

參考文獻(xiàn)2.2Western blot實(shí)驗(yàn)參照[3]步驟進(jìn)行。轉(zhuǎn)染48 h后提取蛋白，利用10%的SDS-PAGE分離蛋白，轉(zhuǎn)膜，封閉，Ⅰ抗(1∶1 000)、Ⅱ抗(1∶1 000)孵育，ECL發(fā)光檢測(cè)，GAPDH作為內(nèi)參照。使用Quantity One 軟件進(jìn)行定量分析。

2.3實(shí)時(shí)熒光定量PCR實(shí)驗(yàn)按照一步法使用Invitrogen的TRIzol試劑來提取總RNA。使用PrimeScript RT Reagent Kit試劑盒逆轉(zhuǎn)錄成單鏈cDNA。使用ABI 7900HT Fast Real-Time PCR體系來進(jìn)行實(shí)時(shí)熒光定量PCR。PAK4的上游引物為5’-ATGTGGTGGAGATGTACAACAGCTA-3’，下游引物為5’-GTTCATCCTGGTGTGGGTGAC-3’；內(nèi)參照U6的上游引物為5’-TGCGGGTGCTCGCTTCGGCAGC-3’，下游引物為 5’-CCAGTGCAGGGTCCGAGGT-3’。

2.4Matrigel侵襲及Transwell遷移實(shí)驗(yàn)Matrigel侵襲實(shí)驗(yàn)：每個(gè)Transwell 小室鋪50 μL Matrigel基質(zhì)蛋白，5×104個(gè)細(xì)胞加入不含血清的Matrigel侵襲小室(BD)，下室加含10%胎牛血清的RPMI-1640培養(yǎng)液，培養(yǎng)24 h后用棉簽擦去小室上層未遷移的細(xì)胞，遷移至小室下層的細(xì)胞用4%多聚甲醛固定、結(jié)晶紫染色。各取5個(gè)400倍視野，顯微鏡下觀察拍照。Transwell遷移實(shí)驗(yàn)不需鋪膠，其余操作同Matrigel侵襲實(shí)驗(yàn)。

2.5免疫共沉淀實(shí)驗(yàn)將A549或NCI-p20細(xì)胞(6×106)溶解在400 μL細(xì)胞裂解液(1% Triton X-100，150 mmol/L NaCl，20 mmol/L Tris-HCl，1 mmol/L EDTA，1 mmol/L Na3VO4，2.5 mmol/L焦磷酸，1 mmol/L磷酸甘油和蛋白酶抑制劑混合物)中4 ℃裂解10 min，于4 ℃、14 000×g離心15 min并迅速轉(zhuǎn)移上清至另一離心管中。上清液中加入4 μg PAK4或LIMK1抗體和60 μL Protein G Plus/Protein A-Agarose 4 ℃搖床振蕩孵育3 h。裂解液清洗3次，每次10 mim，沉淀復(fù)合物加入等體積的2×SDS上樣緩沖液，其余步驟同Western blot實(shí)驗(yàn)。

2.6體外激酶分析實(shí)驗(yàn)PAK4 (WT) cDNA克隆入大腸桿菌表達(dá)載體pET30a，突變載體PAK4 (S445N)和PAK4 (K350M)通過定點(diǎn)突變構(gòu)建并純化相應(yīng)蛋白。將上述等量蛋白及LIMK1純化蛋白與含有100 mmol/L NaCl、10 mmol/L MgCl2、50 mmol/L HEPES(pH 7.5)、1 mmol/L DTT和50 μmol/L ATP的緩沖液30 ℃孵育30 min，3×SDS樣品緩沖液終止。其余步驟同Western blot實(shí)驗(yàn)。

3統(tǒng)計(jì)學(xué)處理

計(jì)量資料數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，數(shù)據(jù)比較應(yīng)用SPSS 17.0 統(tǒng)計(jì)軟件處理，采用單因素方差分析比較組間均數(shù)差異；采用Spearman相關(guān)分析確定PAK4與p-LIMK1的相關(guān)性。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)果

1siRNA抑制轉(zhuǎn)染細(xì)胞PAK4蛋白和mRNA表達(dá)

A549和NCI-p20細(xì)胞分別轉(zhuǎn)染si-PAK4 48 h后，Western blot和實(shí)時(shí)熒光定量PCR方法檢測(cè)PAK4表達(dá)水平。與對(duì)照組比較，轉(zhuǎn)染siPAK4的A549和NCI-p20細(xì)胞PAK4蛋白和mRNA表達(dá)明顯下調(diào)(P<0.05)，見圖1。這表明構(gòu)建的siRNA質(zhì)粒在蛋白和mRNA水平上抑制PAK4表達(dá)。

Figure 1.The protein (A) and mRNA (B) expression of PAK4 in A549 cells and NCI-p20 cells transfected with PAK4-siRNA (si-PAK4) or control siRNA (NC). Mean±SD.n=5.*P<0.05vsNC.

圖1轉(zhuǎn)染siPAK4對(duì)A549細(xì)胞和NCI-p20細(xì)胞PAK4蛋白及mRNA表達(dá)的影響

2沉默PAK4基因?qū)?xì)胞遷移及侵襲能力的影響

轉(zhuǎn)染si-PAK4的A549和NCI-p20細(xì)胞侵襲和遷移出的細(xì)胞數(shù)明顯少于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，表明轉(zhuǎn)染si-PAK4后，A549和NCI-p20細(xì)胞的侵襲及遷移能力下降，見圖2。

Figure 2.PAK4 knockdown suppressed NSCLC cell migration and invasion (×40). Mean±SD.n=5.*P<0.05vsNC.

圖2轉(zhuǎn)染si-PAK4對(duì)A549細(xì)胞和NCI-p20細(xì)胞遷移及侵襲能力的影響

3LIMK1是PAK4的激酶底物

Western blot結(jié)果顯示，si-PAK4轉(zhuǎn)染A549和NCI-p20細(xì)胞后，轉(zhuǎn)染si-PAK4組p-LIMK1和p-cofilin均低于對(duì)照組(P< 0.05)，LIMK1和cofilin的蛋白水平無變化(圖3A)。免疫共沉淀實(shí)驗(yàn)結(jié)果顯示，LIMK1蛋白出現(xiàn)在含有抗PAK4抗體的復(fù)雜免疫沉淀中；PAK4也出現(xiàn)在含有抗LIMK1抗體的免疫沉淀物中，而在與對(duì)照組免疫球蛋白IgG相聯(lián)系的免疫復(fù)合物中未檢測(cè)到PAK4和LIMK1(圖3B)。這表明PAK4與LIMK1在A549和NCI-p20細(xì)胞中相互綁定。

體外激酶分析實(shí)驗(yàn)結(jié)果顯示，激酶缺陷型PAK4(K350M)使LIMK1磷酸化的作用顯著低于野生型PAK4或活化型PAK4(S445N)(P<0.05)，見圖3C。進(jìn)一步在10例NSCLC組織中行Western blot檢測(cè)，發(fā)現(xiàn)PAK4表達(dá)上調(diào)的程度與p-LIMK1含量呈正相關(guān)(P<0.05)，見圖3D。以上結(jié)果提示LIMK1是PAK4的激酶底物。

Figure 3.LIMK1 was the kinase substrate of PAK4. A: the protein levels of LIMK1, p-LIMK1, cofilin, and p-cofilin in the A549 cells and NCI-p20 cells transfected with si-PAK4 or control determined by Western blot. NC: negative control. B: A549 cell and NCI-p20 cell lysates were immunoprecipitated with PAK4 antibody (top panels) or LIMK1 antibody (bottom panels), and subjected to Western blot to ascertain LIMK1 and PAK4 interaction. C:invitrokinase assay using purified activated PAK4 (S445N), kinase-defective PAK4 (K350M), PAK4 (WT), and LIMK1 protein. D: correlation between the protein levels of PAK4 and p-LIMK1 in human NSCLC tissues (n=10).

圖3LIMK1是PAK4的激酶底物

4LIMK1對(duì)沉默PAK4抑制非小細(xì)胞肺癌細(xì)胞遷移和侵襲能力的恢復(fù)性作用

體外遷移及侵襲實(shí)驗(yàn)結(jié)果顯示si-PAK4和LIMK1質(zhì)粒共轉(zhuǎn)染組細(xì)胞遷移和侵襲細(xì)胞數(shù)均顯著高于si-PAK4轉(zhuǎn)染組(P<0.05)；同時(shí)行Western blot檢測(cè)結(jié)果顯示si-PAK4和LIMK1質(zhì)粒共轉(zhuǎn)染組p-LIMK1蛋白表達(dá)水平高于轉(zhuǎn)染si-PAK4組(P<0.05),見圖4，表明LIMK1對(duì)沉默PAK4抑制NSCLC細(xì)胞遷移和侵襲能力的恢復(fù)性作用。進(jìn)一步在功能學(xué)上說明PAK4通過磷酸化LIMK1而促進(jìn)非小細(xì)胞肺癌細(xì)胞的遷移及侵襲能力。

討論

作為癌基因，PAK4參與調(diào)節(jié)正常細(xì)胞基因轉(zhuǎn)錄、遷移、生長(zhǎng)和凋亡等過程，其異常高表達(dá)促進(jìn)腫瘤細(xì)胞增殖、侵襲、轉(zhuǎn)移。研究表明，PAK4上調(diào)在多種癌癥中具有促進(jìn)腫瘤細(xì)胞遷移的作用[6-11]。PAK4在不同腫瘤中可通過不同的途徑促進(jìn)腫瘤細(xì)胞增殖、遷移和侵襲，如在絨毛膜癌中是通過下游的膜型基質(zhì)金屬蛋白1(membrane-type 1 matrix metalloproteinase, MT1 MMP)實(shí)現(xiàn)的[6]；在卵巢癌中是通過c-Src/絲裂原活化蛋白激酶激酶1(mitogen-activated protein kinase kinase-1, MEK1)/細(xì)胞外信號(hào)調(diào)節(jié)激酶(extracellular signal-regulated kinase,ERK)1/2和MMP-2介導(dǎo)的[7]；在前列腺癌中是通過激活LIMK1/cofilin信號(hào)通路完成的[8]，在腦膠質(zhì)瘤中與MMP-2[9]及胃癌中與SCG10或LIMK1/cofilin信號(hào)通路有關(guān)[10-11]。我們前期研究結(jié)果證實(shí)PAK4與NSCLC的惡性特征及預(yù)后有關(guān)，然而，PAK4在NSCLC中的生物學(xué)作用及具體機(jī)制尚不清楚。本研究通過沉默PAK4基因，體外細(xì)胞遷移及侵襲實(shí)驗(yàn)結(jié)果表明PAK4具有促進(jìn)非小細(xì)胞肺癌的遷移及侵襲作用。

Figure 4.LIMK1 overexpression rescued the effects of si-PAK4 on the migration and invasion of A549 cells and NCI-p20 cells (×40). Mean±SD.n=5.*P<0.05vsother groups.

圖4LIMK1對(duì)沉默PAK4抑制非小細(xì)胞肺癌細(xì)胞遷移和侵襲能力的恢復(fù)性作用

目前關(guān)于PAK4上游信號(hào)調(diào)控機(jī)制的研究不多，蛋白激酶D(protein kinase D, PKD)被報(bào)道可以通過色氨酸474位點(diǎn)直接磷酸化PAK4[12]；肝細(xì)胞生長(zhǎng)因子 (hepatocyte growth factor, HGF) 和促卵泡激素 (follicle stimulating hormone, FSH) 被報(bào)道在卵巢癌中可以調(diào)節(jié)PAK4磷酸化從而影響腫瘤的侵襲及轉(zhuǎn)移[13]。Mak等[14]報(bào)道CDK5RAP3可以通過綁定PAK4從而使其活化，促進(jìn)肝癌的轉(zhuǎn)移。

LIMK1是PAK4下游的靶蛋白，通過使cofilin磷酸化和失活調(diào)節(jié)激動(dòng)蛋白細(xì)胞骨架的重組，參與腫瘤血管、細(xì)胞遷移和和遷移等[15]。PAK4-LIMK1-cofilin信號(hào)通路促進(jìn)前列腺癌和胃癌細(xì)胞遷移的作用已被證實(shí)[8-11]。PAK4促進(jìn)NSCLC細(xì)胞遷移和侵襲是否通過LIMK1介導(dǎo)的未見報(bào)道。本研究顯示siRNA介導(dǎo)的PAK4表達(dá)下調(diào)后，LIMK1和cofilin的磷酸化水平均降低，則LIMK1和cofilin的蛋白表達(dá)水平無變化，提示NSCLC中PAK4可以調(diào)節(jié)LIMK1及cofilin的磷酸化。同時(shí)我們通過免疫沉淀發(fā)現(xiàn)PAK4與LIMK1蛋白分別出現(xiàn)在抗對(duì)方抗體的免疫沉淀物中，進(jìn)一步證實(shí)了PAK4可特異地與LIMK1相互作用，最后通過體外激酶分析實(shí)驗(yàn)證實(shí)LIMK1是PAK4的激酶底物。

綜上所述， PAK4通過直接磷酸化LIMK1進(jìn)而促進(jìn)非小細(xì)胞肺癌細(xì)胞的遷移和侵襲，有望為非小細(xì)胞肺癌治療提供新的靶點(diǎn)，PAK4高表達(dá)的上游信號(hào)通路將在下一步研究中探討。

[1]李翔，張曉艷，趙繼敏，等.下調(diào)p21活化蛋白激酶2表達(dá)對(duì)人乳腺癌細(xì)胞增殖和凋亡的影響[J]. 中國(guó)病理生理雜志，2014，30(6)：975-981.

[2]蔡松旺，謝迭來，翁毅敏，等.p21活化激酶6對(duì)人非小細(xì)胞肺癌A549細(xì)胞侵襲及遷移能力的影響[J]. 中國(guó)病理生理雜志，2013，29(9)：1637-1640.

[3]蔡松旺，李冬霞，安軍，等.p21活化激酶6削弱非小細(xì)胞肺癌A549細(xì)胞侵襲及遷移能力的分子機(jī)制[J]. 中國(guó)病理生理雜志，2014，30(1)：67-71.

[4]Radu M, Semenova G, Kosoff R, et al.PAK signalling during the development and progression of cancer[J]. Nat Rev Cancer, 2014, 14(1): 13-25.

[5]Abo A, Qu J, Cammarano MS, et al.PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia[J]. EMBO J, 1998, 17(22): 6527-6540.

[6]Zhang HJ, Siu MK, Yeung MC, et al. Overexpressed PAK4 promotes proliferation, migration and invasion of choriocarcinoma[J]. Carcinogenesis, 2011, 32(5): 765- 771.

[7]Siu MK, Chan HY, Kong DS, et al. p21-activated kinase 4 regulates ovarian cancer cell proliferation, migration, and invasion and contributes to poor prognosis in patients[J]. Proc Natl Acad Sci U S A, 2010, 107(43):18622-18627.

[8]Ahmed T, Shea K, Masters JR, et al. A PAK4-LIMK1 pathway drives prostate cancer cell migration downstream of HGF[J].Cell Signal, 2008, 20(7):1320-1328.

[9]Kesanakurti D, Chetty C, Rajasekhar Maddirela D, et al.Functional coo-perativity by direct interaction between PAK4 and MMP-2 in the regulation of anoikis resistance, migration and invasion in glioma[J]. Cell Death Dis,2012, 3:e445.

[10]Guo Q, Su N, Zhang J, et al. PAK4 kinase-mediated SCG10 phosphorylation involved in gastric cancer metastasis[J]. Oncogene, 2014, 33(25):3277-3287.

[11]Li X, Ke Q, Li Y, et al. DGCR6L, a novel PAK4 interaction protein, regulates PAK4-mediated migration of human gastric cancer cell via LIMK1[J]. Int J Biochem Cell Biol, 2010, 42(1):70-79.

[12]Spratley SJ, Bastea LI, Doppler H, et al. Protein kinase D regulates cofilin activity through p21-activated kinase 4[J]. J Biol Chem, 2011, 286(39): 34254-34261.

[13]Siu MK, Chan HY, Kong DS, et al. p21-activated kinase 4 regulates ovarian cancer cell proliferation, migration, and invasion and contributes to poor prognosis in patients[J]. Proc Natl Acad Sci U S A, 2010, 107(43): 18622-18627.

[14]Mak GW, Chan MM, Leong VY, et al. Overexpression of a novel activator of PAK4, the CDK5 kinase-associated protein CDK5RAP3, promotes hepatocellular carcinoma metastasis[J]. Cancer Res, 2011, 71(8): 2949-2958.

[15]Dan C, Kelly A, Bernard O, et al.Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin[J]. J Biol Chem, 2001, 276(34):32115-32121.

(責(zé)任編輯： 盧萍， 羅森)

*[基金項(xiàng)目]深圳市科技計(jì)劃(No. JCYJ20140414160300592)

Effect of PAK4-LIMK1-cofilin signaling on non-small-cell lung cancer migration and invasionLI Dong-xia1, WANG Yong-ling1, CAI Song-wang2

(1SchoolofBasicMedicalScience,XinxiangMedicalUniversity,Xinxiang453003,China;2DepartmentofCardiothoracicSurgery,TheThirdAffiliatedHospital,SunYat-senUniversity,Guangzhou510630,China.E-mail:songwangcai@yahoo.com)

[ABSTRACT]AIM: To explore the mechanism of p21-activated kinase 4 (PAK4) on non-small-cell lung cancer (NSCLC) migration and invasion.METHODS: After A549 and NCI-p20 cell lines were transfected with PAK4-siRNA or negative control, the expression of PAK4 at mRNA and protein levels was detected by real-time PCR and Western blot， respectively. The invasion and migration of A549 cells and NCI-p20 cells were measured by Matrigel invasion assay and Transwell migration assay. LIMK1, cofilin, and their respective phosphorylation were examined by Western blot. The interaction of PAK4 and LIMK1 was investigated by co-immunoprecipitation assay. The relationship between PAK4 and LIMK1 phosphorylation was examined by a protein kinase assay in the A549 cells and NCI-p20 cells. The expression of PAK4 and p-LIMK1 in 10 human NSCLC tissues was examined by Western blot. A549 cells and NCI-p20 cells were individually or commonly transfected with PAK4-siRNA or LIMK1 plasmid in order to observe the cell migration and invasion. RESULTS: After A549 cells and NCI-p20 cells were transfected with PAK4-siRNA for 48 h, the expression of PAK4 at mRNA and protein levels, and the numbers of invasion and migration cells in PAK4-siRNA group were lower than those in control group. Compared with control group, the phosphorylation of LIMK1 and cofilin was lower in PAK4-siRNA group, whereas the total expression levels of LIMK1 and cofilin did not change. The results of co-immunoprecipitation assays showed that PAK4 specifically interacted with LIMK1 in A549 and NCI-p20 cells. LIMK1 phosphorylation in the presence of PAK4 (K350M) was significantly lower than that in the presence of PAK4 (WT) or PAK4 (S445N) in the protein kinase assay. The PAK4 upregulation was positively correlated with the level of p-LIMK1 (P<0.05). After A549 cells and NCI-p20 cells were co-transfected with PAK4-siRNA and LIMK1 plasmid, the migration and invasion cell numbers in co-transfection group were higher than those in PAK4-siRNA transfection group. CONCLUSION: PAK4 promotes the invasive and migratory abilities of NSCLC, which is mediated by LIMK1 phosphorylation.

[KEY WORDS]p21-activated kinase 4; LIM kinase 1; Non-small-cell lung cancer; Cell migration; Cell invasion

通訊作者△Tel: 020-38688039; E-mail: tangshaohui205@163.com

[收稿日期]2015- 04- 29[修回日期] 2015- 09- 10

[文章編號(hào)]1000- 4718(2015)12- 2136- 08

doi:10.3969/j.issn.1000- 4718.2015.12.004

[中圖分類號(hào)]R363

[文獻(xiàn)標(biāo)志碼]A