陳逸軒，劉燕，婁麗麗，時(shí)妍梅，陳文莉，陳新年*

（蘭州大學(xué)1第一臨床醫(yī)學(xué)院,2基礎(chǔ)醫(yī)學(xué)院病理生理研究所，蘭州 730000）

LKB1抑制人巨細(xì)胞肺癌細(xì)胞的增殖和遷移侵襲、m TOR磷酸化與VEGF和MM P9表達(dá)

陳逸軒1Δ，劉燕2Δ，婁麗麗1，時(shí)妍梅2，陳文莉2，陳新年2*

（蘭州大學(xué)1第一臨床醫(yī)學(xué)院,2基礎(chǔ)醫(yī)學(xué)院病理生理研究所，蘭州 730000）

目的分析肝激酶B1（liver kinase B1, LKB1）對(duì)人巨細(xì)胞肺癌（the people giant cell lung cancer，PGCL3）細(xì)胞增殖、遷移、侵襲的影響及其機(jī)制。方法構(gòu)建真核表達(dá)載體pCMV-LKB1，轉(zhuǎn)染人巨細(xì)胞肺癌細(xì)胞。轉(zhuǎn)染后48h，用免疫印跡法檢測(cè)各組細(xì)胞LKB1蛋白表達(dá)水平，明確轉(zhuǎn)染PGCL3效率。轉(zhuǎn)染后每隔12h、連續(xù)72h檢測(cè)LKB1對(duì)細(xì)胞增殖能力的影響；Transwell小室檢測(cè)轉(zhuǎn)染后各組細(xì)胞遷移、侵襲力。提取各組細(xì)胞蛋白檢測(cè)基質(zhì)金屬蛋白酶2（matrix metalloproteinase 2，MMP2）、基質(zhì)金屬蛋白酶9（matrix metalloproteinase 9，MMP9）、雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）和血管內(nèi)皮生長(zhǎng)因子（vascular endothelial grow th factor, VEGF）的表達(dá)水平。結(jié)果pCMV-LKB1經(jīng)PCR、雙酶切及DNA測(cè)序鑒定后，證實(shí)目的基因片段插入方向正確，核酸序列與NCBI公布的LKB1的核酸序列一致；免疫印跡分析顯示，轉(zhuǎn)染pCMV-LKB1的PGCL3細(xì)胞中LKB1表達(dá)水平明顯增高，表明pCMV-LKB1構(gòu)建成功。增殖曲線(xiàn)分析和5-乙炔基-2，脫氧嘧啶核苷（5-ethynyl-2，-deoxyuridine，EdU）檢測(cè)顯示，轉(zhuǎn)染pCMV-LKB1能顯著抑制PGCL3細(xì)胞的增殖；Transwell小室檢測(cè)顯示，過(guò)表達(dá)LKB1可顯著抑制PGCL3細(xì)胞的遷移與侵襲；免疫印跡分析顯示，過(guò)表達(dá)LKB1的PGCL3細(xì)胞其LKB1下游分子中總mTOR表達(dá)量不變，磷酸化mTOR（p-mTOR）水平降低，mTOR下游分子VEGF表達(dá)量也顯著降低，遷移侵襲相關(guān)蛋白MMP2表達(dá)無(wú)變化，但MMP9水平顯著降低。結(jié)論LKB1可能通過(guò)下調(diào)PGCL3細(xì)胞p-m TOR水平，降低VEGF表達(dá)，從而抑制巨細(xì)胞肺癌細(xì)胞增殖，并可能通過(guò)下調(diào)金屬基質(zhì)蛋白酶MMP9的表達(dá)，降低巨細(xì)胞肺癌細(xì)胞遷移侵襲能力。

肝激酶B1；增殖；遷移；侵襲；基質(zhì)金屬蛋白酶；雷帕霉素靶蛋白；血管內(nèi)皮生長(zhǎng)因子

人巨細(xì)胞肺癌因具極高的轉(zhuǎn)移性而成為一種特殊類(lèi)型的肺癌，被發(fā)現(xiàn)時(shí)往往已出現(xiàn)轉(zhuǎn)移而不能施行常規(guī)的手術(shù)治療，預(yù)后較差[1]，因此如何有效控制其轉(zhuǎn)移是治療人巨細(xì)胞肺癌的關(guān)鍵。人巨細(xì)胞肺癌細(xì)胞株（PGCL3）具有很強(qiáng)的遠(yuǎn)處轉(zhuǎn)移能力[2]。肝激酶B1 （liver kinase B1, LKB1） 即絲氨酸－蘇氨酸激酶11（serine/threonine kinase, STK11）是一個(gè)抑癌基因，于1998年由德國(guó)科學(xué)家在Peutz-Jeghers綜合征（PJ綜合征）患者體內(nèi)發(fā)現(xiàn)[3]，它主要通過(guò)調(diào)節(jié)mTOR的活性來(lái)控制細(xì)胞增殖和生物能量代謝。已有研究表明，LKB1的失活與多種惡性腫瘤的發(fā)生、發(fā)展有關(guān)，表現(xiàn)為使細(xì)胞的遷移侵襲力增加[4-7]，但對(duì)人巨細(xì)胞肺癌的作用如何尚未見(jiàn)報(bào)道。本文以LKB1為靶點(diǎn)，人高轉(zhuǎn)移巨細(xì)胞肺癌細(xì)胞株（PGCL3）為研究對(duì)象，探究 LKB1對(duì)人巨細(xì)胞肺癌增殖、遷移、侵襲的影響，并對(duì)其機(jī)制進(jìn)行探討。

材料與方法

1 pCMV-LKB1真核表達(dá)載體的構(gòu)建

根據(jù)基因文庫(kù)中LKB1登錄號(hào)（NM_000455.4）設(shè)計(jì)LKB1引物: gactgacgtgtagaacaatcg, gaaccggcaggaagactgag。引物由上海捷瑞生物工程有限公司合成。按照Trizol試劑盒（Invitrogen公司）說(shuō)明書(shū)要求提取人胚腎293-T細(xì)胞的總RNA，經(jīng)反轉(zhuǎn)錄獲得cDNA，以此cDNA為模板進(jìn)行PCR擴(kuò)增：Prem ix Taq12.5μl、上下游引物各1μl、模板2μl、加ddH2O至25μl的反應(yīng)體系。反應(yīng)條件為94℃5 m in，94℃30 S，61℃ 30 S，72℃ 2m in，共30個(gè)循環(huán)，72℃延伸10 m in，電泳后可見(jiàn)目的條帶，膠回收目的基因LKB1片段。將回收的LKB1片段與克隆載體pMD-18T（大連寶生物工程有限公司）連接，構(gòu)建克隆載體pMD-18T-LKB1。用限制性?xún)?nèi)切酶SalI、EcoR I（大連寶生物工程有限公司）對(duì)克隆載體pMD-18TLKB1和真核表達(dá)載體pCMV-Blank(天根生物科技有限公司)進(jìn)行雙酶切，將酶切產(chǎn)物用T4 DNA連接試劑盒（大連寶生物工程有限公司）構(gòu)建真核表達(dá)載體pCMV-LKB1。將構(gòu)建的pCMV-LKB1進(jìn)行PCR、雙酶切鑒定，并對(duì)DNA測(cè)序。

2 細(xì)胞培養(yǎng)及轉(zhuǎn)染

PGCL3細(xì)胞株由蘭州大學(xué)病理生理學(xué)研究所保存，用含10%胎牛血清的RPM I-1640培養(yǎng)在37℃、5%CO2的細(xì)胞培養(yǎng)箱內(nèi)培養(yǎng)，每2天換液一次。當(dāng)細(xì)胞生長(zhǎng)至90%的匯合度時(shí)用0.25%的胰酶消化，傳代至新的25cm2的培養(yǎng)瓶中。當(dāng)細(xì)胞生長(zhǎng)處于對(duì)數(shù)期且匯合度達(dá)到80%時(shí)將pCMV-Blank、pCMV-LKB1分別與轉(zhuǎn)染試劑Lipofect以2:1的比例混合轉(zhuǎn)染PGCL3細(xì)胞。

3 細(xì)胞增殖檢測(cè)

轉(zhuǎn)染后24h，將各組細(xì)胞以2×104cells/孔種于24孔板培養(yǎng)，每隔12h，用胰酶消化細(xì)胞，用細(xì)胞計(jì)數(shù)板計(jì)數(shù)每孔中的細(xì)胞總數(shù)，連續(xù)計(jì)數(shù)6次，繪制細(xì)胞增殖曲線(xiàn)，觀察各組細(xì)胞增殖情況。另取各組對(duì)數(shù)生長(zhǎng)期細(xì)胞，以每孔2×103細(xì)胞接種于96孔板中培養(yǎng)24h，按照EdU試劑盒（廣州瑞博）說(shuō)明書(shū)提供的方法將細(xì)胞用EdU孵育2h，4%的多聚甲醛室溫固定30m in，后用Apollo染細(xì)胞，再用Hoechst33342反應(yīng)液染細(xì)胞核，于熒光顯微鏡下觀測(cè)拍照。每個(gè)孔內(nèi)隨機(jī)選取5個(gè)區(qū)域記錄，使用Image J軟件將同一視野下的圖片疊加，計(jì)算增殖細(xì)胞比例。綠色熒光細(xì)胞占總細(xì)胞數(shù)的百分比代表細(xì)胞增殖能力。

4 Transwell小室觀察各組細(xì)胞遷移、侵襲能力

遷移實(shí)驗(yàn)：轉(zhuǎn)染24h后，將各組細(xì)胞消化，以5×104個(gè)/孔接種至Transwell小室（Corning，美國(guó)）上室，下室加入600μl含10%FBS的RPM I-1640培養(yǎng)液。培養(yǎng)24h后取出小室，PBS沖洗2次，用棉簽小心拭去上室細(xì)胞，甲醇固定10m in，風(fēng)干后用0.1%的結(jié)晶紫染色15m in，PBS沖洗2次，晾干后在倒置顯微鏡下（20倍物鏡）隨機(jī)挑選5個(gè)視野拍照并計(jì)數(shù)，以穿過(guò)Transwell小室膜的細(xì)胞數(shù)表示遷移力。

侵襲實(shí)驗(yàn)：接種細(xì)胞前將Matrigel基質(zhì)膠（BD，美國(guó)）用RPM I-1640培養(yǎng)液按1:8比例稀釋?zhuān)?00μl均勻加入小室，置入37℃培養(yǎng)箱1h，使膠變?yōu)楣虘B(tài)，用溫?zé)岬腞PM I-1640培養(yǎng)液清洗1次，接種細(xì)胞，其余步驟與遷移實(shí)驗(yàn)一樣，以穿過(guò)Transwell小室基質(zhì)膠及膜的細(xì)胞數(shù)表示侵襲力。

5 Western blot檢測(cè)LKB1及相關(guān)蛋白表達(dá)水平

轉(zhuǎn)染后48h，收集分別轉(zhuǎn)染pCMV-Blank和pCMV-LKB1的PGCL3細(xì)胞及未轉(zhuǎn)染的PGCL3細(xì)胞提取蛋白進(jìn)行SDS-PAGE凝膠電泳、轉(zhuǎn)膜，用5%的脫脂奶粉封閉1h，分別加入1:1000稀釋的一抗（兔抗人LKB1、m TOR、p-m TOR、VEGF、MMP2、MMP9，Immnoway，美國(guó)），4℃過(guò)夜。次日，三乙醇胺緩沖溶液（Tris buf ered saline Tween-20，TBST）洗膜3次，加入1:5000稀釋的辣根過(guò)氧化物酶標(biāo)記的IgG（Immnoway），室溫?fù)u動(dòng)孵育2h，再用TBST洗膜3次，在暗室加入ECL發(fā)光液后進(jìn)行顯影、定影，掃描膠片后保存圖像。應(yīng)用Image J軟件測(cè)定各蛋白條帶光密度，并用Oringin軟件分析各蛋白的相對(duì)水平。

結(jié) 果

1 重組真核表達(dá)載體pCMV-LKB1鑒定

將重組真核表達(dá)載體pCMV-LKB1用PCR和雙酶切鑒定，結(jié)果顯示PCR擴(kuò)增片段與雙酶切條帶大小相符，約1560bp，與設(shè)計(jì)的LKB1目的基因大小一致（圖1）。將pCMV-LKB1送樣測(cè)序，顯示插入片段與NCBI公布的LKB1序列完全一致，且片段插入方向正確，表明真核表達(dá)載體pCMV-LKB1構(gòu)建成功。

Western blot檢測(cè)顯示，將pCMV-LKB1轉(zhuǎn)染PGCL3細(xì)胞后，PGCL3細(xì)胞內(nèi)LKB1水平顯著高于轉(zhuǎn)染pCMV-LKB1和未轉(zhuǎn)染的PGCL3細(xì)胞，表明pCMV-LKB1能在PGCL3細(xì)胞內(nèi)有效過(guò)表達(dá)LKB1（圖2）。

圖1 重組質(zhì)粒pCMV-LKB1 PCR及雙酶切鑒定Fig.1 Identif cation of the recombinant plasm id pCMV-LKB1 by PCR amplif cation and double digestion

圖2 重組質(zhì)粒pCMV-LKB1 的Western blot鑒定。A，轉(zhuǎn)染pCMV-LKB1后的PGCL3細(xì)胞中LKB1蛋白表達(dá)的Western blot檢測(cè)；B, 統(tǒng)計(jì)學(xué)分析；**，P＜0.01Fig. 2 Identif cation of the recombinant plasm id pCMV-LKB1 by Western blot. A, Western blot detection of LKB1 expression in PGCL3 cells transfected w ith pCMV-LKB1; B, statistical analysis; **, P＜0.01

2 過(guò)表達(dá)LKB1抑制PGCL3細(xì)胞增殖

轉(zhuǎn)染24h后，每12h計(jì)數(shù)各組細(xì)胞數(shù)，繪制分析增殖曲線(xiàn)，結(jié)果顯示在連續(xù)72h檢測(cè)中，轉(zhuǎn)染過(guò)表達(dá)LKB1的PGCL3細(xì)胞增殖能力較未轉(zhuǎn)染的PGCL3細(xì)胞和轉(zhuǎn)染對(duì)照質(zhì)粒的PGCL3細(xì)胞明顯下降（圖3）。

EdU實(shí)驗(yàn)結(jié)果顯示，PGCL3組細(xì)胞增殖率為（61.7±5.6）%、PGCL3-NC組細(xì)胞增殖率為（59.3±6.6）%、PGCL3-LKB1組細(xì)胞增殖率為（29.1±5.7）%，與前兩組相比，轉(zhuǎn)染LKB1組的細(xì)胞增殖能力受到顯著抑制（圖4）。

3 過(guò)表達(dá)LKB1抑制PGCL3細(xì)胞遷移和侵襲

轉(zhuǎn)染24h后用Transwell小室檢測(cè)各組細(xì)胞遷移能力和侵襲力，結(jié)果顯示，與轉(zhuǎn)染對(duì)照質(zhì)粒和未轉(zhuǎn)染的PGCL3細(xì)胞相比，PGCL3-LKB1組細(xì)胞遷移力和侵襲力均顯著下降（圖5，圖6）

圖3 LKB1對(duì)PGCL3細(xì)胞增殖力影響的增殖曲線(xiàn)分析。*，與PGCL3和PGCL3-NC細(xì)胞組比較，P＜0.01Fig. 3 Grow th curve analysis of the ef ect of LKB1 on the proliferation of PGCL3 cells. *, P＜0.01, compared w ith PGCL3 and PGCL3-NC cells

圖4 EdU實(shí)驗(yàn)檢測(cè)LKB1對(duì)PGCL3 細(xì)胞增殖的影響。比例尺，50μmFig. 4 The ef ect of LKB1 on the proliferation of PGCL3 cells detected by EdU assay. Scale bar, 50μm

圖 5 LKB1對(duì)PGCL3細(xì)胞遷移力的影響。A，PGCL3；B，PGCL3-NC；C，PGCL3-LKB1；A-C，轉(zhuǎn)染過(guò)表達(dá)LKB1對(duì)PGCL3細(xì)胞遷移影響的Transwell法檢測(cè)；D，PGCL3細(xì)胞遷移力統(tǒng)計(jì)學(xué)分析；**，與PGCL3和PGCL3-NC細(xì)胞組比較，P＜0.01；比例尺，50μmFig. 5 The ef ect of LKB1 on the migration of PGCL3 cells. A, PGCL3; B, PGCL3-NC; C, PGCL3-LKB1; A to C, Transwell detection of the ef ect of LKB1 overexpression on the m igration of PGCL3 cells; D, statistical analysis of the m igration number of PGCL3 cells; **, P＜0.01, compared w ith PGCL3 and PGCL3-NC cells; scale bar, 50μm

圖 6 LKB1對(duì)PGCL3細(xì)胞侵襲力的影響，A，PGCL3；B，PGCL3-NC；C，PGCL3-LKB1；A-C，轉(zhuǎn)染過(guò)表達(dá)LKB1對(duì)PGCL3細(xì)胞侵襲的Transwell法檢測(cè)；D，PGCL3細(xì)胞侵襲力統(tǒng)計(jì)學(xué)分析；**，與PGCL3和PGCL3-NC細(xì)胞比較，P＜0.01；比例尺，50μmFig. 6 The ef ect of LKB1 on the invasion of PGCL3 cells. A, PGCL3; B, PGCL3-NC; C, PGCL3-LKB1; A to C, Transwell detection of the ef ect of LKB1 overexpression on the invasion of PGCL3 cells; D, statistical analysis of the invasion number of PGCL3 cells; **, P＜0.01, compared w ith PGCL3 and PGCL3-NC cells; scale bar, 50μm

4 過(guò)表達(dá)LKB1降低PGCL3細(xì)胞m TOR磷酸化水平和VEGF表達(dá)

為了探究LKB1抑制PGCL3細(xì)胞增殖能力的機(jī)制，應(yīng)用Western blot檢測(cè)過(guò)表達(dá)LKB1對(duì)LKB1下游與細(xì)胞增殖、代謝等過(guò)程密切相關(guān)的m TOR信號(hào)通路相關(guān)分子的影響。結(jié)果顯示，過(guò)表達(dá)LKB1不改變總m TOR水平，但明顯降低p-m TOR及VEGF水平（圖7），提示LKB1可通過(guò)下調(diào)m TOR信號(hào)通路進(jìn)而抑制PGCL3細(xì)胞的增殖能力。

圖7 過(guò)表達(dá)LKB1對(duì)PGCL3細(xì)胞增殖相關(guān)蛋白的影響。A，增殖相關(guān)蛋白的Western blot檢測(cè)；B，增殖相關(guān)蛋白水平的統(tǒng)計(jì)學(xué)分析；**，與PGCL3和PGCL3-NC細(xì)胞比較，P＜0.01Fig. 7 The ef ect of LKB1 overexpression on proliferation-related proteins in PGCL3 cells. A, Western blot detection of the levels of m TOR, p-mTOR and VEGF; B, statistical analysis of the levels of mTOR, p-mTOR and VEGF; **, P＜0.01, compared w ith PGCL3 and PGCL3-NC cells

5 過(guò)表達(dá)LKB1下調(diào)PGCL3細(xì)胞MMP9水平

為了探討LKB1抑制PGCL3遷移、侵襲的機(jī)制，應(yīng)用Western blot檢測(cè)過(guò)表達(dá)LKB1對(duì)PGCL3細(xì)胞中MMP2及MMP9的影響。結(jié)果顯示，與PGCL3組和PGCL3-NC組比較，PGCL3-LKB1細(xì)胞中MMP2水平無(wú)明顯變化，而MMP9水平顯著下降（圖8），提示LKB1可能通過(guò)下調(diào)MMP9抑制PGCL3細(xì)胞的遷移和侵襲。

圖8 過(guò)表達(dá)LKB1對(duì)PGCL3細(xì)胞MMP2和MMP9水平的影響。A，MMP2和MMP9的Western blot檢測(cè)；B，MMP2和MMP9水平的統(tǒng)計(jì)學(xué)分析；**，與PGCL3和PGCL3-NC細(xì)胞比較，P＜0.01Fig. 8 The ef ect of LKB1 overexpression on the levels of MMP2 and MMP9 in PGCL3 cells. A, Western blot detection of MMP2 and MMP9; B, statistical analysis of the levels of MMP and MMP9; **, P＜0.01, compared w ith PGCL3 and PGCL3-NC cells

討 論

腫瘤的浸潤(rùn)和遠(yuǎn)處轉(zhuǎn)移是惡性腫瘤的主要特征。LKB1是腺苷酸活化蛋白激酶（AMPK）的上游基因。有學(xué)者通過(guò)小鼠體內(nèi)實(shí)驗(yàn)研究表明，LKB1可通過(guò)對(duì)腺苷酸活化蛋白激酶的磷酸化，而使AMPK轉(zhuǎn)化成p-AMPK，從而使AMPK激活進(jìn)而負(fù)向調(diào)節(jié)mTOR的活性[8,9,10]。m TOR是調(diào)節(jié)細(xì)胞代謝和生長(zhǎng)的非常關(guān)鍵的生物大分子，由兩個(gè)分管功能和生物合成的復(fù)合物組成[11]?；罨膍 TOR，即p-m TOR可上調(diào)多種促進(jìn)細(xì)胞生長(zhǎng)和增殖的關(guān)鍵蛋白，如細(xì)胞周期蛋白D1（cyclinD1）、缺氧誘導(dǎo)因子1a（hypoxia inducible factor 1a、HIF-1a）、癌基因c-myc、視網(wǎng)膜母細(xì)胞瘤易感蛋白R(shí)b蛋白、血管內(nèi)皮細(xì)胞生長(zhǎng)因子VEGF等[11-14]。這些下游因子調(diào)控細(xì)胞周期進(jìn)程、細(xì)胞生長(zhǎng)、血管的生成等[13]。在腫瘤形成過(guò)程中，LKB1的表達(dá)降低或缺失，p-AMPK對(duì)m TOR的負(fù)性調(diào)節(jié)消失，這些因子表達(dá)增強(qiáng)，使細(xì)胞周期加速，細(xì)胞增殖能力增強(qiáng)，血管新生加快而最終加速腫瘤的形成及遠(yuǎn)處轉(zhuǎn)移[14]。本研究結(jié)果顯示，過(guò)表達(dá)LKB1不改變PGCL3細(xì)胞過(guò)表達(dá)LKB1使PGCL3細(xì)胞增殖能力減弱，并使其遷移侵襲力顯著下降，提示高水平的LKB1可通過(guò)LKB1+/p-m TOR-/VEGF-調(diào)控使p-m TOR水平降低從而下調(diào) VEGF的表達(dá)，進(jìn)而抑制細(xì)胞增殖力和遷移侵襲力。

金屬基質(zhì)蛋白酶（matrix metalloproteinases, MMPs）幾乎能降解細(xì)胞外基質(zhì)的所有成分，進(jìn)而破壞腫瘤細(xì)胞侵襲過(guò)程中的組織學(xué)屏障，在腫瘤浸潤(rùn)和轉(zhuǎn)移中起重要作用[15,16]。本實(shí)驗(yàn)中，過(guò)表達(dá)LKB1對(duì)MMP2水平無(wú)明顯影響，但使MMP9水平顯著下調(diào)。LV Rhodes等[17]在乳腺癌細(xì)胞中也發(fā)現(xiàn)，LKB1高表達(dá)可以降低MMP1表達(dá)進(jìn)而抑制乳腺癌細(xì)胞的轉(zhuǎn)移能力。可見(jiàn)在PGCL3細(xì)胞中，LKB1可通過(guò)下調(diào)MMP9的表達(dá)而抑制PGCL3細(xì)胞的遷移和侵襲。

已有研究表明，LKB1低表達(dá)或缺失可使腫瘤的遷移侵襲力增強(qiáng)。本研究結(jié)果顯示，通過(guò)轉(zhuǎn)染增強(qiáng)PGCL3細(xì)胞中LKB1的表達(dá)可使PGCL3細(xì)胞的增殖能力、遷移、侵襲力下降。其機(jī)制可能與LKB1下調(diào)p-m TOR、VEGF、MMP9等基因的表達(dá)有關(guān)，但具體作用機(jī)制還需進(jìn)一步研究。

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LKB1 inhibits the proliferation and invasion of human lung giant cell cancer cell line through down-regulating the phosphorylation of m TOR and the exp ression of VEGF and MMP9

Chen Yixuan1Δ, Liu Yan2Δ, Lou Lili1, Shi Yanmei2, Chen Wenli2, Chen Xinnian2*
(1The First Clinical Medical College,2Department of Pathophysiology, Basic Medical College, Lanzhou University, Lanzhou 730000, China)

ObjectiveTo study the ef ect of liver kinase B1 (LKB1) on the proliferation, migration and invasion of human lung giant cell cancer PGCL3 cell line and its mechanism.M ethodsEukaryotic expression vector pCMV-LKB1 was constructed and transfected into giant lung cell cancer cell line PGCL3. 48h after transfection, the level of LKB1 protein was detected by western blotting to determ ine the ef ciency of transfection. The ef ect of LKB1 on the proliferation of the cells was detected every 12h for a total of 72h. Transwell assay was applied to evaluate cell migration and invasion. Western blotting was performed to detect the expression of matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), mammalian target of rapamycin (m TOR) and vascular endothelial grow th factor (VEGF).ResultsPCR, double enzyme digestion and DNA sequencing results conf rmed that the LKB1 fragment was inserted in the right direction into the recombinant eukaryotic vector pCMV-LKB1 and that its nucleic acid sequence was the same aspublished on NCBI. Western blot results showed that the level of LKB1 in the transfected cells increased signif cantly, indicating the success of pCMV-LKB1 construction. Grow th curve analysis and 5-Ethynyl-2’-deoxyuridine (EdU) test showed that the cell proliferative capacity was signif cantly inhibited by pCMV-LKB1 transfection. Transwell assay results showed that LKB1 overexpression obviously inhibited the migration and invasion of PGCL3 cells. Western blot results showed that of the downstream molecules of LKB1 in the transfected PGCL3 cells, the total m TOR level remained unchanged, while the expression of phosphorylated m-TOR (p-m TOR) decreased. VEGF, the downstream protein of mTOR, reduced signif cantly. The level of m igration related protein MMP2 remained unchanged, while that of MMP9 decreased signif cantly.ConclusionLKB1 inhibits the proliferation of PGCL3 cells possibly through down-regulating the levels of p-m TOR and VEGF; and also inhibit its migration by down-regulating MMP9 expression.

Liver kinase B1; proliferation; m igration; invasion; matrix metalloproteinase; mammalian target of rapamycin; vascular endothelial grow th factor

R734.2

A

10.16705/ j. cnki. 1004-1850.2017.03.001

2017-03-02

2017-06-05

甘肅省自然科學(xué)基金（1506RJZA205）

陳逸軒，男（1993年），漢族，本科；劉燕，女（1980年），漢族，講師

Δ共同第一作者：陳逸軒 劉燕

*通訊作者(To whom correspondence should be addressed)：chenxn@lzu.edu.cn