SCD-1在宮頸癌中的表達(dá)及其表達(dá)下調(diào)對(duì)宮頸癌細(xì)胞增殖和凋亡的影響*

楊 君， 張彩鳳， 秦瑞英， 任艷芳， 王慧玲， 華方方

(新鄉(xiāng)醫(yī)學(xué)院第一附屬醫(yī)院 1婦產(chǎn)科， 2消化科，河南 新鄉(xiāng) 453100)

SCD-1在宮頸癌中的表達(dá)及其表達(dá)下調(diào)對(duì)宮頸癌細(xì)胞增殖和凋亡的影響*

楊 君1△， 張彩鳳2， 秦瑞英1， 任艷芳1， 王慧玲1， 華方方1

(新鄉(xiāng)醫(yī)學(xué)院第一附屬醫(yī)院1婦產(chǎn)科，2消化科，河南 新鄉(xiāng) 453100)

目的檢測(cè)正常宮頸組織、宮頸鱗癌組織以及宮頸癌細(xì)胞株HeLa、SiHa和CaSki中?；o酶A去飽和酶1(stearoyl-CoA desaturase-1，SCD-1)蛋白的表達(dá)，分析SCD-1表達(dá)下調(diào)對(duì)宮頸癌細(xì)胞增殖和凋亡的影響。方法采用Western blotting檢測(cè)正常宮頸組織、宮頸鱗癌組織以及宮頸癌細(xì)胞株HeLa、SiHa和CaSki中SCD-1蛋白的表達(dá)。利用脂質(zhì)體2000將SCD-1 siRNA和對(duì)照siRNA轉(zhuǎn)染CaSki細(xì)胞，采用Western blotting檢測(cè)轉(zhuǎn)染SCD-1 siRNA后CaSki細(xì)胞中SCD-1蛋白的表達(dá)。進(jìn)一步利用CCK-8試劑和流式細(xì)胞術(shù)檢測(cè)轉(zhuǎn)染后CaSki細(xì)胞增殖和細(xì)胞凋亡的變化，并利用Caspase-Glo?3/7和9檢測(cè)試劑盒檢測(cè)轉(zhuǎn)染后caspase-3和caspase-9活性的變化，采用Western blotting檢測(cè)Bcl-2和Bax蛋白的表達(dá)。結(jié)果宮頸鱗癌組織中SCD-1蛋白的表達(dá)顯著高于其對(duì)應(yīng)的正常宮頸組織(P<0.05)，而3株宮頸癌細(xì)胞中SCD-1蛋白表達(dá)也顯著高于正常宮頸組織，其中CaSki細(xì)胞中SCD-1蛋白表達(dá)最高(P<0.05)。此外，SCD-1 siRNA組中CaSki細(xì)胞中SCD-1蛋白的表達(dá)顯著低于未處理組和對(duì)照siRNA組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。與未處理組和對(duì)照siRNA組相比，SCD-1 siRNA組中CaSki細(xì)胞的增殖顯著受到抑制(P<0.05)。SCD-1 siRNA組中CaSki細(xì)胞的早期凋亡率顯著高于未處理組和對(duì)照siRNA組(P<0.05)。進(jìn)一步發(fā)現(xiàn)SCD-1 siRNA轉(zhuǎn)染后caspase-3、caspase-9活性和Bax蛋白表達(dá)顯著上升，而B(niǎo)cl-2蛋白表達(dá)顯著下調(diào)(P<0.05)。結(jié)論SCD-1可能在宮頸癌發(fā)生發(fā)展中發(fā)揮重要作用，其表達(dá)下調(diào)介導(dǎo)的細(xì)胞增殖抑制和細(xì)胞凋亡可能與caspase-3、caspase-9活性以及Bcl-2和Bax表達(dá)變化密切相關(guān)。

酰基輔酶A去飽和酶1； 宮頸腫瘤； 細(xì)胞增殖； 細(xì)胞凋亡

腫瘤細(xì)胞能夠增加糖酵解和脂肪合成從而改變其自身的代謝[1]。持續(xù)增殖的腫瘤細(xì)胞不僅在脂質(zhì)從頭生物合成中具有量的改變，而且能修飾脂質(zhì)膜構(gòu)成發(fā)生質(zhì)的改變進(jìn)而影響膜的流動(dòng)性、信號(hào)轉(zhuǎn)導(dǎo)和基因表達(dá)[2]。從轉(zhuǎn)化細(xì)胞的脂質(zhì)分析表明飽和脂肪酸(saturated fatty acid，SFA)向單不飽和脂肪酸(monounsaturated fatty acid，MUFA)的量逐漸增加[3]，?；o酶A去飽和酶1(stearoyl-CoA desaturase-1，SCD-1)是Δ9脂肪酸去飽和酶異構(gòu)體，能將SFA轉(zhuǎn)換為MUFA[4]，提示其可能在腫瘤的脂質(zhì)代謝過(guò)程中發(fā)揮極其重要的作用。研究顯示SCD-1在遺傳上易于發(fā)生肝癌的鼠模型中過(guò)表達(dá)，表明SCD-1可能與腫瘤發(fā)生密切相關(guān)[5]。最近，越來(lái)越多的研究顯示，SCD-1在多種不同的腫瘤包括食管癌、結(jié)腸癌、肝癌以及化學(xué)誘導(dǎo)的乳腺腫瘤中均呈現(xiàn)過(guò)表達(dá)[3, 6-8]，但在前列腺癌中呈現(xiàn)低表達(dá)[9]，提示SCD-1表達(dá)水平可能具有腫瘤類型依賴性。最近，SCD-1在RNAi篩選中被鑒定為腫瘤治療的潛在分子靶點(diǎn)[10]。我們通過(guò)文獻(xiàn)檢索發(fā)現(xiàn)，迄今為止，關(guān)于SCD-1與宮頸癌發(fā)生發(fā)展之間的關(guān)系尚未見(jiàn)報(bào)道。因此在本研究中，我們檢測(cè)宮頸癌細(xì)胞和組織中SCD-1蛋白的表達(dá)，研究其表達(dá)下調(diào)對(duì)宮頸癌細(xì)胞增殖和凋亡的影響，并分析與細(xì)胞凋亡密切相關(guān)的caspase-3和caspase-9的活性，檢測(cè)抑凋亡蛋白Bcl-2和促凋亡蛋白Bax的表達(dá)，初步闡明SCD-1在宮頸癌發(fā)生發(fā)展中可能的作用，為以SCD-1為靶點(diǎn)的宮頸癌分子靶向治療提供理論依據(jù)。

材 料 和 方 法

1材料

宮頸鱗癌組織和正常宮頸組織均通過(guò)病理學(xué)證實(shí)，取自新鄉(xiāng)醫(yī)學(xué)院第一附屬醫(yī)院，并經(jīng)液氮保存?zhèn)溆?。宮頸癌細(xì)胞株HeLa、SiHa和CaSki購(gòu)自中國(guó)典型培養(yǎng)物保藏中心。DMEM培養(yǎng)液、胰酶和胎牛血清均購(gòu)自Gibco；凋亡檢測(cè)試劑盒Annexin V-FITC Kit購(gòu)自Beckman Coulter；兔抗人SCD-1和β-actin抗體、鼠抗人Bcl-2和Bax抗體、SCD-1 siRNA和對(duì)照siRNA均購(gòu)自Santa Cruz；細(xì)胞裂解液購(gòu)自寶生物工程(大連)有限公司；Caspase-Glo?3/7和9試劑盒購(gòu)自Promega。

2細(xì)胞培養(yǎng)、轉(zhuǎn)染及實(shí)驗(yàn)分組

HeLa、SiHa和CaSki細(xì)胞復(fù)蘇后置于含10 %胎牛血清的DMEM培養(yǎng)液中培養(yǎng)，當(dāng)細(xì)胞傳代到狀態(tài)穩(wěn)定后，采用Western blotting法檢測(cè)隨機(jī)選取的2例正常宮頸組織、2例宮頸鱗癌組織和3株宮頸癌細(xì)胞中SCD-1蛋白的表達(dá)。將上述SCD-1表達(dá)量最高的CaSki細(xì)胞培養(yǎng)至細(xì)胞融合度為90%左右時(shí)，將SCD-1 siRNA和對(duì)照siRNA采用脂質(zhì)體介導(dǎo)法轉(zhuǎn)染至CaSki細(xì)胞，轉(zhuǎn)染后將細(xì)胞分為3組進(jìn)行相關(guān)實(shí)驗(yàn)：未處理組(不作任何處理)、對(duì)照siRNA組(利用對(duì)照siRNA轉(zhuǎn)染)和SCD-1 siRNA組(采用SCD-1 siRNA轉(zhuǎn)染)。

3細(xì)胞增殖分析

分別收集轉(zhuǎn)染后24 h、48 h、72 h及96 h的3組宮頸癌CaSki細(xì)胞，并將每孔2 000個(gè)細(xì)胞接種于96孔板，每個(gè)時(shí)點(diǎn)設(shè)置5個(gè)復(fù)孔，當(dāng)測(cè)定生長(zhǎng)速率時(shí)，加入含10% CCK-8的等量新鮮培養(yǎng)基于37 ℃繼續(xù)培養(yǎng)2 h，最后采用酶標(biāo)儀測(cè)定450 nm的吸光度。

4流式細(xì)胞術(shù)檢測(cè)宮頸癌CaSki細(xì)胞凋亡

收集轉(zhuǎn)染后48 h的3組宮頸癌CaSki細(xì)胞，于預(yù)冷PBS中洗滌。每組1×109/L的細(xì)胞重懸，取100 μL細(xì)胞置于試管中，加入Annexin V-FITC和碘化丙啶(propidium iodide，PI)各5 μL，避光孵育15 min后，采用流式細(xì)胞術(shù)檢測(cè)1×104個(gè)細(xì)胞，并利用CellQuest軟件分析結(jié)果。

5Caspase-3和caspase-9的活性分析

采用caspase-Glo?3/7和9檢測(cè)試劑盒分析3組宮頸癌CaSki細(xì)胞中caspase-3和caspase-9的活性。SCD-1 siRNA和對(duì)照siRNA轉(zhuǎn)染后于48 h收獲3組宮頸癌CaSki細(xì)胞，分別置于緩沖液(25 mmol/L HEPES,pH 7.5，5 mmol/L MgCl2，1 mmol/L EGTA，1 mmol/L Pefabloc?絲氨酸蛋白酶抑制劑，胃酶抑素、亮肽素和抑肽酶各1 mg/L)中充分懸浮后，于4 ℃、13 000 r/min離心15 min，澄清提取液。將澄清的提取液中的蛋白濃度預(yù)先調(diào)整為1 g/L，于-80 ℃下凍存。為了測(cè)定caspase活性，將稀釋后的提取液(10 mg/L)與caspase-Glo?檢測(cè)試劑等體積混合，置于96孔白壁培養(yǎng)板中，室溫孵育1 h，然后采用發(fā)光檢測(cè)儀獲取數(shù)據(jù)。

6Westernblotting檢測(cè)

Western blotting采用吳裕丹等[11]報(bào)道的方法。收集轉(zhuǎn)染后48 h的3組宮頸癌CaSki細(xì)胞，利用細(xì)胞裂解液提取總蛋白，并采用Bradford法測(cè)定各組蛋白濃度。將50 μg總蛋白在上樣緩沖液中煮5～10 min，采用10% SDS-PAGE進(jìn)行蛋白分離，接著將凝膠上的蛋白轉(zhuǎn)移至硝酸纖維素(nitrocellulose,NC)膜上。將NC膜用含5%脫脂奶粉的TBST于室溫封閉2 h，分別加Ⅰ抗(SCD-1、Bcl-2、Bax和β-actin;按1∶200稀釋)于含5%脫脂奶粉的TBST中4 ℃過(guò)夜孵育，接著用TBST洗膜3次，每次5 min，洗膜結(jié)束后加Ⅱ抗室溫孵育2 h，然后曝光成像。最后用Gene Tools軟件分析蛋白相對(duì)表達(dá)量(目的蛋白表達(dá)量與內(nèi)參照β-actin表達(dá)量的比值)。

7統(tǒng)計(jì)學(xué)處理

采用統(tǒng)計(jì)學(xué)軟件SPSS 13.0分析，數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，3組之間比較采用單因素方差分析(One-way ANOVA)，以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1SCD-1在宮頸癌組織和細(xì)胞中的表達(dá)

用Western blotting方法檢測(cè)宮頸不同組織和細(xì)胞中SCD-1蛋白的表達(dá)，結(jié)果表明，2例宮頸鱗癌組織中SCD-1蛋白表達(dá)均顯著高于2例正常宮頸組織，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；與正常宮頸組織相比，3株宮頸癌細(xì)胞系中SCD-1蛋白表達(dá)均顯著上調(diào)(P<0.05)；在所有檢測(cè)的組織和細(xì)胞中，CaSki細(xì)胞中SCD-1蛋白表達(dá)顯著高于其它各組(P<0.05)，見(jiàn)圖1。因此，在后續(xù)的研究中我們選用CaSki細(xì)胞作為研究對(duì)象。

2SCD-1siRNA抑制宮頸癌CaSki細(xì)胞中SCD-1蛋白的表達(dá)

SCD-1 siRNA組中SCD-1蛋白表達(dá)顯著低于未處理組和對(duì)照siRNA組(P<0.05)，而未處理組與對(duì)照siRNA組之間的SCD-1蛋白表達(dá)無(wú)顯著差異(P>0.05)，見(jiàn)圖2。

Figure 1. Expression of SCD-1 protein in cervical carcinoma tissues and cells detected by Western blotting.N: normal cervical tissues;T: cervical carcinoma tissues. β-actin was utilized as internal control.Mean±SD.n=3.*P<0.05vs1N;△P<0.05vs1N and 2N;#P<0.05vs1N, 2N and 1T;▲P<0.05vs1N, 2N, 1T and 2T;○P<0.05vs1N, 2N, 1T, 2T and HeLa;●P<0.05vsother groups.

圖1SCD-1蛋白在宮頸癌組織和細(xì)胞中的表達(dá)

Figure 2. Effect of SCD-1 siRNA on the protein expression of SCD-1 in cervical carcinoma CaSki cells.Mean±SD.n=3.*P<0.05vsuntreated group and control siRNA group.

圖2SCD-1siRNA對(duì)宮頸癌CaSki細(xì)胞中SCD-1蛋白表達(dá)的影響

3SCD-1表達(dá)下調(diào)抑制宮頸癌CaSki細(xì)胞增殖

收集SCD-1 siRNA和對(duì)照siRNA處理后不同時(shí)間點(diǎn)的3組宮頸癌CaSki細(xì)胞，采用CCK-8檢測(cè)試劑盒分析不同時(shí)間點(diǎn)CaSki細(xì)胞增殖的變化，結(jié)果表明，與未處理組和對(duì)照siRNA組相比，SCD-1 siRNA組中CaSki細(xì)胞在轉(zhuǎn)染后的24 h、48 h、72 h和96 h，其細(xì)胞增殖均受到明顯抑制(P<0.05)，而未處理組和對(duì)照siRNA組之間CaSki細(xì)胞的增殖無(wú)顯著差異(P>0.05)，見(jiàn)圖3。

Figure 3. Effect of down-regulation of SCD-1 on the proliferation in cervical carcinoma CaSki cells. Mean±SD.n=3.*P<0.05vsuntreated group and control siRNA group.

圖3SCD-1表達(dá)下調(diào)對(duì)宮頸癌CaSki細(xì)胞增殖的影響

4SCD-1表達(dá)下調(diào)誘導(dǎo)宮頸癌CaSki細(xì)胞凋亡

SCD-1 siRNA組中CaSki細(xì)胞的早期凋亡率(19.32%±0.93%)顯著高于未處理組(8.25%±0.35%)和對(duì)照siRNA組(8.29%±0.22%)，而未處理組和對(duì)照siRNA組之間CaSki細(xì)胞早期凋亡率無(wú)顯著差異。此外，SCD-1 siRNA組(66.59%±1.28%)中CaSki細(xì)胞的活細(xì)胞率顯著低于未處理組(87.35%±0.89%)和對(duì)照siRNA組(86.87%±0.93%)，而未處理組和對(duì)照siRNA組之間CaSki細(xì)胞的活細(xì)胞率無(wú)顯著差異，見(jiàn)表1。

表1SCD-1表達(dá)下調(diào)對(duì)宮頸癌CaSki細(xì)胞凋亡的影響

Table 1. Effect of down-regulation of SCD-1 on the apoptosis of cervical carcinoma CaSki cells(%.Mean±SD.n=3)

GroupEarly-phaseapoptoticratio ViablecellratioUntreated8.25±0.3587.35±0.89ControlsiRNA8.29±0.2286.87±0.93SCD-1siRNA19.32±0.93*66.59±1.28*

*P<0.05vsuntreated group and control siRNA group.

5SCD-1表達(dá)下調(diào)對(duì)宮頸癌CaSki細(xì)胞caspase-3和caspase-9活性的影響

收集轉(zhuǎn)染后48 h的3組宮頸癌CaSki細(xì)胞，采用Caspase-Glo?3/7和9檢測(cè)試劑盒分析3組細(xì)胞中caspase-3和caspase-9的活性，結(jié)果顯示，與未處理組和對(duì)照siRNA組相比，SCD-1 siRNA組中caspase-3和caspase-9的活性均顯著上升(P<0.05)，而未處理組和對(duì)照siRNA組中caspase-3和caspase-9的活性無(wú)顯著差異(P>0.05)，見(jiàn)圖4。

Figure 4. Down-regulation of SCD-1 significantly enhanced the activities of caspase-3 and caspase-9 in cervical carcinoma CaSki cells.Mean±SD.n=3.*P<0.05vsuntreated group and control siRNA group.

圖4SCD-1表達(dá)下調(diào)顯著提高宮頸癌CaSki細(xì)胞caspase-3和caspase-9的活性

6SCD-1表達(dá)下調(diào)對(duì)宮頸癌CaSki細(xì)胞Bcl-2和Bax蛋白表達(dá)的影響

SCD-1 siRNA組中Bcl-2蛋白的表達(dá)顯著低于未處理組和對(duì)照siRNA組，而B(niǎo)ax蛋白表達(dá)顯著高于未處理組和對(duì)照siRNA組(P<0.05)，而未處理組和對(duì)照siRNA組之間Bcl-2和Bax蛋白的表達(dá)無(wú)顯著差異(P>0.05)，見(jiàn)圖5。

討 論

對(duì)于正常細(xì)胞和腫瘤細(xì)胞的增殖、死亡和衰老的控制是與代謝的調(diào)控密切相關(guān)，特別是脂質(zhì)信號(hào)和結(jié)構(gòu)的合成與重建[12]。SCD-1是脂肪酸合成的關(guān)鍵調(diào)控基因，與腫瘤的發(fā)生發(fā)展關(guān)系十分密切，并牽涉腫瘤發(fā)展的多方面，包括：細(xì)胞增殖、細(xì)胞周期、凋亡、炎癥反應(yīng)和轉(zhuǎn)化等[12]。Falvella等[5]研究發(fā)現(xiàn)SCD-1 mRNA在小鼠和大鼠肝癌中的表達(dá)水平分別是正常肝組織的10倍和4倍，提示SCD-1可能與肝癌發(fā)生發(fā)展關(guān)系密切。Mauvoisin等[13]在研究SCD-1在乳腺癌MCF7和MDA-MB-231細(xì)胞中的功能時(shí)，發(fā)現(xiàn)轉(zhuǎn)染SCD-1 siRNA其表達(dá)水平顯著低于對(duì)照組，并且其表達(dá)下調(diào)能明顯抑制β-catenin信號(hào)途徑，抑制細(xì)胞的增殖和降低細(xì)胞的侵襲能力，提示SCD-1在乳腺癌中可能扮演癌基因的作用。盡管上述研究支持SCD-1在這些腫瘤中作為癌基因發(fā)揮作用，但是Zhang等[14]研究SCD-1在白血病干細(xì)胞中的作用時(shí)，發(fā)現(xiàn)SCD-1在白血病干細(xì)胞中表達(dá)下調(diào)，在白血病干細(xì)胞中發(fā)揮腫瘤抑制的作用，并對(duì)正常造血干細(xì)胞無(wú)影響，進(jìn)一步研究發(fā)現(xiàn)缺失SCD-1能加速慢性髓細(xì)胞樣白血病的進(jìn)展，相反SCD-1過(guò)表達(dá)延遲慢性髓細(xì)胞樣白血病的進(jìn)展，這一研究提示SCD-1在白血病中發(fā)揮抑癌基因的作用。SCD-1在宮頸癌中的作用如何？目前尚不清楚，因此在本研究中，我們檢測(cè)了宮頸鱗癌組織和細(xì)胞中SCD-1蛋白的表達(dá)，發(fā)現(xiàn)SCD-1蛋白在隨機(jī)選擇的2例宮頸鱗癌組織中比其相應(yīng)的正常宮頸組織顯著升高(P<0.05)，并且3株宮頸癌細(xì)胞HeLa、SiHa和CaSki中SCD-1蛋白的表達(dá)也明顯高于其它組織中SCD-1蛋白的表達(dá)，且CaSki細(xì)胞中SCD-1蛋白水平最高(P<0.05)，我們這里呈現(xiàn)的結(jié)果初步提示SCD-1可能在宮頸癌的發(fā)生發(fā)展過(guò)程中發(fā)揮重要作用，但其確切的表達(dá)模式尚需進(jìn)一步探討。

Figure 5. Effect of down-regulation of SCD-1 on the protein expression of Bcl-2 and Bax in cervical carcinoma CaSki cells.Mean±SD.n=3.*P<0.05vsuntreated group and control siRNA group.

圖5SCD-1表達(dá)下調(diào)對(duì)宮頸癌CaSki細(xì)胞中Bcl-2和Bax蛋白表達(dá)的影響

活化的脂肪生成，尤其是膜脂的合成對(duì)于腫瘤細(xì)胞的連續(xù)增殖起關(guān)鍵的調(diào)控作用，是避免腫瘤細(xì)胞進(jìn)行凋亡程序的關(guān)鍵機(jī)制[15]。已有的研究結(jié)果顯示SCD-1能調(diào)控腫瘤細(xì)胞的增殖和存活，通過(guò)siRNA沉默SCD-1能顯著降低多種人類腫瘤細(xì)胞系的存活[10]。在本研究中，我們首先通過(guò)SCD-1 siRNA轉(zhuǎn)染宮頸癌CaSki細(xì)胞，結(jié)果發(fā)現(xiàn)，與未處理組和對(duì)照siRNA組相比，SCD-1 siRNA能顯著下調(diào)宮頸癌CaSki細(xì)胞中SCD-1蛋白水平(P<0.05)。進(jìn)一步我們采用CCK-8試劑分析SCD-1表達(dá)下調(diào)對(duì)宮頸癌CaSki細(xì)胞增殖的影響，結(jié)果顯示，與未處理組和對(duì)照siRNA組相比，SCD-1 siRNA組中宮頸癌CaSki細(xì)胞的增殖明顯受到抑制，這一結(jié)果提示SCD-1能調(diào)控宮頸癌細(xì)胞的增殖，但其確切的分子機(jī)制尚待進(jìn)一步鑒定。

研究表明，抑制SCD-1表達(dá)能顯著增加腫瘤細(xì)胞的凋亡[16]，提示腫瘤細(xì)胞的存活需要適量的SCD-1水平去維持。在多種不同類型的腫瘤中，抑制SCD-1的表達(dá)能夠?qū)е录?xì)胞凋亡[10]。Minville-Walz等[17]發(fā)現(xiàn)，在人骨肉瘤U2OS細(xì)胞中抑制SCD-1的表達(dá)能誘導(dǎo)高水平的caspase-3活性，從而促進(jìn)腫瘤細(xì)胞凋亡。為了進(jìn)一步探討是否抑制宮頸癌細(xì)胞中SCD-1的表達(dá)能誘導(dǎo)細(xì)胞凋亡？在本研究中，我們采用流式細(xì)胞術(shù)分析SCD-1 siRNA處理前后宮頸癌CaSki細(xì)胞凋亡水平，結(jié)果表明，SCD-1 siRNA組中細(xì)胞早期凋亡數(shù)顯著高于未處理組和對(duì)照siRNA組，而活細(xì)胞數(shù)顯著低于未處理組和對(duì)照siRNA組(P<0.05)，提示SCD-1表達(dá)下調(diào)能明顯誘導(dǎo)宮頸癌細(xì)胞發(fā)生凋亡。進(jìn)一步分析與細(xì)胞凋亡密切相關(guān)的caspase-3、caspase-9、Bcl-2和Bax蛋白的活性和表達(dá)水平，結(jié)果發(fā)現(xiàn)SCD-1表達(dá)下調(diào)能顯著提升caspase-3和caspase-9的活性，增加Bax蛋白的表達(dá)水平，但降低Bcl-2蛋白的水平，這一結(jié)果提示SCD-1表達(dá)下調(diào)介導(dǎo)的細(xì)胞凋亡可能與caspase-3和caspase-9活性的升高、Bax蛋白表達(dá)上調(diào)以及Bcl-2表達(dá)下調(diào)密切相關(guān)。

總之，我們的結(jié)果顯示，SCD-1表達(dá)下調(diào)能顯著抑制宮頸癌細(xì)胞增殖和誘導(dǎo)細(xì)胞凋亡，其凋亡發(fā)生可能與凋亡相關(guān)蛋白表達(dá)變化密切相關(guān)，未來(lái)進(jìn)一步研究SCD-1在宮頸癌發(fā)生發(fā)展中的分子機(jī)制有望為宮頸癌的分子靶向治療提供新的實(shí)驗(yàn)依據(jù)。

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ExpressionofSCD-1incervicalcarcinomaandeffectofitsdown-regulationonproliferationandapoptosisofcervicalcarcinomacells

YANG Jun1, ZHANG Cai-feng2, QIN Rui-ying1, REN Yan-fang1, WANG Hui-ling1, HUA Fang-fang1

(1DepartmentofObstetricsandGynecology,2DepartmentofGastroenterology,theFirstAffiliatedHospitalofXinxiangMedicalUniversity,Xinxiang453100,China.E-mail:wylyangjun888@126.com)

AIM: To examine the expression of stearoyl-CoA desaturase-1 (SCD-1) in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki, and to investigate the effect of down-regulation of SCD-1 on the proliferation and apoptosis of cervical carcinoma cells.METHODSThe expression of SCD-1 was detected by Western blotting in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki. SCD-1 siRNA and control siRNA were utilized to transfect CaSki cells by Lipofectamine 2000, and SCD-1 protein level was determined by Western blotting after transfection. Furthermore, CCK-8 and flow cytometry were utilized to investigate the changes of cell proliferation and apoptosis after transfection with SCD-1 siRNA in CaSki cells. Subsequently, the activities of caspase-3 and caspase-9 were analyzed by Caspase-Glo?3/7 and 9 detection kit after transfection with SCD-1 siRNA in CaSki cells. Finally, the protein expression of Bcl-2 and Bax was detected by Western blotting.RESULTSThe protein expression of SCD-1 in cervical squamous cell carcinoma tissues was significantly higher than that in normal cervical tissues, and the protein expression of SCD-1 in the 3 cervical carcinoma cell lines was obviously higher than that in normal cervical tissues, in which CaSki cells displayed the highest SCD-1 protein level. In addition, the protein expression of SCD-1 in SCD-1 siRNA group was significantly lower than that in untreated group and control siRNA group. Compared with untreated group and control siRNA group, the proliferation of CaSki cells was markedly inhibited in SCD-1 siRNA group. Early apoptotic rate in SCD-1 siRNA group was evidently higher than that in untreated group and control siRNA group. The activities of caspase-3 and caspase-9, and the level of Bax protein were significantly elevated, and the protein level of Bcl-2 was obviously reduced after transfection with SCD-1 siRNA in CaSki cells.CONCLUSIONSCD-1 may play an important role in the occurrence and development of cervical carcinoma, and its down-regulation, which mediates cell proliferation inhibition and apoptosis, may be tightly associated with the activities of caspase-3 and caspase-9, and the protein expression of Bcl-2 and Bax.

Stearoyl-CoA desaturase-1; Uterine cervical neoplasms; Cell proliferation; Apoptosis

R363

A

10.3969/j.issn.1000- 4718.2013.11.010

1000- 4718(2013)11- 1972- 06

2013- 07- 01

2013- 10- 08

河南省人口和計(jì)劃生育科學(xué)技術(shù)研究課題(No. 20121026)

△通訊作者 Tel： 0373-4402424； E-mail： wylyangjun888@163.com