Rab23對(duì)鱗狀細(xì)胞癌Sa3細(xì)胞增殖的抑制作用及相關(guān)機(jī)制

劉曦霖 簡(jiǎn)強(qiáng) 苗葉 黃敏 李承新

·論著·

Rab23對(duì)鱗狀細(xì)胞癌Sa3細(xì)胞增殖的抑制作用及相關(guān)機(jī)制

劉曦霖 簡(jiǎn)強(qiáng) 苗葉 黃敏 李承新

目的觀察Rab23分子對(duì)鱗狀細(xì)胞癌(簡(jiǎn)稱(chēng)鱗癌)細(xì)胞Sa3增殖的影響及機(jī)制。 方法 將鱗狀細(xì)胞癌Sa3細(xì)胞分為4組:正常對(duì)照組[轉(zhuǎn)染綠色熒光蛋白(eGFP)]、Rab23過(guò)表達(dá)組(轉(zhuǎn)染eGFP標(biāo)記的Rab23過(guò)表達(dá)質(zhì)粒)、Rab23沉默組(轉(zhuǎn)染Rab23 shRNA質(zhì)粒)、Rab23空載體組(轉(zhuǎn)染空載體)。平板克隆形成實(shí)驗(yàn)、流式細(xì)胞儀檢測(cè)Rab23過(guò)表達(dá)和沉默對(duì)Sa3細(xì)胞增殖能力的影響。Western印跡檢測(cè)Rab23過(guò)表達(dá)和沉默對(duì)Sa3細(xì)胞Erk/p-Erk表達(dá)水平的變化。兩組間均數(shù)比較采用t檢驗(yàn),多組間均數(shù)比較采用單因素方差分析,多組資料兩兩比較使用Bonferroni's多重比較分析。結(jié)果通過(guò)構(gòu)建質(zhì)粒、慢病毒感染,獲得Rab23過(guò)表達(dá)和Rab23沉默的穩(wěn)定Sa3細(xì)胞株。平板克隆形成實(shí)驗(yàn)結(jié)果顯示,Rab23過(guò)表達(dá)組克隆形成率為2.3%±0.2%,與正常對(duì)照組(3.6%±0.3%)相比,Sa3細(xì)胞增殖能力降低(P＜0.05),而Rab23沉默組克隆形成率(4.1%±0.2%)較空載體組(1.8%±0.0%)增殖能力明顯提高(P＜0.01)。細(xì)胞周期檢測(cè)顯示,Rab23過(guò)表達(dá)可引起Sa3細(xì)胞G1期阻滯,Rab23過(guò)表達(dá)組增殖指數(shù)(0.581±0.035)較正常對(duì)照組(0.698±0.018)降低(P＜0.05),而Rab23沉默組增殖指數(shù)(0.567±0.015)較空載體組(0.444±0.014)明顯提高(P＜0.01)。Western印跡結(jié)果顯示,與正常對(duì)照組相比,Rab23過(guò)表達(dá)組和Rab23沉默組Sa3細(xì)胞Erk表達(dá)水平無(wú)變化,但Rab23過(guò)表達(dá)組磷酸化Erk表達(dá)水平較正常對(duì)照組降低,Rab23沉默組磷酸化Erk表達(dá)水平較空載體組升高。結(jié)論Rab23對(duì)鱗癌Sa3細(xì)胞增殖起抑制作用,這種抑制作用可能與Erk通路有關(guān)。

Rab23;慢病毒感染;癌,鱗狀細(xì)胞;細(xì)胞系,腫瘤;細(xì)胞增殖

皮膚鱗狀細(xì)胞癌(簡(jiǎn)稱(chēng)鱗癌)是起源于表皮、皮膚附屬器和復(fù)層扁平上皮黏膜角質(zhì)形成細(xì)胞的惡性腫瘤,發(fā)病率僅次于基底細(xì)胞上皮瘤[1],具有侵襲性,可發(fā)生早期轉(zhuǎn)移。因此對(duì)鱗癌發(fā)生發(fā)展、侵襲轉(zhuǎn)移的研究具有重要臨床意義。Rab23屬于小GTP酶超家族Rab家族成員,不僅與腦、骨骼、四肢的發(fā)育有關(guān)[2],而且具有囊泡轉(zhuǎn)運(yùn)功能。研究發(fā)現(xiàn),Rab23參與多種腫瘤的發(fā)生發(fā)展,可促進(jìn)胃癌[3]、肝癌[4]、肺癌[5]細(xì)胞的侵襲,促進(jìn)肝癌[6]細(xì)胞的增殖,抑制乳腺癌細(xì)胞的生長(zhǎng)[7]。我們前期研究發(fā)現(xiàn),Rab23在鱗癌中高表達(dá),并明顯促進(jìn)鱗癌細(xì)胞的侵襲[8],而其對(duì)鱗癌細(xì)胞生長(zhǎng)增殖的影響尚不清楚。本實(shí)驗(yàn)通過(guò)慢病毒轉(zhuǎn)染技術(shù),建立過(guò)表達(dá)和干涉Rab23的鱗癌細(xì)胞株,研究Rab23對(duì)鱗癌細(xì)胞增殖的作用及可能機(jī)制。

材料和方法

1.材料:慢病毒載體質(zhì)粒(上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司),人鱗癌Sa3細(xì)胞(日本Reken Cell Bank公司),胎牛血清(北京Solarbio公司),高糖DMEM培養(yǎng)基(美國(guó)Hyclone公司),胰酶(美國(guó)Sigma公司),嘌呤霉素(德國(guó)Merck公司),β肌動(dòng)蛋白抗體(北京康為世紀(jì)生物公司),Rab23抗體(美國(guó)Proteintech公司),Erk抗體、磷酸化Erk(p-Erk)抗體(美國(guó)Cell signaling公司)。

2.構(gòu)建穩(wěn)定的Rab23過(guò)表達(dá)和沉默的Sa3細(xì)胞模型:構(gòu)建Rab23分子過(guò)表達(dá)質(zhì)粒和shRNA質(zhì)粒,進(jìn)行慢病毒包裝,將包裝好的病毒感染人鱗癌Sa3細(xì)胞系。試驗(yàn)分為4組:正常對(duì)照組[轉(zhuǎn)染綠色熒光蛋白(eGFP)]、Rab23過(guò)表達(dá)組(轉(zhuǎn)染eGFP標(biāo)記的 Rab23過(guò)表達(dá)質(zhì)粒)、Rab23沉默組[轉(zhuǎn)染Rab23短發(fā)卡RNA(shRNA)質(zhì)粒]、Rab23空載體組(轉(zhuǎn)染空載體)。按照Lentiviral Vector Particle使用操作手冊(cè)進(jìn)行感染。將Sa3細(xì)胞接種于96孔板,細(xì)胞融合度達(dá)到70%時(shí),按1×108轉(zhuǎn)導(dǎo)單位(TU)/ml(MOI=100,即病毒 2 μl加入 98 μl含血清 DMEM培養(yǎng)基)加入病毒感染細(xì)胞,8～12 h后換成鱗癌細(xì)胞常規(guī)培養(yǎng)基培養(yǎng)2～3 d,可觀察到eGFP熒光。再繼續(xù)培養(yǎng)1～2周,當(dāng)熒光表達(dá)細(xì)胞超過(guò)70%時(shí),將細(xì)胞種于24孔板,在含嘌呤霉素(1 mg/L)的常規(guī)培養(yǎng)基中培養(yǎng)1～2周,得到穩(wěn)定株。Western印跡檢測(cè)經(jīng)慢病毒轉(zhuǎn)染鱗癌Sa3細(xì)胞株Rab23的表達(dá)水平。

3.平板克隆實(shí)驗(yàn)檢測(cè)Rab23對(duì)Sa3細(xì)胞增殖能力的影響:無(wú)血清培養(yǎng)穩(wěn)轉(zhuǎn)細(xì)胞24 h后,常規(guī)消化離心。細(xì)胞計(jì)數(shù),調(diào)整為104個(gè)/ml細(xì)胞懸液。取200 μl細(xì)胞懸液接種6孔板,補(bǔ)加培養(yǎng)液至8 ml,放入孵箱常規(guī)培養(yǎng)2周。當(dāng)出現(xiàn)肉眼可見(jiàn)克隆時(shí)棄培養(yǎng)液。磷酸鹽緩沖液(PBS)洗滌,甲醇1 ml固定15 min,棄去固定液,加入結(jié)晶紫染液1 ml染色15 min,自來(lái)水沖洗,空氣干燥。計(jì)數(shù)肉眼可見(jiàn)克隆數(shù),計(jì)算克隆形成率?？寺⌒纬陕?(克隆數(shù)/接種細(xì)胞數(shù))×100%。

4.流式細(xì)胞儀檢測(cè)Rab23對(duì)Sa3細(xì)胞增殖能力的影響:取同步化的穩(wěn)轉(zhuǎn)細(xì)胞常規(guī)消化離心,重懸細(xì)胞沉淀,調(diào)整細(xì)胞數(shù)為6×106個(gè)/ml,預(yù)冷的PBS洗3次,加入-20℃預(yù)冷的75%乙醇吹打均勻固定。檢測(cè)前,PBS洗滌2次,沉淀重懸于200 μl PBS,加入Rnase A 37℃水浴1 h,再加入0.5%碘化丙錠染色,4℃避光30 min,上流式細(xì)胞儀檢測(cè):在488 nm激發(fā)波長(zhǎng)下測(cè)定細(xì)胞各周期DNA含量,并計(jì)算增殖指數(shù)(PI)。PI=S期與G2期細(xì)胞比例之和/G1期和S期及G2期細(xì)胞比例之和。重復(fù)3次。

5.Western印跡檢測(cè)Sa3細(xì)胞Erk、磷酸化Erk表達(dá)水平:常規(guī)培養(yǎng)穩(wěn)轉(zhuǎn)細(xì)胞,傳至3代以上,收集細(xì)胞,加入RIPA蛋白裂解液,提取細(xì)胞總蛋白,使用BCA蛋白定量試劑盒進(jìn)行蛋白定量;每泳道按30 μg上樣進(jìn)行蛋白電泳;轉(zhuǎn)0.45 μm硝酸纖維素(NC)膜,恒流300 mA,25 min;5%牛血清白蛋白(BSA)封閉液封閉1 h,加1∶10 000稀釋的兔Erk1/2、p-Erk1/2單克隆抗體和1∶2 000稀釋的鼠β肌動(dòng)蛋白單克隆抗體4℃孵育過(guò)夜,1×TBST(TrisbufferedsalineandTween20)緩沖液洗膜,每次15 min,共3次,加1∶2 000稀釋的辣根過(guò)氧化物酶(HRP)-鼠抗人、兔抗人抗體,37℃孵育1 h,1×TBST緩沖液洗膜,使用ECL化學(xué)發(fā)光試劑盒進(jìn)行發(fā)光。

6.統(tǒng)計(jì)學(xué)處理:采用GraphPad Prism 5.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。數(shù)據(jù)采用±s表示,兩組間均數(shù)比較使用t檢驗(yàn),多組間均數(shù)比較使用單因素方差分析,而多組資料兩兩比較使用Bonferroni多重比較分析。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1.建立穩(wěn)定過(guò)表達(dá)或沉默Rab23的鱗癌Sa3細(xì)胞:倒置熒光顯微鏡觀察,轉(zhuǎn)染細(xì)胞內(nèi)可見(jiàn)eGFP熒光,正常對(duì)照組、Rab23沉默組、空載體組Rab23分布于胞質(zhì)中;Rab23過(guò)表達(dá)組Rab23分布于細(xì)胞膜上。Western印跡檢測(cè)Rab23過(guò)表達(dá)組、Rab23沉默組轉(zhuǎn)染成功,Rab23沉默組Rab23的表達(dá)與空載體組相比下降。見(jiàn)圖1。光鏡下觀察穩(wěn)轉(zhuǎn)后各組細(xì)胞形態(tài),發(fā)現(xiàn)與未做任何處理的Sa3細(xì)胞相比未發(fā)生明顯變化。

圖1 Western印跡檢測(cè)Sa3細(xì)胞Rab23表達(dá)水平 過(guò)表達(dá)Rab23組融合蛋白轉(zhuǎn)染成功,Rab23沉默組較空載體組相比,Rab23表達(dá)水平降低

表1 Rab23對(duì)Sa3細(xì)胞周期的影響(±s)

表1 Rab23對(duì)Sa3細(xì)胞周期的影響(±s)

注:n=3

組別 細(xì)胞周期(%)G0/G1 G2/M S正常對(duì)照組 30.17±1.83 20.46±0.31 49.37±1.78 Rab23過(guò)表達(dá)組 41.91±3.55 16.90±0.99 41.19±3.22空載體組 55.60±1.36 21.37±1.62 22.83±0.60 Rab23沉默組 43.27±1.48 24.07±1.82 32.66±0.47

2.平板克隆形成實(shí)驗(yàn)證實(shí)Rab23抑制鱗癌Sa3細(xì)胞增殖:Rab23過(guò)表達(dá)組克隆形成率(2.3%±0.2%)較正常對(duì)照組(3.6%±0.3%)降低(t=4.02,P＜0.05),Rab23沉默組(4.1%±0.2%)較空載體組(1.8%±0.0%)明顯增高(t=10.00,P＜0.01)。提示Rab23對(duì)Sa3細(xì)胞增殖具有抑制作用。見(jiàn)圖2。

3.流式細(xì)胞儀檢測(cè)顯示Rab23抑制鱗癌Sa3細(xì)胞的增殖:見(jiàn)表1。Rab23過(guò)表達(dá)組細(xì)胞增殖指數(shù)(0.581±0.035)較正常對(duì)照組(0.698±0.018)降低(t=2.94,P＜0.05),穩(wěn)定轉(zhuǎn)染Rab23沉默組(0.567±0.015)較空載體組(0.444±0.014)顯著增高(t=6.12,P＜0.01)。表明過(guò)表達(dá)Rab23可引起Sa3細(xì)胞G1期阻滯,影響細(xì)胞周期進(jìn)程。

4.Sa3細(xì)胞Erk/p-Erk表達(dá)水平:Rab23過(guò)表達(dá)和Rab23沉默的Sa3細(xì)胞Erk蛋白水平較正常對(duì)照組和空載體組均無(wú)明顯變化。與正常對(duì)照組相比,Rab23過(guò)表達(dá)的Sa3細(xì)胞p-Erk蛋白水平降低,而Rab23沉默的Sa3細(xì)胞p-Erk蛋白水平較空載體組明顯增高。見(jiàn)圖3。

圖3 Western印跡檢測(cè)Erk/p-Erk表達(dá)水平

圖2 平板克隆實(shí)驗(yàn)檢測(cè)Rab23對(duì)Sa3細(xì)胞增殖的影響 Rab23過(guò)表達(dá)組克隆形成率較正常對(duì)照組降低,Rab23沉默組較空載體組增高

討 論

Eggenschwiler等[9]發(fā)現(xiàn),Rab23是Sonic Hedgehog(Hh)信號(hào)通路重要的負(fù)調(diào)控因子,Rab23作用位點(diǎn)在Hh信號(hào)通路的SMO和Gli分子之間,通過(guò)組織SMO分子進(jìn)入纖毛而負(fù)調(diào)控Hh信號(hào)通路[10]。研究發(fā)現(xiàn),在甲狀腺癌惡性類(lèi)型中Rab23的mRNA水平低于濾泡狀腺瘤[11];在乳腺癌細(xì)胞中,過(guò)表達(dá)Rab23可降低Hh信號(hào)通路的激活狀態(tài),并且抑制乳腺癌細(xì)胞的增殖[7]。而隨著研究的廣泛,發(fā)現(xiàn)Rab23在不同腫瘤細(xì)胞中對(duì)生長(zhǎng)進(jìn)程的影響并不一致,甚至呈相反的作用。在胃癌組織中,與非轉(zhuǎn)移型胃癌細(xì)胞相比,Rab23顯著高表達(dá)于轉(zhuǎn)移型胃癌細(xì)胞,研究者通過(guò)比較基因組雜交和基因表達(dá)分析將Rab23確定為胃癌侵襲介導(dǎo)因素;在肝癌組織中,Rab23表現(xiàn)為核陽(yáng)性,其陽(yáng)性率與腫瘤尺寸大小成正比,RNA干涉Rab23表達(dá)后癌細(xì)胞增殖能力下降,說(shuō)明Rab23可促進(jìn)肝癌細(xì)胞的增殖[6]。

侵襲性研究表明,Rab23促進(jìn)鱗癌細(xì)胞的侵襲[8]。與此同時(shí),Rab23對(duì)鱗癌細(xì)胞生長(zhǎng)增殖的作用尚不清楚,因此我們首先通過(guò)慢病毒感染,構(gòu)建穩(wěn)定過(guò)表達(dá)、敲除Rab23的鱗癌Sa3細(xì)胞株。細(xì)胞周期檢測(cè)發(fā)現(xiàn),Rab23可以引起鱗癌Sa3細(xì)胞G1期阻滯而阻礙細(xì)胞周期進(jìn)程;平板克隆形成實(shí)驗(yàn)發(fā)現(xiàn),過(guò)表達(dá)Rab23組與對(duì)照組相比,Sa3細(xì)胞增殖水平降低,而敲除Rab23的Sa3細(xì)胞增殖能力提高,表明Rab23抑制鱗癌Sa3細(xì)胞增殖,這與Rab23對(duì)乳腺癌細(xì)胞生長(zhǎng)的作用一致。

目前研究已明確,Hh信號(hào)通路過(guò)度激活可引發(fā)多種腫瘤。研究者通過(guò)免疫組化和原位雜交的方法,已明確在口腔和咽鱗癌[12]、肺鱗癌[5]組織、細(xì)胞中發(fā)現(xiàn)Hh信號(hào)通路的激活,并參與腫瘤的侵襲和生長(zhǎng)。MAPK/Erk信號(hào)級(jí)聯(lián)反應(yīng)也是影響多種癌細(xì)胞生長(zhǎng)增殖的重要信號(hào)通路,可被表皮生長(zhǎng)因子受體(EGFR)、整合素(integrins)、離子通道等激活。在多種上皮源性惡性腫瘤如頭頸部鱗癌細(xì)胞中,均有EGFR表達(dá)異常,并在鱗癌細(xì)胞的生長(zhǎng)、侵襲、血管形成中起關(guān)鍵作用[13]。Morozevich等[14]發(fā)現(xiàn),在皮膚鱗癌細(xì)胞中,EGFR-Erk通路的激活可促進(jìn)鱗癌細(xì)胞增殖。而最近研究發(fā)現(xiàn),在人髓母細(xì)胞瘤細(xì)胞中,Hh信號(hào)通路和EGFR信號(hào)通路存在交互作用[15]。我們初步研究發(fā)現(xiàn),過(guò)表達(dá)Rab23的Sa3細(xì)胞,p-Erk表達(dá)水平較對(duì)照組降低;敲除Rab23的Sa3細(xì)胞,p-Erk表達(dá)水平較對(duì)照組升高,提示Rab23抑制鱗癌Sa3細(xì)胞增殖的機(jī)制可能與MAPK/Erk通路相關(guān)。而作為Hh信號(hào)通路必要的負(fù)調(diào)控因子,Rab23對(duì)鱗癌細(xì)胞增殖的抑制作用是單純通過(guò)MAPK/Erk通路還是同時(shí)通過(guò)Hh與EGFR信號(hào)通路有待進(jìn)一步研究。

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2014-01-16)

(本文編輯:尚淑賢)

Inhibitory effect of Rab23 on the proliferation of a squamous cell carcinoma cell line Sa3 and its mechanisms

Liu Xilin,Jian Qiang,Miao Ye,Huang Min,Li Chengxin.Department of Dermatology,Xijing Hospital,Fourth Military Medical University,Xi'an 710032,China

Li Chengxin,Email:chengxinderm@163.com

ObjectiveTo evaluate the effect of Rab23 on the proliferation of a squamous cell carcinoma cell line Sa3,and to investigate its mechanisms.MethodsCultured Sa3 cells were classified into four groups:normal control group transfected with green fluorescent protein(GFP),Rab23-overexpressing group transfected with a GFP-labelled Rab23-overexpressing plasmid,Rab23-silencing group transfected with a plasmid carrying a Rab23-targeting shRNA,empty vector group transfected with an empty vector.After additional culture for different durations,plate colony formation assay and flow cytometry were performed to evaluate the proliferative activity of Sa3 cells,and Western blot was conducted to detect the expression of Erk/phosphorylated-Erk in Sa3 cells.Statistical analysis was carried out by t test,one-way analysis of variance and Bonferroni's multiple comparison test.ResultsStable Sa3 cell lines with overexpression or silencing of Rab23 were established by plasmid construction andlentivirus-mediated transfection.The plate colony formation assay showed that the colony formation rate was significantly lower in the Rab23-overexpressing group than in the normal control group(2.3%±0.2%vs.3.6%±0.3%,P＜0.05),but higher in the Rab23-silencing group than in the empty vector group(4.1%±0.2%vs.1.8%±0.03%,P＜0.01).Rab23 overexpression induced G1 phase arrest in Sa3 cells.The proliferation index was significantly decreased in the Rab23-overexpressing group compared with the normal control group(0.581±0.035 vs.0.698±0.018,P＜0.05),but increased in the Rab23-silencing group compared with the empty vector group(0.567±0.015 vs.0.444±0.014,P＜0.01).As Western blot showed,there were no significant changes in the expression of Erk in the Rab23-silencing or-overexpressing group compared with the normal control group,whereas the expression of p-Erk was attenuated in the Rab23-overexpressing group compared with the normal control group,but enhanced in the Rab23-silencing group compared with the empty vector group.ConclusionsRab23 could inhibit the proliferation of Sa3 cells,which may be associated with the Erk pathway.

Rab23;Lentivirus infections;Carcinoma,squamous cell;Cell line,tumor;Cell proliferation

10.3760/cma.j.issn.0412-4030.2014.07.014

710032西安,第四軍醫(yī)大學(xué)西京皮膚醫(yī)院

李承新,Email:chengxinderm@163.com