沉默GOLPH3基因?qū)ξ赴┘?xì)胞增殖、凋亡的影響及機(jī)制探討

謝芳，賴銘裕，農(nóng)云翠，蘇婷

(1 廣西醫(yī)科大學(xué)第一附屬醫(yī)院，南寧530021；2 廣西民族醫(yī)院；3南寧市第二人民醫(yī)院)

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沉默GOLPH3基因?qū)ξ赴┘?xì)胞增殖、凋亡的影響及機(jī)制探討

謝芳1，賴銘裕1，農(nóng)云翠2，蘇婷3

(1 廣西醫(yī)科大學(xué)第一附屬醫(yī)院，南寧530021；2 廣西民族醫(yī)院；3南寧市第二人民醫(yī)院)

摘要：目的觀察特異性沉默高爾基體磷蛋白3(GOLPH3)基因?qū)ξ赴┘?xì)胞增殖、凋亡的影響，并探討其機(jī)制。方法將人胃癌SGC-7901細(xì)胞隨機(jī)分為3組，A、B組分別轉(zhuǎn)染LV-GOLPH3-RNAi-1、無(wú)義鏈，C組不轉(zhuǎn)染。轉(zhuǎn)染72 h后，采用MTT法檢測(cè)細(xì)胞增殖能力，流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡，Western blot法檢測(cè)細(xì)胞GOLPH3、磷酸化哺乳動(dòng)物雷帕霉素靶蛋白(p-mTOR)和mTOR。結(jié)果A組細(xì)胞增殖的OD值較B、C組低，細(xì)胞凋亡率(2.70%±0.11%)高于B組(0.42%±0.02%)和C組(0.31%±0.02%)，細(xì)胞GOLPH3、p-mTOR蛋白表達(dá)量較B、C組少，P均<0.05；B、C組間各指標(biāo)比較，P均>0.05。結(jié)論沉默GOLPH3基因能抑制胃癌細(xì)胞增殖、促進(jìn)胃癌細(xì)胞凋亡，其機(jī)制可能與下調(diào)mTOR信號(hào)通路中關(guān)鍵因子p-mTOR表達(dá)有關(guān)。

關(guān)鍵詞：胃癌細(xì)胞；高爾基體磷蛋白3；小干擾核糖核酸；細(xì)胞增殖；細(xì)胞凋亡；哺乳動(dòng)物雷帕霉素靶蛋白

高爾基體磷蛋白3(GOLPH3)是一個(gè)新發(fā)現(xiàn)的癌基因[1]，在多種惡性腫瘤中存在過(guò)量表達(dá)[2～6]，能通過(guò)激活哺乳動(dòng)物雷帕霉素靶蛋白(mTOR)信號(hào)通路促進(jìn)腫瘤發(fā)生。有研究發(fā)現(xiàn)，GOLPH3在胃癌組織中高表達(dá)，提示其在胃癌發(fā)生、發(fā)展中起重要作用，但機(jī)制尚不清楚[7]。2013年3月～2015年1月，我們觀察了特異性沉默GOLPH3基因?qū)ξ赴┘?xì)胞增殖、凋亡的影響，為尋找胃癌的基因靶向治療奠定實(shí)驗(yàn)基礎(chǔ)?，F(xiàn)報(bào)告如下。

1材料與方法

1.1材料人胃癌細(xì)胞株SGC-7901購(gòu)自中國(guó)科學(xué)院上海細(xì)胞所；本實(shí)驗(yàn)室共設(shè)計(jì)3條針對(duì)GOLPH3基因的siRNA序列，選擇其中沉默效率最高的LV-GOLPH3-RNAi-1進(jìn)行實(shí)驗(yàn)；胎牛血清(FBS)購(gòu)自杭州四季青生物工程有限公司；RPMI 1640培養(yǎng)基購(gòu)自Hyclone公司；逆轉(zhuǎn)錄試劑盒及實(shí)時(shí)熒光定量PCR試劑均購(gòu)自日本TaKaRa公司；MTT購(gòu)自北京索來(lái)寶生物科技有限公司；Annexin V-PE/7-AAD凋亡試劑盒購(gòu)自美國(guó)BD公司；GOLPH3兔抗人多克隆抗體購(gòu)自美國(guó)Abcam公司、 mTOR和磷酸化mTOR(p-mTOR)兔抗人單克隆抗體購(gòu)自美國(guó)Cell Signaling Technology公司。

1.2方法

1.2.1細(xì)胞培養(yǎng)與分組處理將SGC-7901置于含10% FBS的RPMI 1640培養(yǎng)基中，37 ℃ 5% CO2條件下培養(yǎng)，常規(guī)更換培養(yǎng)液、傳代。取第5代細(xì)胞，隨機(jī)分為3組。A、B組分別采用Lipofectamine 2000轉(zhuǎn)染LV-GOLPH3-RNAi-1、無(wú)義鏈，C組不轉(zhuǎn)染。A、B組細(xì)胞均轉(zhuǎn)染72 h。

1.2.2細(xì)胞增殖觀察采用MTT法。收集各組細(xì)胞，以5×103/孔接種于96孔板，每組設(shè)5個(gè)復(fù)孔。于接種后24、48、72、96、120、144、168 h在培養(yǎng)基中加入20 μL的 MTT溶液，使MTT終濃度為0.5 mg/mL，置于培養(yǎng)箱中繼續(xù)培養(yǎng)4 h。吸凈上清后，加入等體積DMSO，置于搖床振蕩10 min；酶標(biāo)儀檢測(cè)492 nm波長(zhǎng)處各孔光密度(OD)值，以此表示細(xì)胞增殖能力。

1.2.3細(xì)胞凋亡觀察收集各組細(xì)胞，按照Annexin V-PE/7-AAD凋亡試劑盒使用說(shuō)明書操作，采用流式細(xì)胞術(shù)檢測(cè)凋亡細(xì)胞，計(jì)算細(xì)胞凋亡率。

1.2.4細(xì)胞GOLPH3、mTOR和p-mTOR蛋白檢測(cè)采用Western blot法。收集各組細(xì)胞，用RIPA及PMSF裂解液提取各組細(xì)胞總蛋白；將總蛋白液及上樣緩沖液按5∶1混合，沸水煮5 min變性后制成蛋白質(zhì)樣品；配制5%濃縮膠和10%分離膠，將等量樣品加入凝膠孔道中電泳；100 mA穩(wěn)流狀態(tài)下轉(zhuǎn)膜,將蛋白轉(zhuǎn)至聚偏二氟乙烯膜；用5%蛋白封閉液室溫于搖床上搖動(dòng)封閉1 h，用TBST漂洗后置于一抗(GOLPH3、β-actin均1∶10 000稀釋) 中4 ℃孵育過(guò)夜；再次TBST漂洗后加入稀釋的山羊抗兔二抗(1∶5 000) ，室溫置于搖床上孵育1 h，TBST洗膜(5次×5 min)后進(jìn)行顯影定影。以目的蛋白與β-actin條帶的OD比值表示目的蛋白表達(dá)量。

2結(jié)果

2.1各組細(xì)胞增殖能力比較A組各時(shí)點(diǎn)細(xì)胞增殖較B、C組緩慢(P均<0.05)，而B(niǎo)、C組比較無(wú)統(tǒng)計(jì)學(xué)意義(P均>0.05)。見(jiàn)表1。

表1　各組細(xì)胞增殖能力比較±s)

注：與C組比較，*P<0.05；與B組比較，#P<0.05。2.2各組細(xì)胞凋亡率比較A組細(xì)胞凋亡率(2.70%±0.11%)高于B組(0.42%±0.02%)和C組(0.31%±0.02%)，P均<0.05；B、C組間比較，P>0.05。

2.3各組GOLPH3、mTOR和p-mTOR蛋白表達(dá)比較 見(jiàn)表2。

表2　各組GOLPH3、mTOR和p-mTOR蛋白表達(dá)

注：與C組比較，*P<0.05；與B組比較，#P<0.05。

3討論

GOLPH3蛋白是一種高度保守的高爾基體基質(zhì)蛋白，相對(duì)分子量為34 kD，其編碼基因位于人類染色體5p13區(qū)域上。2000年，Wu等[8]首次發(fā)現(xiàn)位于高爾基體反面網(wǎng)絡(luò)結(jié)構(gòu)(GN)的GOLPH3。GOLPH3與高爾基體正常形態(tài)保持有關(guān)，并參與蛋白質(zhì)的加工、修飾和轉(zhuǎn)運(yùn)：GOLPH3與磷脂酰肌醇-4-磷酸結(jié)合[ptdlns(4)p]，影響高爾基體的分泌和轉(zhuǎn)運(yùn)功能[9]；GOLPH3與糖基酰轉(zhuǎn)移酶的N末端相連接，使蛋白質(zhì)錨定于高爾基體上，從而影響蛋白質(zhì)的糖基化修飾[10,11]；GOLPH3與肌球蛋白MY018A、ptdlns(4)p及細(xì)胞骨架肌動(dòng)蛋白F-actin相連組成復(fù)合體，參與高爾基體正常形態(tài)的保持和囊泡轉(zhuǎn)運(yùn)[12]。有研究者發(fā)現(xiàn)，將橫紋肌肉瘤細(xì)胞中的GOLPH3基因敲除后可抑制橫紋肌肉瘤細(xì)胞的增殖。體外實(shí)驗(yàn)發(fā)現(xiàn)，慢病毒介導(dǎo)的GOLPH3沉默有明顯的抗食管鱗癌作用，是治療食管癌的潛在分子靶點(diǎn)[13]。Hu等[7]研究發(fā)現(xiàn)，GOLPH3表達(dá)與胃癌的腫瘤大小、浸潤(rùn)深度、淋巴結(jié)轉(zhuǎn)移、遠(yuǎn)處轉(zhuǎn)移、病理分級(jí)等顯著相關(guān)，提示GOLPH3過(guò)表達(dá)有可能是胃癌預(yù)后不良的獨(dú)立預(yù)測(cè)指標(biāo)。

RNAi技術(shù)是利用外源性或內(nèi)源性的雙鏈RNA(dsRNA)特異性的將細(xì)胞內(nèi)的同源mRNA降解為21～23個(gè)核苷酸的小片段，從而導(dǎo)致靶基因沉默的技術(shù)，具有特異性、高效性等特點(diǎn)，目前廣泛應(yīng)用于惡性腫瘤的基因治療、病毒感染性疾病等的研究[14]。本研究結(jié)果表明，RNAi沉默GOLPH3表達(dá)能顯著抑制胃癌細(xì)胞SGC-7901的增殖，但機(jī)制尚不清楚。

細(xì)胞增殖、凋亡受多種信號(hào)通路的調(diào)控,信號(hào)傳導(dǎo)系統(tǒng)異?？梢鸺?xì)胞增殖、凋亡改變，甚至惡變[15]。Scott等[1]認(rèn)為，GOLPH3能通過(guò)激活mTOR信號(hào)通路誘導(dǎo)正常細(xì)胞發(fā)生轉(zhuǎn)化，促使細(xì)胞增殖、分化過(guò)程失去平衡，最終導(dǎo)致細(xì)胞惡性轉(zhuǎn)化以及腫瘤發(fā)生，并增加腫瘤細(xì)胞對(duì)雷帕霉素的敏感性。國(guó)內(nèi)有研究發(fā)現(xiàn)，PI3K/mTOR雙重抑制劑PF-04691502能通過(guò)阻滯細(xì)胞周期來(lái)抑制SGC-7901細(xì)胞增殖[16]。本研究結(jié)果表明，抑制GOLPH3基因可下調(diào)mTOR信號(hào)轉(zhuǎn)導(dǎo)通路中關(guān)鍵因子p-mTOR的表達(dá)，GOLPH3可能是通過(guò)mTOR信號(hào)轉(zhuǎn)導(dǎo)通路影響胃癌細(xì)胞的增殖、凋亡。因此，GOLPH3可作為潛在靶點(diǎn)在胃癌早期治療中發(fā)揮重要作用，也為進(jìn)一步闡明GOLPH3與胃癌發(fā)生的可能機(jī)制提供了實(shí)驗(yàn)基礎(chǔ)。

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Effect of silencing GOLPH3 on proliferation and apoptosis of gastric cancer cells

XIEFang1,LAIMingyu,NONGYuncui,SUTing

(1TheFirstAffiliatedHospitalofGuangxiMedicalUniversity,Nanning530021,China)

Abstract:ObjectiveTo observe the effects of specifically silencing Golgi phosphoprotein 3 (GOLPH3 ) gene on proliferation and apoptosis of gastric cancer cells, and to investigate the mechanism.MethodsThe human gastric carcinoma cells SGC-7901 were randomly divided into 3 groups. Cells of group A were transfected with lentiviral-mediated siRNA sequences while cells of group B were transfected with lentiviral-mediated scramble sequences and group C without transfection. The cell proliferation was detected by methyl thiazol tetrazolium (MTT) at 72 h after transfection. The apoptotic rate was detected by flow cytometry. The protein expression of GOLPH3, mammalian target of rapamycin (mTOR) and p-mTOR was examined by Western blotting. ResultsThe OD value of group A was lower than those of groups B and C at different time points, the apoptotic rate of A group (2.70%±0.11%) was statistically higher than that of group B (0.42%±0.02%) and group C (0.31%±0.02%), and the relative expression of GOLPH3 and p-mTOR protein of group A was lower than that of groups B and C (all P<0.05); no significant differences in the above indexes were found between groups B and C (all P>0.05). ConclusionSilencing GOLPH3 can inhibit the cell proliferation and promote the apoptosis of gastric cancer cells and the mechanism may be related with the down-regulated expression of p-mTOR in SGC-7901 cells.

Key words:gastric carcinoma cells; Golgi phosphoprotein 3; small interfering RNA; cell proliferation; cell apoptosis; mammalian target of rapamycin

(收稿日期：2015-10-21)

中圖分類號(hào)：R735.2

文獻(xiàn)標(biāo)志碼：A

文章編號(hào)：1002-266X(2016)10-0007-03

doi：10.3969/j.issn.1002-266X.2016.10.003

通信作者簡(jiǎn)介：賴銘裕(1969-)，男，主任醫(yī)師,主要研究方向?yàn)槲赴┌l(fā)病機(jī)制。E-mail: 2411423770@qq.com

作者簡(jiǎn)介：第一謝芳(1986-)，女，在讀碩士研究生，主要研究方向?yàn)槲赴┌l(fā)病機(jī)制。E-mail: 993661989@qq.com

基金項(xiàng)目：廣西醫(yī)療衛(wèi)生適宜技術(shù)研究與開(kāi)發(fā)資金資助項(xiàng)目(S201415-01)。