孫 磊 劉雨清 李小龍 劉 菲 張麗娜 李洪利 張寶剛

Gab2-Akt-ARK5通路在膠質(zhì)瘤侵襲中的研究*

孫 磊①劉雨清①李小龍①劉 菲①張麗娜①李洪利②張寶剛①

目的：探討Gab2-Akt-ARK5通路在膠質(zhì)瘤侵襲中的意義。方法：采用免疫組織化學(xué)SP法檢測(cè)90例膠質(zhì)瘤組織中ARK5及Gab2表達(dá)。采用小RNA干擾轉(zhuǎn)染LN-229細(xì)胞株，Western Blot檢測(cè)瞬時(shí)轉(zhuǎn)染后ARK5及Gab2表達(dá)。體外侵襲實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后侵襲能力變化及Western Blot檢測(cè)Gab2下降后Akt和ARK5的磷酸化。結(jié)果：膠質(zhì)瘤組織中ARK5和Gab2免疫組織化學(xué)陽性結(jié)果呈正相關(guān)且在高級(jí)別膠質(zhì)瘤（WHO分級(jí)為Ⅲ、Ⅳ級(jí)）中表達(dá)明顯高于低級(jí)別膠質(zhì)瘤（WHO分級(jí)為Ⅰ、Ⅱ級(jí)）。轉(zhuǎn)染ARK5、Gab2、ARK5-Gab2及SCR質(zhì)粒的LN-229細(xì)胞分別稱siARK5/LN-229、siGab2/LN-229、siARK5-siGab2/LN-229和SCR/LN-229。其中siARK5干擾效率為70%，siGab2的干擾效率為75%。轉(zhuǎn)染后，與SCR/LN-229相比，siARK5/LN-229中ARK5表達(dá)降低，siGab2/LN-229中Gab2表達(dá)降低，siARK5-siGab2/LN-229中ARK5和Gab2表達(dá)均降低。siARK5/LN-229和siGab2/LN-229侵襲并穿透Matrigel膜基質(zhì)的細(xì)胞數(shù)均比對(duì)照組少（P＜0.01），且siARK5-siGab2/LN-229細(xì)胞數(shù)減少更顯著（P＜0.01）。在IGF-1刺激下，siGab2/LN-229中Akt和ARK5的磷酸化減弱。結(jié)論：應(yīng)用小RNA干擾技術(shù)降低ARK5或Gab2表達(dá)使LN-229細(xì)胞侵襲轉(zhuǎn)移能力降低，同時(shí)Gab2表達(dá)降低抑制ARK5和Akt磷酸化，提示Gab2-Akt-ARK5通路參與膠質(zhì)瘤細(xì)胞的侵襲。

膠質(zhì)瘤 ARK5 Gab2 LN-229細(xì)胞 小RNA干擾 侵襲

膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)常見的一類腫瘤，具有高侵襲性、難治愈、易復(fù)發(fā)等特點(diǎn)，深入研究膠質(zhì)瘤的侵襲過程涉及的分子機(jī)制對(duì)有效治療此病有重要作用。ARK5作為AMPK亞家族成員之一，是Akt下游信號(hào)分子［1］。ARK5被認(rèn)為在腫瘤侵襲中起關(guān)鍵作用［2］。Grb2 協(xié)同結(jié) 合蛋 白 2（binding protein-2，Gab2），Gabs家族成員之一，可參與細(xì)胞內(nèi)多種信號(hào)轉(zhuǎn)導(dǎo)通路，在細(xì)胞增殖、分化、凋亡及遷移等生理過程發(fā)揮重要作用［3-5］。研究表明Gab2在人類白血病和卵巢癌中高表達(dá)［6-7］。本課題組前期實(shí)驗(yàn)已發(fā)現(xiàn)Gab2和ARK5均與膠質(zhì)瘤的侵襲密切相關(guān)［8-9］。因此本實(shí)驗(yàn)用小RNA干擾技術(shù)降低ARK5和Gab2表達(dá)，通過體外實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后LN-229細(xì)胞侵襲能力變化及通過Western Blot檢測(cè)Akt及ARK5的磷酸化，探討Gab2-Akt-ARK5通路參與膠質(zhì)瘤細(xì)胞侵襲的作用機(jī)制。

1 材料與方法

1.1 材料

1.1.1 病例資料 收集濰坊醫(yī)學(xué)院病理學(xué)教研室2008年2月至2012年3月期間臨床及病理資料齊全，且病理已證實(shí)為膠質(zhì)瘤的90例蠟塊標(biāo)本為研究對(duì)象，其中低級(jí)別膠質(zhì)瘤共計(jì)42例，高級(jí)別膠質(zhì)瘤共計(jì)48例。所有患者術(shù)前均未經(jīng)放療、化療。

1.1.2 主要試劑 免疫組織化學(xué)一抗ARK5和Gab2購于美國Santa Cruz公司；通用性二抗二步法檢測(cè)試劑盒、PBS和枸櫞酸鈉均購自北京中杉金橋公司；1640培養(yǎng)液購自美國Hyclone公司；IGF-1購于美國R&D systems公司。胰蛋白酶、彩色預(yù)染蛋白、DOO18質(zhì)粒中量抽提試劑盒均購自碧云天公司；胎牛血清為杭州四季青公司；侵襲實(shí)驗(yàn)所用24孔細(xì)胞培養(yǎng)板、Matrigel膜基質(zhì)均購自美國Corning公司；24孔趨化小室、細(xì)胞轉(zhuǎn)染試劑購自康為世紀(jì)公司。

1.2 方法

1.2.1 免疫組織化學(xué) 膠質(zhì)瘤蠟塊標(biāo)本連續(xù)切片3張，每張5 μm厚。以PBS代替一抗做陰性對(duì)照，其余2張切片，滴加的一抗分別為ARK5和Gab2工作液，采用免疫組織化學(xué)SP法染色。高倍鏡（×400）下每張切片至少5個(gè)隨機(jī)視野中計(jì)數(shù)500個(gè)腫瘤細(xì)胞，陽性細(xì)胞占同類計(jì)數(shù)細(xì)胞的百分比為陽性細(xì)胞率。陽性結(jié)果的判定根據(jù)陽性細(xì)胞的百分率及顯色深淺分級(jí)［14］。

1.2.2 細(xì)胞培養(yǎng) 膠質(zhì)瘤細(xì)胞株LN-229購自美國ATCC細(xì)胞庫。LN-229細(xì)胞常規(guī)培養(yǎng)于含10%胎牛血清中1 640液中。當(dāng)細(xì)胞密度70%～85%時(shí)，分別轉(zhuǎn)染插入ARK5目標(biāo)片段5'-GAAGTTATGCTTTAT TCAC-3'、Gab2目標(biāo)片段5'-GTGAGAACGATGAGAA ATA-3'的小RNA干擾質(zhì)粒和SCR序列的小RNA干擾質(zhì)粒，轉(zhuǎn)染步驟參照轉(zhuǎn)染試劑說明書。轉(zhuǎn)染細(xì)胞株分別為實(shí)驗(yàn)組siARK5/LN-229、siGab2/LN-229，siARK5-siGab2/LN-229和對(duì)照組SCR/LN-229。

1.2.3 Western Blot實(shí)驗(yàn) 將轉(zhuǎn)染后的實(shí)驗(yàn)組和對(duì)照組細(xì)胞培養(yǎng)72 h后提取蛋白，制備SDS-PAGE凝膠，蛋白質(zhì)變性后電泳，轉(zhuǎn)膜、封閉，分別滴加一抗為ARK5、Gab2進(jìn)行孵育，二抗孵育后顯影。當(dāng)轉(zhuǎn)染成功后進(jìn)行侵襲實(shí)驗(yàn)。將所得結(jié)果分別進(jìn)行分析。

1.2.4 體外癌細(xì)胞侵襲能力檢測(cè) 按文獻(xiàn)［6］操作，結(jié)果置400倍光鏡下觀察，5個(gè)高倍鏡視野，計(jì)數(shù)Boyden小室下室面的細(xì)胞數(shù)即為穿透人工基膜的細(xì)胞數(shù)，每個(gè)實(shí)驗(yàn)重復(fù)3次，取平均數(shù)作為實(shí)驗(yàn)結(jié)果。

1.3 統(tǒng)計(jì)學(xué)分析

運(yùn)用SPSS 16.0進(jìn)行統(tǒng)計(jì)學(xué)處理。對(duì)所有定量資料數(shù)據(jù)結(jié)果用±s表示，進(jìn)行χ2檢驗(yàn)、Spearman相關(guān)分析和兩獨(dú)立樣本t檢驗(yàn)。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 膠質(zhì)瘤組織中ARK5、Gab2蛋白的表達(dá)

在顯微鏡下觀察，結(jié)果顯示：ARK5和Gab2在高級(jí)別膠質(zhì)瘤中陽性表達(dá)高于低級(jí)別膠質(zhì)瘤，差異具有統(tǒng)計(jì)學(xué)意義（P＜0.05，圖1，表1）。90例膠質(zhì)瘤患者中ARK5和Gab2表達(dá)呈正相關(guān)（r=0.418，P＜0.05）。

2.2 siARK5/LN-229、siGab2/LN-229、siARK5、siGab2/LN-229和SCR/LN-229細(xì)胞中ARK5及Gab2的表達(dá)

應(yīng)用質(zhì)粒轉(zhuǎn)染細(xì)胞，轉(zhuǎn)染成功后siARK5/LN-229、siARK5-siGab2/LN-229細(xì)胞中ARK5表達(dá)減少。siGab2/LN-229、siARK5-siGab2/LN-229細(xì)胞中Gab2表達(dá)降低，灰度與SCR/LN-229相比：siARK5/LN-229為0.3，siGab2/LN-229為0.25（圖2）。

圖1 ARK5和Gab2在低級(jí)別膠質(zhì)瘤及高級(jí)別膠質(zhì)瘤中的表達(dá)（SP×400）Figure 1 Expression of ARK5 and Gab2 proteins in low-grade and high-grade gliomas（SP×400）

表1 ARK5和Gab2的表達(dá)與臨床病理分型的關(guān)系Table 1 Relationship between ARK5 and Gab2 expressions and clinicopathological types

圖2 Western Blot法檢測(cè)實(shí)驗(yàn)組和對(duì)照組細(xì)胞中ARK5及Gab2蛋白的表達(dá)Figure 2 Western blot results of ARK5 and Gab2 protein expression in experimental group cells and SCR/LN-229 cells（control group）

圖3 ARK5和Gab2降低表達(dá)對(duì)LN-229細(xì)胞侵襲性的影響Figure 3 Effect of decreased ARK5 and Gab2 expressions on the invasiveness in LN-229 cells

圖4 Western Blot檢測(cè)siGab2/LN-229細(xì)胞和SCR/LN-229細(xì)胞中Akt和ARK5的磷酸化Figure 4 Western Blot results of phosphorylation of Akt and ARK5 in siGab2/LN-229 cells and SCR/LN-229 cells

2.3 體外侵襲實(shí)驗(yàn)

轉(zhuǎn)染成功后，進(jìn)行侵襲實(shí)驗(yàn)。轉(zhuǎn)染對(duì)LN-229細(xì)胞的影響：與SCR/LN-229相比，siARK5/LN-229、siGab2/LN-229、siARK5-siGab2/LN-229穿過8μm微孔濾膜細(xì)胞數(shù)減少，且siARK5-siGab2/LN-229細(xì)胞數(shù)目減少更甚，差異有統(tǒng)計(jì)學(xué)意義（P＜0.01，圖3）。

2.4 降低Gab2的LN-229細(xì)胞Akt和ARK5磷酸化的變化

為了研究ARK5、Gab2表達(dá)降低引起膠質(zhì)瘤侵襲能力下調(diào)的原因及ARK5和Gab2間的相互關(guān)系，再次用Western Blot檢測(cè)，結(jié)果顯示：在IGF-1刺激下，與SCR/LN-229相比，siGab2/LN-229中Akt和ARK5的磷酸化減弱（圖4）。

3 討論

膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)常見的一類腫瘤。其平均生存時(shí)間12～15個(gè)月［10］，被稱為人類最具危害性的腫瘤。而腫瘤惡性程度與腫瘤細(xì)胞的侵襲有關(guān)，因此研究膠質(zhì)瘤細(xì)胞的侵襲對(duì)提高治療功效具有重要的作用［11］。已有研究表明：ARK5在膠質(zhì)瘤及人類乳腺癌的侵襲中起到不同程度的作用［12-13］。Gab2具有潛在的原癌基因特性，在人類卵巢癌和乳腺癌中高表達(dá)［14-15］但鮮見ARK5和Gab2與膠質(zhì)瘤侵襲相關(guān)研究。因此明確ARK5和Gab2在膠質(zhì)瘤侵襲中的作用及ARK5與Gab2在膠質(zhì)瘤侵襲相關(guān)的信號(hào)轉(zhuǎn)導(dǎo)通路之間的相互關(guān)系，對(duì)抑制膠質(zhì)瘤的侵襲具有重要作用。

本文通過免疫組織化學(xué)表明：ARK5與Gab2在高級(jí)膠質(zhì)瘤中的表達(dá)明顯高于在低級(jí)膠質(zhì)瘤中的表達(dá)。這一結(jié)果與本課題組前期研究結(jié)果相一致［8，12］。同時(shí)實(shí)驗(yàn)還證實(shí)，ARK5與Gab2在膠質(zhì)瘤中的表達(dá)呈正相關(guān)。此外，通過小RNA干擾技術(shù)將相關(guān)質(zhì)粒轉(zhuǎn)染進(jìn)膠質(zhì)瘤LN-229細(xì)胞株，培養(yǎng)72 h后，Western Blot檢測(cè)顯示ARK5和Gab2蛋白量降低。隨后的體外癌細(xì)胞侵襲實(shí)驗(yàn)也證實(shí)ARK5和Gab2參與了癌細(xì)胞侵襲過程且ARK5和Gab2同時(shí)存在時(shí)癌細(xì)胞侵襲更明顯。為了進(jìn)一步研究ARK5和Gab2表達(dá)降低后引起膠質(zhì)瘤侵襲下調(diào)的原因并明確ARK5與Gab2在膠質(zhì)瘤侵襲相關(guān)的信號(hào)轉(zhuǎn)導(dǎo)通路之間的相互關(guān)系，再次做Western Blot檢測(cè)。結(jié)果發(fā)現(xiàn)：在IGF-1刺激下，與SCR/LN-229相比，siGab2/LN-229中Akt和ARK5的磷酸化減弱。說明ARK5和Gab2表達(dá)降低后引起膠質(zhì)瘤侵襲下調(diào)的原因是由于降低了ARK5和Akt的磷酸化，同時(shí)也驗(yàn)證ARK5和Akt在信號(hào)轉(zhuǎn)導(dǎo)通路中位于Gab2下游，結(jié)合相關(guān)研究證明的ARK5作為Akt的下游信號(hào)分子參與細(xì)胞的侵襲［12］，最終得出Gab2和ARK5通過Gab2-Akt-ARK5通路參與膠質(zhì)瘤侵襲。

綜上所述，本研究證實(shí)Gab2-Akt-ARK5通路是抑制膠質(zhì)瘤侵襲的重要信號(hào)通路，糾正膠質(zhì)瘤細(xì)胞中Gab2和ARK5蛋白高表達(dá)，可有效阻斷其侵襲過程，為膠質(zhì)瘤的治療有望提供新的靶點(diǎn)。

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（2013-08-07收稿）

（2013-10-20修回）

Gab2-Akt-ARK5 signaling pathway is associated with the invasion of glioma cells

Lei SUN,Yuqing LIU,Xiaolong LI,Fei LIU,Lina ZHANG,Hongli LI,Baogang ZHANG

Baogang ZHANG;E-mail:zbg@hotmail.com
Department of Pathology,Wei-fang Medical College,Weifang 261053,China.

Objective:This study aimed to investigate the effect and significance of a binding protein-2(Gab2)-Akt-ARK5 signaling pathway on the invasion of glioma cells.Methods:Immunohistochemical methods were used to detect the expressions of Gab2 andARK5 in 45 cases of glioma tissue.siRNA plasmid was used to transfect LN-229 cells,and western blot was performed to analyze the protein expressions of Gab2 andARK5.In vitro Matrigel invasion assay was conducted to detect variations in the invasiveness of transfected cells.Western blot was also conducted to analyze the protein phosphorylation ofAkt andARK5 in the cells transfected with Gab2 plasmid.Results:Immunohistochemical assay revealed that the expressions ofARK5 and Gab2 in glioma cells were positively correlated,and both expressions were higher in high-grade glioma(WHO gradeⅢ,Ⅳ)than in low-grade glioma(WHO gradeⅠ,Ⅱ).LN-229 cells transfected withARK5 plasmid,Gab2 plasmid,ARK5 and Gab2 plasmid,and control plasmid were named siARK5/LN-229,siGab2/LN-229,siARK5 and siGab2/LN-229,and SCR/LN-229,respectively.After transfection was performed,the protein expressions of ARK5 and Gab2 were respectively decreased in siARK5/LN-229 and siGab2/LN-229.The protein expressions of ARK5 and Gab2 in siARK5 and siGab2/LN-229 were also respectively decreased.After ARK5 or Gab2 was downregulated,the number of glioma cells,which invaded and penetrated Matrigel,was decreased(P＜0.01).The number of glioma cells also decreased significantly afterARK5 and Gab2 were downregulated.The phosphorylation ofAkt andARK5 in siGab2/LN-229 cells was decreased after these cells were stimulated by insulin-like growth factor-1.Conclusion:The silencing of ARK5 or Gab2 impaired glioma cell invasiveness.The decreased protein expression of Gab2 inhibited the phosphorylation of Akt andARK5.These results suggested that the Gab2-Akt-ARK5 signaling pathway could be relevantly involved in glioma cell invasion.

glioma,ARK5,Gab2,LN-229 cell,siRNA,invasiveness

10.3969/j.issn.1000-8179.20131276

①濰坊醫(yī)學(xué)院病理學(xué)教研室（山東省濰坊市261053）；②醫(yī)學(xué)研究實(shí)驗(yàn)中心

*本文課題受國家自然科學(xué)基金項(xiàng)目（編號(hào)：81072068）、山東省中青年科學(xué)家科研獎(jiǎng)勵(lì)基金項(xiàng)目（編號(hào)：2010BSB14050）、山東省中青年科學(xué)家獎(jiǎng)勵(lì)基金項(xiàng)目（編號(hào)：BS2011YY060）、山東省高等學(xué)?？萍加?jì)劃項(xiàng)目（編號(hào)：J12LK03，J13LK03，J10LF64）和濰坊醫(yī)學(xué)院科技創(chuàng)新研究基金青年基金項(xiàng)目（編號(hào)：K11QC1002）資助

張寶剛 zbg@hotmail.com

This study was supported by the National Natural Science Foundation of China(Grant No.81072068),Shandong Provincial Award Fund for Scientific Research of Young and Middle-Aged Scientists(Dr.Fund;Grant No.2010BSB14050),Shandong Province Awards Foundation for Young and Middle-Aged Scientists(Grant No.BS2011YY060),Shandong Provincial Scientific Plan Foundation for Institutions of Higher Learning(Grant Nos.J12LK03,J10LF64,and J13LK03),and the Youth Fund Project of Scientific and Technological Innovation Research Foundation,Wei-fang Medical College(Grant No.K11QC1002).

（本文編輯：賈樹明）

孫磊 在讀碩士研究生。研究方向?yàn)槟[瘤病理的臨床研究。

E-mail：rizhao-ruby@126.com