李莉娜，肖鵬，王云峰，王翠蓮

（長(zhǎng)治醫(yī)學(xué)院病理學(xué)教研室，長(zhǎng)治 046000）

Flot-2高表達(dá)促進(jìn)食管鱗狀細(xì)胞癌的生長(zhǎng)和侵襲

李莉娜*，肖鵬，王云峰，王翠蓮

（長(zhǎng)治醫(yī)學(xué)院病理學(xué)教研室，長(zhǎng)治 046000）

目的探究fotillin-2（Flot-2）在人食管鱗狀細(xì)胞癌中的表達(dá)及基本功能。方法利用免疫組織化學(xué)技術(shù)檢測(cè)76例人食管鱗狀細(xì)胞癌組織及相應(yīng)癌旁組織中Flot-2的免疫反應(yīng)陽(yáng)性水平，并分析其與食管鱗狀細(xì)胞癌的相關(guān)性。以小分子siRNA降低人食管鱗狀細(xì)胞癌細(xì)胞系KYSE150細(xì)胞中Flot-2表達(dá)后，利用CKK-8及Transwell小室實(shí)驗(yàn)，檢測(cè)細(xì)胞生長(zhǎng)和侵襲能力的變化。結(jié)果Flot-2在人食管鱗狀細(xì)胞癌組織中免疫反應(yīng)性明顯升高，其在轉(zhuǎn)移組織中的免疫反應(yīng)水平顯著高于非轉(zhuǎn)移組織。敲低Flot-2表達(dá)后，KYSE150細(xì)胞的生長(zhǎng)和侵襲能力明顯降低。結(jié)論Flot-2能促進(jìn)細(xì)胞的生長(zhǎng)和侵襲，參與人食管鱗狀細(xì)胞癌的發(fā)生與轉(zhuǎn)移過(guò)程。

Flotillin-2；食管鱗狀細(xì)胞癌；轉(zhuǎn)移

食管癌惡性程度高，五年生存率低，其致死率在全球惡性腫瘤致死率中位列第八。在中國(guó)等發(fā)展中國(guó)家，鱗狀細(xì)胞癌為食管癌的主要病理類型。在我國(guó)其發(fā)病率和致死率位居各類惡性腫瘤前列，已成為嚴(yán)重影響我國(guó)人民群眾生命健康的重大疾病[1]。因此，深入研究食管癌，特別是食管鱗狀細(xì)胞癌的分子病理機(jī)制意義重大。

Flotillins是一類重要的脂筏蛋白，是細(xì)胞膜表面脂筏結(jié)構(gòu)的重要組成成分，包含fotillin-1(Flot-1)和fotillin-2(Flot-2)兩個(gè)成員，在細(xì)胞信號(hào)傳導(dǎo)，物質(zhì)運(yùn)輸?shù)冗^(guò)程中發(fā)揮重要作用，參與細(xì)胞連接、細(xì)胞惡性轉(zhuǎn)化等一系病理生理進(jìn)程的調(diào)節(jié)[2]。Flot-1和Flot-2已被證實(shí)參與肺癌、口腔癌、胃癌、鼻咽癌等腫瘤的惡性進(jìn)程[3-7]。對(duì)Flot-1與食管癌關(guān)系的研究發(fā)現(xiàn)，F(xiàn)lot-1在食管鱗狀細(xì)胞癌組織和細(xì)胞中高表達(dá)，并能促進(jìn)食管鱗狀細(xì)胞癌細(xì)胞的生長(zhǎng)[8]。Flotillins蛋白之間能形成異源或同源二聚體，是其穩(wěn)定和發(fā)揮作用的結(jié)構(gòu)基礎(chǔ)[2]。因此，作為另一個(gè)fotillins，F(xiàn)lot-2在食管鱗狀細(xì)胞癌是否存在類似于Flot-1的表達(dá)水平和功能，目前尚不清楚。本研究觀察了Flot-2在人食管鱗狀細(xì)胞癌中的表達(dá)及降低人食管鱗狀細(xì)胞癌細(xì)胞系KYSE150細(xì)胞中Flot-2表達(dá)對(duì)該細(xì)胞系生長(zhǎng)和侵襲的影響。

材料和方法

1 臨床標(biāo)本與細(xì)胞系

收集長(zhǎng)治醫(yī)學(xué)院附屬和平醫(yī)院病理科2016年1月至5月手術(shù)切除的食管鱗狀細(xì)胞癌患者的癌組織和癌旁組織標(biāo)本蠟塊，共76對(duì)。所有病例術(shù)前均無(wú)化療、放療及免疫治療史。人食管鱗狀細(xì)胞癌細(xì)胞系KYSE150細(xì)胞購(gòu)自中南大學(xué)細(xì)胞中心。

2 試劑

SP法免疫組織化學(xué)試劑盒及DAB顯示試劑盒均購(gòu)自福州邁新生物技術(shù)有限公司，F(xiàn)lot-2兔多抗、GAPDH兔多抗及相應(yīng)的二抗購(gòu)自武漢三鷹生物技術(shù)有限公司。Flot-2 siRNA（si-Flot-2）和對(duì)照siRNA（si-NC）由廣州銳博生物科技有限公司合成，F(xiàn)lot-2 siRNA序列為5’-ATGACAAAGTGGACTATCT-3’[7]。RPMI-1640、胎牛血清和0.25%胰蛋白酶購(gòu)自以色列BI公司， LipofectamineTM2000和Transwell 小室購(gòu)自美國(guó)Thermo公司，ECL顯色液購(gòu)自Millipore，RIPA蛋白裂解液、5×SDS上樣緩沖液、CCK-8購(gòu)自南京諾唯瓚生物有限公司。

3 免疫組織化學(xué)染色

組織蠟塊4μm切片，檸檬酸緩沖液高壓修復(fù)3min，F(xiàn)lot-2抗體的工作濃度1∶100，詳細(xì)步驟按照SP試劑盒說(shuō)明書(shū)操作。陽(yáng)性對(duì)照取乳腺癌組織，陰性對(duì)照用PBS代替一抗設(shè)立。結(jié)果判斷標(biāo)準(zhǔn)：先按陽(yáng)性細(xì)胞占上皮細(xì)胞或腫瘤細(xì)胞的百分比計(jì)分：無(wú)陽(yáng)性細(xì)胞為0分，陽(yáng)性細(xì)胞≤10%計(jì)1分，11%～50%計(jì)2分，51% ～ 75%計(jì)3分，＞75%計(jì)4分；再按染色強(qiáng)度計(jì)分：淺黃色計(jì)1分，棕黃色計(jì)2分，棕褐色計(jì)3分。以兩者乘積為最后得分：0～3分為陰性或弱陽(yáng)性，4～6為陽(yáng)性，6～12分為強(qiáng)陽(yáng)性。

4 細(xì)胞轉(zhuǎn)染

轉(zhuǎn)染前24h，將KYSE150細(xì)胞消化接種于6孔板中，使第二天轉(zhuǎn)染時(shí)，細(xì)胞匯合度在70%左右。根據(jù)LipofectamineTM2000說(shuō)明書(shū)，制備si-Flot-2脂質(zhì)體混合液及相應(yīng)的si-NC脂質(zhì)體混合液，室溫靜置20min；隨后將混合好的siRNA脂質(zhì)體懸液均勻滴加至細(xì)胞中，繼續(xù)培養(yǎng)6h后更換培養(yǎng)基；48h后，抽提細(xì)胞總蛋白，檢測(cè)Flot-2敲低效率。

5 Western blot

處于對(duì)數(shù)生長(zhǎng)期的細(xì)胞，經(jīng)PBS漂洗后，加入適量RIPA蛋白裂解液，于冰上裂解30min后，用細(xì)胞刮將細(xì)胞裂解液刮下，經(jīng)超聲破碎，低溫高速離心后，得到細(xì)胞總蛋白。經(jīng)SDS-PAGE(聚丙烯酰胺凝膠)電泳、轉(zhuǎn)膜、封閉、一抗和二抗孵育后，進(jìn)行ECL化學(xué)發(fā)光顯色，利用Gel-Pro analyzer軟件分析條帶灰度值，經(jīng)內(nèi)參校正后，比較蛋白表達(dá)差異。

6 CCK-8實(shí)驗(yàn)

將分別轉(zhuǎn)染si-Flot-2和si-NC的KYSE150細(xì)胞，按照2000個(gè)/孔的密度接種于96孔板，于接種后24h、48h、72h、96h，按10μl CCK-8/孔（每孔含100μl培養(yǎng)基）的比例，加入CCK-8溶液，每組設(shè)置5個(gè)復(fù)孔，37℃孵育1h后，于酶標(biāo)儀（Epoch，Bio Tek公司）450nm波長(zhǎng)下，檢測(cè)每孔的吸光值，計(jì)算細(xì)胞生長(zhǎng)速率。

7 Transwell侵襲實(shí)驗(yàn)

按2.5×104個(gè)/孔的密度，將分別轉(zhuǎn)染si-Flot-2和si-NC的KYSE150細(xì)胞，接種于包被有基質(zhì)膠（Matrigel）的Transwell小室中（細(xì)胞懸液總體積保持在500μl），下層為750μl 5%FBS+RPMI1640培養(yǎng)基，保證小室和下層培養(yǎng)基的接觸面沒(méi)有氣泡，正常培養(yǎng)48h后，棄培養(yǎng)基，經(jīng)緩沖液清洗、甲醇固定后，0.1%結(jié)晶紫染色20min，自來(lái)水沖洗干凈后，用棉簽小心刮掉上層膜側(cè)未穿過(guò)的細(xì)胞，于倒置顯微鏡下觀察細(xì)胞穿過(guò)情況，每組設(shè)置3個(gè)復(fù)孔，每孔隨機(jī)選取5個(gè)視野，計(jì)數(shù)穿過(guò)細(xì)胞數(shù)，以平均數(shù)作為此孔的細(xì)胞穿過(guò)數(shù)，取3個(gè)復(fù)孔的細(xì)胞穿過(guò)數(shù)進(jìn)行統(tǒng)計(jì)學(xué)分析。

8 統(tǒng)計(jì)學(xué)分析

利用SPSS 18.0 統(tǒng)計(jì)分析軟件進(jìn)行數(shù)據(jù)分析，免疫組化結(jié)果采用Kruskal-Wallis H 檢驗(yàn)比較組間總體差異，Nemenyi秩和檢驗(yàn)比較組間相互差異。計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差（s）表示，采用兩樣本t檢驗(yàn)比較實(shí)驗(yàn)組和對(duì)照組間的差異， P＜ 0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 食管鱗狀細(xì)胞癌組織中Flot-2免疫反應(yīng)性增強(qiáng)

免疫組織化學(xué)檢測(cè)顯示，F(xiàn)lot-2免疫反應(yīng)產(chǎn)物主要定位于食管鱗狀細(xì)胞癌癌組織和配對(duì)癌旁組織細(xì)胞質(zhì)和細(xì)胞膜。Flot-2在鱗狀細(xì)胞癌組織中的免疫反應(yīng)性明顯強(qiáng)于相應(yīng)癌旁組織 (圖1A，圖1B，表1)。對(duì)Flot-2在無(wú)淋巴結(jié)轉(zhuǎn)移和有淋巴結(jié)轉(zhuǎn)移的食管鱗狀細(xì)胞癌中的免疫反應(yīng)性比較發(fā)現(xiàn)，相較于無(wú)淋巴結(jié)轉(zhuǎn)移的食管鱗狀細(xì)胞癌組織，F(xiàn)lot-2在發(fā)生淋巴結(jié)轉(zhuǎn)移的食管鱗狀細(xì)胞癌組織中的免疫反應(yīng)性更強(qiáng)(圖1C，表1)，提示Flot-2可能參與食管鱗狀細(xì)胞癌的轉(zhuǎn)移過(guò)程。

圖1 食管鱗狀細(xì)胞癌及癌旁組織中Flot-2的免疫組織化學(xué)表達(dá)。A，癌旁組織中；B，無(wú)轉(zhuǎn)移的食管鱗狀細(xì)胞癌組織；C，有轉(zhuǎn)移的食管鱗狀細(xì)胞癌組織；比例尺，100μmFig. 1 Immunohistochemical expression of Flot-2 in ESCC tissue and adjacent tissue. A, adjacent tissue; B, non-metastatic ESCC tissue; C, metastatic ESCC tissue; scale bar, 100μm

表1 Flot-2在食管鱗狀細(xì)胞癌組織和癌旁組織中的免疫反應(yīng)性比較Tab. 1 Comparison of Flot-2 immunoreactivity in ESCC tissue and adjacent tissue

2 沉默F(xiàn)lot-2抑制KYSE150細(xì)胞生長(zhǎng)和侵襲

為了探討Flot-2在人食管鱗狀細(xì)胞癌中的作用，利用脂質(zhì)體轉(zhuǎn)染技術(shù)，將靶向Flot-2的si-Flot-2和相應(yīng)的對(duì)照siRNA（si-NC）轉(zhuǎn)入人食管鱗狀細(xì)胞癌細(xì)胞系KYSE150細(xì)胞，建立低表達(dá)Flot-2的細(xì)胞模型。收集轉(zhuǎn)染si-RNA 48h后的KYSE150細(xì)胞，提取總蛋白，進(jìn)行Flot-2蛋白水平的免疫印記檢測(cè)。結(jié)果顯示，與未轉(zhuǎn)染siRNA的空白細(xì)胞和轉(zhuǎn)染si-NC的細(xì)胞相比，轉(zhuǎn)染si-Flot-2的KYSE150細(xì)胞中，F(xiàn)lot-2d蛋白水平明顯降低（圖2），表明成功建立了敲低Flot-2的KYSE150細(xì)胞模型。

圖2 Western blot檢測(cè)Si-Flot-2沉默KYSE150細(xì)胞中Flot-2表達(dá)Fig. 2 Western blot detection of the efciency of Flot-2 knockdown in KYSE150 cells by Si-Flot-2

利用CCK-8實(shí)驗(yàn)檢測(cè)沉默F(xiàn)lot-2的KYSE150細(xì)胞生長(zhǎng)速率，結(jié)果顯示：與轉(zhuǎn)染si-NC的細(xì)胞相比，轉(zhuǎn)染si-Flot-2的細(xì)胞生長(zhǎng)速率明顯減慢，在轉(zhuǎn)染72h和96h后差異尤其顯著（圖3）。由此提示，F(xiàn)lot-2可能通過(guò)促進(jìn)人食管鱗狀細(xì)胞癌細(xì)胞的生長(zhǎng)而參與腫瘤進(jìn)程。

圖3 沉默F(xiàn)lot-2抑制KYSE150細(xì)胞的生長(zhǎng)速率。*，與轉(zhuǎn)染si-NC的細(xì)胞比較，P ＜0.01Fig. 3 Statistical analysis for the effect of Flot-2 knockdown on the proliferative rate of KYSE150 cells detected by CCK-8. *, P＜0.01, compared with cells transfected with si-NC

圖4 沉默F(xiàn)lot-2對(duì)KYSE150細(xì)胞侵襲力的影響。A， 穿過(guò)Transwell膜細(xì)胞的結(jié)晶紫染色；B，穿過(guò)Transwell膜的細(xì)胞數(shù)統(tǒng)計(jì)學(xué)分析； *，與轉(zhuǎn)染si-NC細(xì)胞比較，P ＜0.01Fig. 4 The efect of Flot-2 knockdown on the invasion ability of KYSE150 cells detected by Transwell assay. A, crystal violet staining of the cells having migrated through the Transwell membrane; B, statistical analysis for the number of cells having migrated through the Transwell membrane; *, P＜0.01, compared with cells transfected with si-NC

利用Transwell小室實(shí)驗(yàn)對(duì)沉默F(xiàn)lot-2對(duì)KYSE150細(xì)胞侵襲能力檢測(cè)顯示，與轉(zhuǎn)染si-NC的對(duì)照細(xì)胞相比，抑制Flot-2表達(dá)后，KYSE150細(xì)胞消化基質(zhì)膠、穿過(guò)小室的細(xì)胞數(shù)目明顯降低（35.00 ± 2.89 vs 14.67 ± 1.45, P ＜ 0.05），細(xì)胞的侵襲能力顯著降低（圖4）。因此，F(xiàn)lot-2可能通過(guò)促進(jìn)人食管鱗狀細(xì)胞癌細(xì)胞的侵襲而參與其轉(zhuǎn)移進(jìn)程。

討 論

脂筏是細(xì)胞表面信號(hào)分子聚集的超微結(jié)構(gòu)，是細(xì)胞信號(hào)匯集、整理和傳導(dǎo)的分子平臺(tái)[9]。Flot-2作為重要的脂筏結(jié)構(gòu)蛋白，在上述過(guò)程中發(fā)揮重要作用。已知Flot-2與神經(jīng)再生分化、細(xì)胞內(nèi)吞、細(xì)胞增殖、胰島素信號(hào)傳導(dǎo)等生命過(guò)程密切相關(guān)，其表達(dá)異常與包括腫瘤在內(nèi)的多種病理過(guò)程相關(guān)[2,10-12]。目前，F(xiàn)lot-2已被證實(shí)在多種實(shí)體腫瘤中表達(dá)異常升高，且與腫瘤早期診斷、轉(zhuǎn)移及預(yù)后判斷等因素相關(guān)。例如：在乳腺癌和非小細(xì)胞肺癌中，F(xiàn)lot-2異?；罨c乳腺癌的病理分期、轉(zhuǎn)移能力呈正相關(guān)，與患者生存期呈負(fù)相關(guān)[5,13-15]；在宮頸癌及腎細(xì)胞癌等腫瘤中，F(xiàn)lot-2呈漸行性升高[16,17]；Flot-2的表達(dá)水平，可以作為鼻咽癌淋巴結(jié)轉(zhuǎn)移的分子標(biāo)記[18]；在口腔鱗狀細(xì)胞癌及胃癌等消化道相關(guān)腫瘤中，F(xiàn)lot-2同樣與腫瘤生長(zhǎng)、轉(zhuǎn)移等密切相關(guān)[4,19]。基于Flot-2在腫瘤中的關(guān)鍵作用，在本研究中，我們檢測(cè)了其在食管鱗狀細(xì)胞癌中的水平，結(jié)果顯示Flot-2在食管鱗狀細(xì)胞癌中的水平明顯升高，且在存在淋巴結(jié)或遠(yuǎn)端轉(zhuǎn)移的組織中更高。由此提示，F(xiàn)lot-2可能作為人食管鱗狀細(xì)胞癌早期診斷和靶向治療的潛在靶標(biāo)。

已有研究顯示，F(xiàn)lot-2促進(jìn)腫瘤進(jìn)程的主要分子機(jī)制是促進(jìn)腫瘤細(xì)胞生長(zhǎng)和侵襲。降低Flot-2的表達(dá)，能抑制Akt/FOXO等信號(hào)軸的活性，進(jìn)而抑制乳腺癌的生長(zhǎng)、遷移和侵襲[14]；在鼻咽癌中，降低5-8F細(xì)胞中Flot-2的表達(dá)，能抑制Akt3/NF-kB通路的活性，進(jìn)而抑制其侵襲和轉(zhuǎn)移[7]；降低Flot-2表達(dá)，同樣能抑制胃癌細(xì)胞的生長(zhǎng)、遷移和侵襲能力[20]。本研究中，我們同樣探討了Flot-2對(duì)人食管癌鱗狀細(xì)胞癌細(xì)胞生長(zhǎng)和侵襲能力的影響。在SiRNA下調(diào)Flot-2表達(dá)后，KYSE150細(xì)胞的生長(zhǎng)和侵襲能力均受到明顯抑制，由此提示Flot-2可以通過(guò)促進(jìn)人食管鱗狀細(xì)胞癌細(xì)胞的生長(zhǎng)和侵襲，而促進(jìn)其發(fā)生和轉(zhuǎn)移。

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High expression of Flot-2 promotes the development and invasion of esophageal squamous cell carcinoma

Li Lina*, Xiao Peng, Wang Yunfeng, Wang Cuilian
(Department of Pathology, Changzhi Medical College, Changzhi 046000)

ObjectiveTo explore the expression and functions of fotillin-2 (Flot-2) in human esophageal squamous cell carcinoma (ESCC).MethodsThe immunoreactivity of Flot-2 in the cancerous and adjacent tissue from 76 ESCC cases was detected by immunohistochemistry, and its correlation with ESCC was analyzed. Small RNA interference (siRNA) was applied to reduce the expression of Flot-2 in KYSE150 ESCC cells, after which the proliferation and invasion abilities of KYSE150 cells were examined by CCK-8 and Transwell tests.ResultsFlot-2 immunoreactivity was signifcantly higher in ESCC tissue than in adjacent tissue, and in metastatic ESCC than in non-metastatic ESCC. The proliferation and invasion abilities of KYSE150 cells were obviously inhibited after Flot-2 knockdown.ConclusionFlot-2 plays a role in the development and metastasis of ESCC by promoting cell proliferation and invasion.

Flotillin-2; esophageal squamous cell carcinoma; metastasis

R730.2

A

10.16705/ j. cnki. 1004-1850. 2017.02.011

2016-11-07

2017-03-30

長(zhǎng)治醫(yī)學(xué)院科研啟動(dòng)基金普及項(xiàng)目（201508）

李莉娜，女（1981年），漢族，講師

*通訊作者(To whom correspondence should be addressed)：lilina9825@163. com