Raptor通過上皮-間質(zhì)轉(zhuǎn)化促進(jìn)乳腺癌的侵襲與轉(zhuǎn)移

徐新偉，王照巖，王瑞鴿，李洪利，尹崇高，劉雨清

(濰坊醫(yī)學(xué)院1.病理學(xué)教研室、2.醫(yī)學(xué)研究實(shí)驗(yàn)中心、3.護(hù)理學(xué)院，山東 濰坊 261053)

Raptor通過上皮-間質(zhì)轉(zhuǎn)化促進(jìn)乳腺癌的侵襲與轉(zhuǎn)移

徐新偉1，王照巖1，王瑞鴿1，李洪利2，尹崇高3，劉雨清1

(濰坊醫(yī)學(xué)院1.病理學(xué)教研室、2.醫(yī)學(xué)研究實(shí)驗(yàn)中心、3.護(hù)理學(xué)院，山東 濰坊 261053)

目的 探討Raptor在乳腺癌侵襲與轉(zhuǎn)移過程中的作用。方法 采用Western blot檢測(cè)乳腺癌細(xì)胞MCF-7和MDA-MB-231(MDA231)中Raptor蛋白的表達(dá)情況。應(yīng)用小干擾RNA技術(shù)下調(diào)乳腺癌細(xì)胞MDA231中Raptor的表達(dá)量，同時(shí)將過表達(dá)質(zhì)粒轉(zhuǎn)入乳腺癌細(xì)胞MCF-7中，應(yīng)用Western blot法檢測(cè)轉(zhuǎn)染后各組細(xì)胞中上皮性鈣黏著蛋白(E-cadherin)和波形蛋白(Vimentin)的表達(dá)；應(yīng)用Transwell體外侵襲實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后各組細(xì)胞的侵襲能力；應(yīng)用細(xì)胞核質(zhì)分離實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后各組細(xì)胞中Snail蛋白的轉(zhuǎn)核情況。結(jié)果 MCF-7細(xì)胞中Raptor蛋白的表達(dá)量明顯低于MDA231細(xì)胞中的表達(dá)量；轉(zhuǎn)染后，與對(duì)照組Scr/MDA231相比，siRaptor/MDA231組中Raptor蛋白的表達(dá)量明顯降低，Vimentin蛋白表達(dá)量降低，而E-cadherin 蛋白表達(dá)量升高；與對(duì)照組MCF-7/Con相比，MCF-7/Raptor組中Raptor蛋白的表達(dá)量明顯升高，并伴有Vimentin蛋白表達(dá)量升高，而E-cadherin 蛋白表達(dá)量降低。siRaptor/MDA231細(xì)胞組中穿過Matrigel小室的細(xì)胞數(shù)量明顯減少(P<0.05)；MCF-7/Raptor細(xì)胞組中穿過Matrigel小室的細(xì)胞數(shù)量明顯升高(P<0.05)。細(xì)胞核質(zhì)分離實(shí)驗(yàn)結(jié)果顯示，siRaptor/MDA231組中Snail蛋白表達(dá)量較Scr/MDA231組中明顯降低；MCF-7/Raptor組中Snail蛋白表達(dá)量較MCF-7/Con組中明顯升高。結(jié)論 Raptor能夠促進(jìn)乳腺癌EMT的發(fā)生，對(duì)乳腺癌的侵襲與轉(zhuǎn)移有重要作用。

Raptor；上皮-間質(zhì)轉(zhuǎn)化；乳腺癌；侵襲；轉(zhuǎn)移；核質(zhì)分離

乳腺癌在女性惡性腫瘤中最為常見，其發(fā)病率連年升高，盡管各種治療乳腺癌的方法有實(shí)質(zhì)性進(jìn)展，但病人的臨床療效仍然達(dá)不到預(yù)期的效果。因此，提高患者的治愈率是非常重要的[1]。

Raptor是mTOR的一種沒有酶活性的調(diào)控蛋白。Aylett等[2]通過冷凍電子顯微鏡結(jié)合技術(shù)對(duì)嗜熱毛殼菌中Raptor的晶體進(jìn)行研究，揭示Raptor是作為mTORC1重要的結(jié)構(gòu)支持而發(fā)揮作用的。mTORC1-Raptor復(fù)合物在細(xì)胞中發(fā)揮重要的作用，控制蛋白質(zhì)與核糖體的合成，影響細(xì)胞的生長(zhǎng)與增殖[3]。多項(xiàng)研究發(fā)現(xiàn)，mTORC1-Raptor復(fù)合物與垂體腺瘤[4]、皮膚癌[5]的發(fā)展與轉(zhuǎn)移密切相關(guān)。上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition，EMT)在癌癥的侵襲與轉(zhuǎn)移中起重要作用。EMT涉及上皮細(xì)胞程序重排、失去極性，常發(fā)生在腫瘤的發(fā)生發(fā)展過程中[6]。目前，Raptor影響乳腺癌的分子機(jī)制并無定論。因此，本實(shí)驗(yàn)對(duì)乳腺癌細(xì)胞中Raptor的表達(dá)進(jìn)行調(diào)控，通過檢測(cè)EMT相關(guān)標(biāo)志物E-cadherin、Vimentin的表達(dá)情況，探討Raptor在乳腺癌EMT中的作用。

1 材料與方法

1.1 材料 Raptor、Snail單抗購自Abcam；Vimentin和E-cadherin單抗購自Santa Cruz Biotechnology；Raptor過表達(dá)質(zhì)粒與敲除質(zhì)粒均購自吉?jiǎng)P基因；Matrigel購自比迪醫(yī)療器械有限公司；Lipofectamine 2000脂質(zhì)體轉(zhuǎn)染試劑購自Invitrogen公司。

1.2 方法

1.2.1 細(xì)胞培養(yǎng) MDA231于10% FBS的RPMI 1640培養(yǎng)基、空氣、37 ℃培養(yǎng)。乳腺癌細(xì)胞MCF-7于10% FBS的MEM培養(yǎng)基、5% CO2、37 ℃培養(yǎng)。將轉(zhuǎn)染小RNA干擾質(zhì)粒的MDA-MB-231乳腺癌細(xì)胞作為siRaptor/MDA231組，轉(zhuǎn)染空載質(zhì)粒的Scr/MDA231細(xì)胞作為對(duì)照組，轉(zhuǎn)染過表達(dá)質(zhì)粒的MCF-7乳腺癌細(xì)胞作為MCF-7/Raptor組，轉(zhuǎn)染空載質(zhì)粒的MCF-7/Con細(xì)胞作為對(duì)照組。

1.2.2 MTT實(shí)驗(yàn) 參照文獻(xiàn)[7]進(jìn)行實(shí)驗(yàn)。

1.2.3 Western blot檢測(cè) 細(xì)胞轉(zhuǎn)染后培養(yǎng)并裂解，提取總蛋白，配制12% SDS-PAGE，然后電泳、轉(zhuǎn)膜、封閉，加入一抗中4℃過夜，TBST洗膜，二抗孵育，顯影，曝光。

1.2.4 細(xì)胞轉(zhuǎn)染 按照Lipofectamine 2000的說明書操作。

1.2.5 Transwell侵襲實(shí)驗(yàn) 將各組懸浮細(xì)胞加入小室上室、培養(yǎng)、染色、計(jì)數(shù)。實(shí)驗(yàn)至少獨(dú)立重復(fù)3次。

1.2.6 核質(zhì)分離 參照先前課題組發(fā)表的文獻(xiàn)[8]進(jìn)行實(shí)驗(yàn)。

1.3 統(tǒng)計(jì)學(xué)分析 全部數(shù)據(jù)資料用SPSS17.0分析，計(jì)量資料采用獨(dú)立樣本t檢驗(yàn)或單因素方差分析。

2 結(jié)果

2.1 Raptor對(duì)各組乳腺癌細(xì)胞增殖能力的影響 MTT實(shí)驗(yàn)結(jié)果顯示，轉(zhuǎn)染后各組乳腺癌細(xì)胞的增殖變化無統(tǒng)計(jì)學(xué)意義(Fig 1)。

2.2 乳腺癌細(xì)胞MCF-7、MDA231中Raptor蛋白的表達(dá) Western blot結(jié)果顯示，Raptor蛋白在MDA231細(xì)胞中表達(dá)量明顯高于在MCF-7細(xì)胞中的表達(dá)量(Fig 2)。

2.3 轉(zhuǎn)染后各組細(xì)胞中Raptor蛋白的表達(dá) β-actin作為內(nèi)參，Raptor蛋白在siRaptor/MDA231組中的表達(dá)明顯低于Scr/MDA231和MDA231組中的表達(dá)；Raptor蛋白在MCF-7/Raptor組中的表達(dá)明顯高于MCF-7/Con和MCF-7組中的表達(dá)，表明轉(zhuǎn)染成功(Fig 3)。

Fig 1 Influence of Raptor on cell proliferation of each breast cancer cell

MTT results showed that Raptor had no significant effect on cell proliferation of breast cancer cells. The MTT assay was repeated at least three times.

Fig 2 Expression of Raptor protein in MCF-7 and MDA231

Total protein was isolated from MCF-7 and MDA231.β-actin was used as a loading control. Western blot was repeated at least three times.

2.4 Raptor蛋白對(duì)各組乳腺癌細(xì)胞侵襲能力的影響 siRaptor/MDA231細(xì)胞組穿過Matrigel小室的細(xì)胞數(shù)量比Scr/MDA231組明顯減少，差異有顯著性(P<0.05)；MCF-7/Raptor細(xì)胞組穿過Matrigel小室的細(xì)胞數(shù)量比MCF-7/Con組明顯增多，差異有顯著性(P<0.05)，見Fig 4。

2.5 各組乳腺癌細(xì)胞中EMT標(biāo)志物的表達(dá) 如Fig 5所示，Vimentin在siRaptor/MDA231組的表達(dá)明顯低于MDA231組和Scr/MDA231組，而E-cadherin在siRaptor/MDA231組的表達(dá)與MDA231組和Scr/MDA231組相比明顯升高，差異有統(tǒng)計(jì)學(xué)意義；Vimentin在MCF-7/Raptor組的表達(dá)明顯高于MCF-7組和MCF-7/Con組，而E-cadherin在MCF-7/Raptor組的表達(dá)與MCF-7和MCF-7/Con組相比明顯降低，差異有統(tǒng)計(jì)學(xué)意義。

Fig 3 Expression of Raptor protein in various breast cancer cells

2.6 各組乳腺癌細(xì)胞中Snail蛋白的表達(dá) siRaptor/MDA231組中細(xì)胞核中Snail的表達(dá)量比Scr/MDA231組明顯降低，差異有統(tǒng)計(jì)學(xué)意義。MCF-7/Raptor組中細(xì)胞核中Snail表達(dá)量與MCF-7/Con組相比明顯升高，差異有統(tǒng)計(jì)學(xué)意義(Fig 6)。

3 討論

乳腺癌嚴(yán)重威脅女性健康，盡管乳腺癌的治療已經(jīng)取得很大進(jìn)展，但是轉(zhuǎn)移仍然是乳腺癌患者死亡的主要原因。

Raptor通過與mTORC1結(jié)合形成復(fù)合體來行使功能，是mTORC1的主要組成成分。Raptor作為橋梁分子，可以將mTOR與下游的靶分子連接起來，如S6K1(S6 kinase 1)和4E-BP1(eukaryotic initiation factor 4E binding protein 1)。Misra等[9]發(fā)現(xiàn)，Raptor能影響mTORC1下游S6K和4EBP1兩種蛋白，沉默 Raptor也可引起細(xì)胞形態(tài)的改變，Raptor在前列腺癌中與Akt1關(guān)系密切。多項(xiàng)研究表明，mTOR與Raptor的高表達(dá)與惡性淋巴瘤[10]等的侵襲和轉(zhuǎn)移有關(guān)。本實(shí)驗(yàn)結(jié)果顯示，MDA231細(xì)胞株中Raptor蛋白高表達(dá)，而MCF-7細(xì)胞株中Raptor蛋白低表達(dá)，結(jié)果提示Raptor蛋白的表達(dá)與乳腺癌的侵襲和轉(zhuǎn)移關(guān)系密切。

EMT正常情況下發(fā)生在胚胎發(fā)育時(shí)期，它可以使上皮細(xì)胞從某些部位遷移到其他部位，當(dāng)細(xì)胞惡變后，上皮細(xì)胞層喪失極性，黏附性減弱，細(xì)胞骨架發(fā)生重構(gòu)[11]。在惡性腫瘤中，EMT使腫瘤細(xì)胞轉(zhuǎn)移浸潤(rùn)到其他部位。EMT與多種腫瘤的原位浸潤(rùn)和遠(yuǎn)處轉(zhuǎn)移密切相關(guān)。已有研究發(fā)現(xiàn)EMT與胰腺癌、結(jié)腸癌等的侵襲與轉(zhuǎn)移有關(guān)[12-13]。本實(shí)驗(yàn)中，Transwell結(jié)果顯示，與Scr/MDA231組相比，siRaptor/MDA231細(xì)胞組穿過小室細(xì)胞數(shù)量明顯減少；與MCF-7/Con組相比，MCF-7/Raptor細(xì)胞組穿過小室的細(xì)胞數(shù)量明顯增多，結(jié)果提示Raptor促進(jìn)了乳腺癌細(xì)胞的侵襲能力。

Fig 4 Influence of Raptor on invasion capacity in various breast cancer cells

The number of cells in siRaptor/MDA231 group was significantly reduced (A) and in MCF-7/Raptor group, the number of cells was significantly increased (B).*P<0.05vsScr/MDA231 or MCF-7/Con

Fig 5 The protein levels of Vimentin and E-cadherin in various breast cancer cells

β-actin was used as a loading control.Western blot was repeated at least three times.

Fig 6 The protein levels of Snail in groups of various breast cancer cells

Nucleolin was used as a loading control. Western blot was repeated at least three times.

EMT的標(biāo)志物有多種，如E-cadherin、Vimentin、N-cadherin等，當(dāng)發(fā)生EMT時(shí)各種蛋白標(biāo)志物的表達(dá)會(huì)發(fā)生改變。波形蛋白的異常增加常被作為細(xì)胞發(fā)生 EMT 的標(biāo)志。本課題組先前研究發(fā)現(xiàn)，Vimentin作為EMT的重要標(biāo)志物在浸潤(rùn)性導(dǎo)管癌中高表達(dá)[14]。E-cadherin是鈣黏附蛋白家族中重要成員之一，與腫瘤組織的分化、侵襲和轉(zhuǎn)移相關(guān)，是腫瘤進(jìn)展及預(yù)后重要標(biāo)志物之一。研究表明，mTORC1與Raptor可以降低E-cadherin的表達(dá)，并誘導(dǎo)乳腺癌細(xì)胞發(fā)生EMT[15]。本實(shí)驗(yàn)結(jié)果顯示，用 Western blot檢測(cè)E-cadherin、Vimentin的表達(dá)，當(dāng)下調(diào)乳腺癌細(xì)胞MDA231中Raptor的表達(dá)時(shí)，Vimentin的表達(dá)量明顯降低，而E-cadherin蛋白的表達(dá)則明顯升高；當(dāng)上調(diào)乳腺癌細(xì)胞MCF-7中Raptor的表達(dá)時(shí)，Vimentin的表達(dá)量明顯升高，而E-cadherin蛋白的表達(dá)量則明顯降低，說明Raptor通過EMT促進(jìn)乳腺癌的侵襲和轉(zhuǎn)移。通過檢測(cè)各組乳腺癌細(xì)胞中Snail蛋白的表達(dá)發(fā)現(xiàn)，siRaptor/MDA231組中細(xì)胞核中Snail蛋白的表達(dá)量與Scr/MDA231組相比明顯降低，MCF-7/Raptor組中細(xì)胞核中Snail蛋白表達(dá)量與MCF-7/Con組相比明顯升高，提示Raptor促進(jìn)Snail蛋白的轉(zhuǎn)核。

綜上所述，本實(shí)驗(yàn)證實(shí)Raptor通過影響E-cadherin和Vimentin蛋白的表達(dá)，誘導(dǎo)EMT的發(fā)生，進(jìn)而影響乳腺癌細(xì)胞的侵襲和轉(zhuǎn)移。因此，通過對(duì)Raptor在乳腺癌中分子機(jī)制的研究，為進(jìn)一步控制乳腺癌的侵襲與轉(zhuǎn)移提供重要參考。

(致謝：感謝濰坊醫(yī)學(xué)院醫(yī)學(xué)研究實(shí)驗(yàn)中心為本實(shí)驗(yàn)提供平臺(tái)。)

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Raptor induces migration and invasion of breast cancer through epithelial-mesenchymal transition

XU Xin-wei1,WANG Zhao-yan1,WANG Rui-ge1,LI Hong-li2,YIN Chong-gao3,LIU Yu-qing1
(1.DeptofPathology, 2.MedicineResearchCenter,3.CollogeofNursing,WeifangMedicalUniversity,WeifangShandong261053,China)

Aim To study the molecular mechanism of Raptor in the migration and invasion of breast cancer cells and provide the clinical theory basis for prevention of breast cancer invasion and metastasis.Methods Western blot was used to detect the expression of Raptor protein in MCF-7 cells and MDA231 cells.The siRNA plasmids were used to transfect MDA231 cells. At the same time, the plasmid pcDNA3.1-Raptor was transfected into MCF-7 breast cancer cells. And Western blot was used to analyze the protein expression level of E-cadherin and Vimentin.Transwell was used to test the ability of invasion. Nucleus mass separation experiment was used to test the expression of Snail.Results The expression of Raptor protein in MDA231 cells was higher than in the MCF-7 cell. When the control group of Scr/MDA231 cells compared with siRaptor/MDA231, Raptor protein expression was decreased obviously after plasmid transfection interference, accompanied by reduction of Vimentin protein expression and increase of E-cadherin protein expression. Compared with MCF-7/Con, Raptor protein expression significantly increased in MCF-7/Raptor, accompanied by increase of Vimentin protein expression and reduction of E-cadherin protein expression .The number of cells through the artificial basilemma Transwell in siRaptor/MDA231 cells was significantly reduced(P<0.05), and the number of cells through the Transwell chambers artificial basilemma was significantly increased in MCF-7/Raptor(P<0.05). Nucleus mass separation experiment results showed that the expression of Snail decreased obviously in the siRaptor/MDA231 than in the Scr/MDA231, however,the expression of Snail was obviously higher in the MCF-7/Raptor.Conclusions Raptor can promote the occurrence of EMT in breast cancer, thus it promotes invasion and metastasis of breast cancer.

Raptor;epithelial-mesenchymal transition;breast cancer; invasion; metastasis; nucleus mass separation

2017-04-22,

2017-05-19

國(guó)家自然科學(xué)基金青年基金項(xiàng)目(No 81402389，81641111)；山東省自然科學(xué)基金資助項(xiàng)目(No ZR2014HL077，ZR 2015HL065)；山東省高等學(xué)?？萍加?jì)劃(No J12LK03，J13LK03)

徐新偉(1991-)，女，碩士，研究方向：腫瘤病理學(xué)，E-mail:18765693066@163.com; 劉雨清(1962-)，女，碩士，教授，研究方向：腫瘤病理學(xué)，通訊作者，E-mail: yuqingliu89@hotmail.com; 尹崇高(1979-)，男，碩士，副教授，研究方向：乳腺腫瘤，通訊作者，E-mail: ycglihongli@163.com

時(shí)間：2017-7-7 11:04 網(wǎng)絡(luò)出版地址：http://kns.cnki.net/kcms/detail/34.1086.R.20170707.1104.022.html

10.3969/j.issn.1001-1978.2017.08.011

A

1001-1978(2017)08-1091-05

R329.24；R341；R737.902.2；R977.6