Raptor通过上皮-间质转化促进乳腺癌的侵袭与转移

徐新伟，王照岩，王瑞鸽，李洪利，尹崇高，刘雨清

(潍坊医学院1.病理学教研室、2.医学研究实验中心、3.护理学院，山东 潍坊 261053)

Raptor通过上皮-间质转化促进乳腺癌的侵袭与转移

徐新伟1，王照岩1，王瑞鸽1，李洪利2，尹崇高3，刘雨清1

(潍坊医学院1.病理学教研室、2.医学研究实验中心、3.护理学院，山东 潍坊 261053)

目的 探讨Raptor在乳腺癌侵袭与转移过程中的作用。方法 采用Western blot检测乳腺癌细胞MCF-7和MDA-MB-231(MDA231)中Raptor蛋白的表达情况。应用小干扰RNA技术下调乳腺癌细胞MDA231中Raptor的表达量，同时将过表达质粒转入乳腺癌细胞MCF-7中，应用Western blot法检测转染后各组细胞中上皮性钙黏着蛋白(E-cadherin)和波形蛋白(Vimentin)的表达；应用Transwell体外侵袭实验检测转染后各组细胞的侵袭能力；应用细胞核质分离实验检测转染后各组细胞中Snail蛋白的转核情况。结果 MCF-7细胞中Raptor蛋白的表达量明显低于MDA231细胞中的表达量；转染后，与对照组Scr/MDA231相比，siRaptor/MDA231组中Raptor蛋白的表达量明显降低，Vimentin蛋白表达量降低，而E-cadherin 蛋白表达量升高；与对照组MCF-7/Con相比，MCF-7/Raptor组中Raptor蛋白的表达量明显升高，并伴有Vimentin蛋白表达量升高，而E-cadherin 蛋白表达量降低。siRaptor/MDA231细胞组中穿过Matrigel小室的细胞数量明显减少(P<0.05)；MCF-7/Raptor细胞组中穿过Matrigel小室的细胞数量明显升高(P<0.05)。细胞核质分离实验结果显示，siRaptor/MDA231组中Snail蛋白表达量较Scr/MDA231组中明显降低；MCF-7/Raptor组中Snail蛋白表达量较MCF-7/Con组中明显升高。结论 Raptor能够促进乳腺癌EMT的发生，对乳腺癌的侵袭与转移有重要作用。

Raptor；上皮-间质转化；乳腺癌；侵袭；转移；核质分离

乳腺癌在女性恶性肿瘤中最为常见，其发病率连年升高，尽管各种治疗乳腺癌的方法有实质性进展，但病人的临床疗效仍然达不到预期的效果。因此，提高患者的治愈率是非常重要的[1]。

Raptor是mTOR的一种没有酶活性的调控蛋白。Aylett等[2]通过冷冻电子显微镜结合技术对嗜热毛壳菌中Raptor的晶体进行研究，揭示Raptor是作为mTORC1重要的结构支持而发挥作用的。mTORC1-Raptor复合物在细胞中发挥重要的作用，控制蛋白质与核糖体的合成，影响细胞的生长与增殖[3]。多项研究发现，mTORC1-Raptor复合物与垂体腺瘤[4]、皮肤癌[5]的发展与转移密切相关。上皮-间质转化(epithelial-mesenchymal transition，EMT)在癌症的侵袭与转移中起重要作用。EMT涉及上皮细胞程序重排、失去极性，常发生在肿瘤的发生发展过程中[6]。目前，Raptor影响乳腺癌的分子机制并无定论。因此，本实验对乳腺癌细胞中Raptor的表达进行调控，通过检测EMT相关标志物E-cadherin、Vimentin的表达情况，探讨Raptor在乳腺癌EMT中的作用。

1 材料与方法

1.1 材料 Raptor、Snail单抗购自Abcam；Vimentin和E-cadherin单抗购自Santa Cruz Biotechnology；Raptor过表达质粒与敲除质粒均购自吉凯基因；Matrigel购自比迪医疗器械有限公司；Lipofectamine 2000脂质体转染试剂购自Invitrogen公司。

1.2 方法

1.2.1 细胞培养 MDA231于10% FBS的RPMI 1640培养基、空气、37 ℃培养。乳腺癌细胞MCF-7于10% FBS的MEM培养基、5% CO2、37 ℃培养。将转染小RNA干扰质粒的MDA-MB-231乳腺癌细胞作为siRaptor/MDA231组，转染空载质粒的Scr/MDA231细胞作为对照组，转染过表达质粒的MCF-7乳腺癌细胞作为MCF-7/Raptor组，转染空载质粒的MCF-7/Con细胞作为对照组。

1.2.2 MTT实验 参照文献[7]进行实验。

1.2.3 Western blot检测 细胞转染后培养并裂解，提取总蛋白，配制12% SDS-PAGE，然后电泳、转膜、封闭，加入一抗中4℃过夜，TBST洗膜，二抗孵育，显影，曝光。

1.2.4 细胞转染 按照Lipofectamine 2000的说明书操作。

1.2.5 Transwell侵袭实验 将各组悬浮细胞加入小室上室、培养、染色、计数。实验至少独立重复3次。

1.2.6 核质分离 参照先前课题组发表的文献[8]进行实验。

1.3 统计学分析 全部数据资料用SPSS17.0分析，计量资料采用独立样本t检验或单因素方差分析。

2 结果

2.1 Raptor对各组乳腺癌细胞增殖能力的影响 MTT实验结果显示，转染后各组乳腺癌细胞的增殖变化无统计学意义(Fig 1)。

2.2 乳腺癌细胞MCF-7、MDA231中Raptor蛋白的表达 Western blot结果显示，Raptor蛋白在MDA231细胞中表达量明显高于在MCF-7细胞中的表达量(Fig 2)。

2.3 转染后各组细胞中Raptor蛋白的表达 β-actin作为内参，Raptor蛋白在siRaptor/MDA231组中的表达明显低于Scr/MDA231和MDA231组中的表达；Raptor蛋白在MCF-7/Raptor组中的表达明显高于MCF-7/Con和MCF-7组中的表达，表明转染成功(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蛋白对各组乳腺癌细胞侵袭能力的影响 siRaptor/MDA231细胞组穿过Matrigel小室的细胞数量比Scr/MDA231组明显减少，差异有显著性(P<0.05)；MCF-7/Raptor细胞组穿过Matrigel小室的细胞数量比MCF-7/Con组明显增多，差异有显著性(P<0.05)，见Fig 4。

2.5 各组乳腺癌细胞中EMT标志物的表达 如Fig 5所示，Vimentin在siRaptor/MDA231组的表达明显低于MDA231组和Scr/MDA231组，而E-cadherin在siRaptor/MDA231组的表达与MDA231组和Scr/MDA231组相比明显升高，差异有统计学意义；Vimentin在MCF-7/Raptor组的表达明显高于MCF-7组和MCF-7/Con组，而E-cadherin在MCF-7/Raptor组的表达与MCF-7和MCF-7/Con组相比明显降低，差异有统计学意义。

Fig 3 Expression of Raptor protein in various breast cancer cells

2.6 各组乳腺癌细胞中Snail蛋白的表达 siRaptor/MDA231组中细胞核中Snail的表达量比Scr/MDA231组明显降低，差异有统计学意义。MCF-7/Raptor组中细胞核中Snail表达量与MCF-7/Con组相比明显升高，差异有统计学意义(Fig 6)。

3 讨论

乳腺癌严重威胁女性健康，尽管乳腺癌的治疗已经取得很大进展，但是转移仍然是乳腺癌患者死亡的主要原因。

Raptor通过与mTORC1结合形成复合体来行使功能，是mTORC1的主要组成成分。Raptor作为桥梁分子，可以将mTOR与下游的靶分子连接起来，如S6K1(S6 kinase 1)和4E-BP1(eukaryotic initiation factor 4E binding protein 1)。Misra等[9]发现，Raptor能影响mTORC1下游S6K和4EBP1两种蛋白，沉默 Raptor也可引起细胞形态的改变，Raptor在前列腺癌中与Akt1关系密切。多项研究表明，mTOR与Raptor的高表达与恶性淋巴瘤[10]等的侵袭和转移有关。本实验结果显示，MDA231细胞株中Raptor蛋白高表达，而MCF-7细胞株中Raptor蛋白低表达，结果提示Raptor蛋白的表达与乳腺癌的侵袭和转移关系密切。

EMT正常情况下发生在胚胎发育时期，它可以使上皮细胞从某些部位迁移到其他部位，当细胞恶变后，上皮细胞层丧失极性，黏附性减弱，细胞骨架发生重构[11]。在恶性肿瘤中，EMT使肿瘤细胞转移浸润到其他部位。EMT与多种肿瘤的原位浸润和远处转移密切相关。已有研究发现EMT与胰腺癌、结肠癌等的侵袭与转移有关[12-13]。本实验中，Transwell结果显示，与Scr/MDA231组相比，siRaptor/MDA231细胞组穿过小室细胞数量明显减少；与MCF-7/Con组相比，MCF-7/Raptor细胞组穿过小室的细胞数量明显增多，结果提示Raptor促进了乳腺癌细胞的侵袭能力。

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的标志物有多种，如E-cadherin、Vimentin、N-cadherin等，当发生EMT时各种蛋白标志物的表达会发生改变。波形蛋白的异常增加常被作为细胞发生 EMT 的标志。本课题组先前研究发现，Vimentin作为EMT的重要标志物在浸润性导管癌中高表达[14]。E-cadherin是钙黏附蛋白家族中重要成员之一，与肿瘤组织的分化、侵袭和转移相关，是肿瘤进展及预后重要标志物之一。研究表明，mTORC1与Raptor可以降低E-cadherin的表达，并诱导乳腺癌细胞发生EMT[15]。本实验结果显示，用 Western blot检测E-cadherin、Vimentin的表达，当下调乳腺癌细胞MDA231中Raptor的表达时，Vimentin的表达量明显降低，而E-cadherin蛋白的表达则明显升高；当上调乳腺癌细胞MCF-7中Raptor的表达时，Vimentin的表达量明显升高，而E-cadherin蛋白的表达量则明显降低，说明Raptor通过EMT促进乳腺癌的侵袭和转移。通过检测各组乳腺癌细胞中Snail蛋白的表达发现，siRaptor/MDA231组中细胞核中Snail蛋白的表达量与Scr/MDA231组相比明显降低，MCF-7/Raptor组中细胞核中Snail蛋白表达量与MCF-7/Con组相比明显升高，提示Raptor促进Snail蛋白的转核。

综上所述，本实验证实Raptor通过影响E-cadherin和Vimentin蛋白的表达，诱导EMT的发生，进而影响乳腺癌细胞的侵袭和转移。因此，通过对Raptor在乳腺癌中分子机制的研究，为进一步控制乳腺癌的侵袭与转移提供重要参考。

(致谢：感谢潍坊医学院医学研究实验中心为本实验提供平台。)

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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

国家自然科学基金青年基金项目(No 81402389，81641111)；山东省自然科学基金资助项目(No ZR2014HL077，ZR 2015HL065)；山东省高等学校科技计划(No J12LK03，J13LK03)

徐新伟(1991-)，女，硕士，研究方向：肿瘤病理学，E-mail:18765693066@163.com; 刘雨清(1962-)，女，硕士，教授，研究方向：肿瘤病理学，通讯作者，E-mail: yuqingliu89@hotmail.com; 尹崇高(1979-)，男，硕士，副教授，研究方向：乳腺肿瘤，通讯作者，E-mail: ycglihongli@163.com

时间：2017-7-7 11:04 网络出版地址：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