孫振亞梁琳何潔婷王玲玲曾濤
·論著·
沉默Kin17表達(dá)抑制乳腺癌細(xì)胞MDA-MB-231的增殖
孫振亞1梁琳2何潔婷2王玲玲2曾濤3,4★
目的探討沉默Kin17表達(dá)對(duì)乳腺癌細(xì)胞生長(zhǎng)增殖的影響,為評(píng)估該分子作為未來(lái)藥物靶點(diǎn)的潛能提供依據(jù)。方法分別用攜帶Kin17基因特異性siRNA與正常對(duì)照siRNA的慢病毒重組載體感染MDA-MB-231細(xì)胞,再用嘌呤霉素進(jìn)行抗性篩選,然后在熒光顯微鏡下觀察陽(yáng)性細(xì)胞的比例;用Real-time PCR與Western blot方法檢測(cè)穩(wěn)定轉(zhuǎn)染了慢病毒重組載體的MDA-MB-231細(xì)胞中的Kin17 mRNA與蛋白表達(dá)水平;CCK-8實(shí)驗(yàn)檢測(cè)細(xì)胞生長(zhǎng)增殖狀況。結(jié)果通過(guò)感染攜帶Kin17特異性siRNA的病毒重組載體,穩(wěn)定、明顯敲減了MDA-MB-231細(xì)胞中的Kin17mRNA與蛋白表達(dá)水平,而且顯著減緩了該乳腺癌細(xì)胞的生長(zhǎng)增殖速度。結(jié)論沉默Kin17表達(dá)能抑制三陰表型乳腺癌細(xì)胞的增殖,并有可能成為一種潛在的乳腺癌治療新策略。
Kin17;基因沉默;乳腺癌細(xì)胞;細(xì)胞增殖;三陰型
乳腺癌是女性的頭號(hào)腫瘤殺手,嚴(yán)重威脅著女性的生命健康[1]。早期患者進(jìn)行腫瘤組織手術(shù)切除為較好的選擇,而進(jìn)展期的患者的放化療效果還不是很理想。目前,分子分型及相應(yīng)的靶向治療在乳腺癌中已取得了令人振奮的療效[2]。內(nèi)分泌療法能明顯改善雌激素(estrogen receptor,ER)或孕激素受體(progestrone receptor,PR)陽(yáng)性的患者的預(yù)后生存[3]。人表皮生長(zhǎng)因子受體2(human epidermal grow th factor receptor-2,HER-2)陽(yáng)性的患者對(duì)抗HER-2抗體藥物的臨床療效喜人[4]。而最近發(fā)現(xiàn),這些靶向藥物在臨床應(yīng)用中也出現(xiàn)了較多的耐藥性。特別是三陰表型(ER-PRHER-2-)的患者,暫時(shí)沒(méi)有明顯有效的靶向藥物選擇,預(yù)后極差[5]。因此,挖掘更多的藥物靶點(diǎn),對(duì)乳腺癌患者的有效治療非常緊要。
Kin17是一個(gè)物種進(jìn)化中十分保守的蛋白質(zhì),在細(xì)胞中主要參與DNA復(fù)制和DNA修復(fù)功能[6,7]。最近,多項(xiàng)研究顯示,該蛋白與肝癌、結(jié)腸癌、肺癌、膠質(zhì)瘤等多種腫瘤的進(jìn)展及預(yù)后療效有關(guān)[8-11]。本課題組前期研究發(fā)現(xiàn),Kin17在乳腺癌組織中表達(dá)明顯升高,很可能參與該腫瘤的發(fā)生發(fā)展[12]。本文將探討沉默Kin17表達(dá)對(duì)三陰表型乳腺癌細(xì)胞MDA-MB-231生長(zhǎng)增殖的影響,為評(píng)估該分子作為未來(lái)藥物靶點(diǎn)的潛能提供依據(jù)。
1.1 材料
MDA-MB-231細(xì)胞購(gòu)自上海中科院細(xì)胞庫(kù),慢病毒載體GV115與siRNA購(gòu)自上海吉?jiǎng)P基因有限公司,胎牛血清FBS、Leibovitz-15培養(yǎng)基和Trizol購(gòu)自美國(guó)Invitrogen公司,M-MLV逆轉(zhuǎn)錄酶購(gòu)自美國(guó)Promega公司,PrimerSTAR DNA聚合酶購(gòu)自大連寶生物公司,CCK-8購(gòu)自于美國(guó)sigma公司,鼠抗人Kin17單抗、羊抗鼠IgG-HRP二抗購(gòu)于美國(guó)Santa cruz公司,PCR引物由廣州華大基因有限公司合成,其他試劑為國(guó)產(chǎn)或進(jìn)口分析純?cè)噭?/p>
1.2 細(xì)胞培養(yǎng)
MDA-MB-231細(xì)胞用Leibovitz-15培養(yǎng)基(添加10%胎牛血清、100 U/m L青霉素、100mg/L鏈霉素)在37℃、含5%CO2的孵箱中培養(yǎng)。
1.3 穩(wěn)定轉(zhuǎn)染慢病毒重組載體的細(xì)胞株篩選
先針對(duì)Kin17基因(GenBank number NM_ 012311,1 182 bp)靶序列設(shè)計(jì)siRNA,然后分別把合成的相應(yīng)Kin17_siRNA和正常對(duì)照siRNA雙鏈DNA與慢病毒載體GV115進(jìn)行定向拼接,形成重組載體GV-siRNA_Kin17與GV-siRNA_NC,經(jīng)測(cè)序鑒定正確。重組病毒載體與輔助包裝原件質(zhì)粒用Lipofectamine 2000共轉(zhuǎn)染293T細(xì)胞,轉(zhuǎn)染8 h后更換為完全培養(yǎng)基,繼續(xù)培養(yǎng)48 h,收集細(xì)胞上清液,然后進(jìn)行慢病毒顆粒濃縮與病毒滴度測(cè)定。接著,把包裝好的病毒感染MDA-MB-231細(xì)胞,用嘌呤霉素篩選,在熒光顯微鏡下觀察攜帶熒光細(xì)胞的比例,當(dāng)陽(yáng)性感染率達(dá)到80%以上時(shí)停止抗性篩選,得到穩(wěn)定轉(zhuǎn)染沉默載體的細(xì)胞株MDA-MB-231KD與對(duì)照細(xì)胞株MDA-MB-231NC(圖1)。
1.4 Real-time PCR
根據(jù)已知的人Kin17 cDNA序列自行設(shè)計(jì)上游引物(5′-CAACTATTGCTGGCTTCA-3′)與下游引物(5′-TGTCTCGTCCACTTTGC-3′),PCR產(chǎn)物片段的理論值為243 bp。GAPDH作為內(nèi)參對(duì)照,上游引物(5′-TGACTTCAACAGCGACACCCA-3′)與下游引物(5′-CACCCTGTTGCTGTAGCCAAA-3′)的理論上PCR產(chǎn)物片段為121 bp。收集生長(zhǎng)狀態(tài)良好的細(xì)胞,先用Trizol抽提總RNA,再用M-MLV逆轉(zhuǎn)錄酶把總RNA反轉(zhuǎn)錄而獲得cDNA,接著配制PCR反應(yīng)體系,混勻后稍離心,于Takara TP800實(shí)時(shí)定量PCR儀上進(jìn)行擴(kuò)增測(cè)定。擴(kuò)增參數(shù)為:95℃預(yù)變性30 s;95℃5 s,60℃30 s,共進(jìn)行40個(gè)循環(huán)。
1.5 Western blot分析
收集生長(zhǎng)狀態(tài)較好的細(xì)胞,用含蛋白酶抑制劑的蛋白裂解液溶解細(xì)胞,離心取上清進(jìn)行SDS-PAGE電泳分離,隨后電轉(zhuǎn)至PVDF膜上。PVDF膜用5%脫脂奶粉于室溫封閉1 h,再加入鼠抗人Kin17單抗,GAPDH作為內(nèi)參。次日,滴加羊抗鼠IgG-HRP二抗孵育45m in,經(jīng)PBST漂洗后,最后用ECL底物曝光及拍照。
1.6 CCK-8細(xì)胞增殖檢測(cè)
將處于對(duì)數(shù)生長(zhǎng)期的細(xì)胞用胰酶消化并收集,再用完全培養(yǎng)基制成細(xì)胞懸液(2×104個(gè)/m L);按照每孔100μL種植于96孔板中,每組設(shè)置6孔重復(fù);待細(xì)胞完全沉降下來(lái)后,在顯微鏡下觀察各組的細(xì)胞密度與均勻程度適可后,放入細(xì)胞培養(yǎng)箱中培養(yǎng)。20 h后,加入10μL CCK-8試劑于培養(yǎng)板孔中,無(wú)需換液。繼續(xù)培養(yǎng)4 h后,把96孔板置于振蕩器上振蕩5m in,再用酶標(biāo)儀在450 nm波長(zhǎng)下檢測(cè)OD值。本實(shí)驗(yàn)重復(fù)3次,數(shù)值用均值±標(biāo)準(zhǔn)差表示。
1.7 統(tǒng)計(jì)學(xué)處理
實(shí)驗(yàn)數(shù)據(jù)用SPSS 13.0軟件進(jìn)行統(tǒng)計(jì)分析,不同組細(xì)胞間的生長(zhǎng)曲線用配對(duì)T檢驗(yàn)來(lái)比較分析,P<0.05時(shí)認(rèn)為差異具有統(tǒng)計(jì)學(xué)意義。
2.1 成功篩選獲得穩(wěn)轉(zhuǎn)慢病毒載體GV-siRNA_Kin17與GV-siRNA_NC的細(xì)胞株
分別將已構(gòu)建的慢病毒重組載體GV-siRNA_Kin17與GV-siRNA_NC感染MDA-MB-231細(xì)胞,用嘌呤霉素進(jìn)行抗性篩選,攜帶有綠色熒光載體的陽(yáng)性細(xì)胞比例逐漸增加。當(dāng)陽(yáng)性細(xì)胞率達(dá)到80%以上(圖1)及細(xì)胞匯合度達(dá)到約80%時(shí),我們即得到了穩(wěn)定轉(zhuǎn)染重組病毒載體的細(xì)胞株MDA-MB-231KD與對(duì)照細(xì)胞株MDA-MB-231NC,并把它們作為下一步細(xì)胞功能研究的實(shí)驗(yàn)材料。
2.2 用GV-siRNA_Kin17載體有效沉默MDA-MB -231細(xì)胞的Kin17表達(dá)
Real-time PCR檢測(cè)結(jié)果顯示,轉(zhuǎn)染了GV-siRNA_Kin17病毒載體的MDA-MB-231KD細(xì)胞中的Kin17mRNA表達(dá)水平明顯下調(diào),比正常對(duì)照細(xì)胞MDA-MB-231NC減低了77.5%(圖2A)。Western blot實(shí)驗(yàn)結(jié)果也表明,MDA-MB-231KD細(xì)胞中的Kin17蛋白表達(dá)水平也比正常對(duì)照細(xì)胞明顯下降(圖2B)。這提示該病毒重組載體的基因沉默效果較好。
2.3 沉默Kin17表達(dá)對(duì)MDA-MB-231細(xì)胞增殖的影響
正常對(duì)照細(xì)胞MDA-MB-231NC的生長(zhǎng)狀態(tài)良好。而CCK-8實(shí)驗(yàn)顯示,MDA-MB-231KD細(xì)胞的生長(zhǎng)增殖速度比對(duì)照細(xì)胞MDA-MB-231NC要明顯減緩,P=0.002 6<0.01(圖3)。
已有多項(xiàng)研究表明,Kin17是細(xì)胞中DNA復(fù)制復(fù)合物的一個(gè)重要成員,用Kin17抗體作用于細(xì)胞,會(huì)抑制細(xì)胞DNA復(fù)制水平[6]。紫外線或離子輻射細(xì)胞,能引起該分子的表達(dá)上調(diào)及定位改變[7];它很像一個(gè)DNA維持蛋白,參與晚期的DNA修復(fù)[13]。最近研究發(fā)現(xiàn),該蛋白與一些腫瘤的發(fā)生發(fā)展密切相關(guān)[8,14]。上調(diào)Kin17水平能促進(jìn)肝癌細(xì)胞的生長(zhǎng)增殖[8]。而沉默Kin17表達(dá)能促進(jìn)結(jié)直腸癌細(xì)胞對(duì)常用化療藥物的敏感性[9]。
據(jù)報(bào)道,Kin17在機(jī)體各種正常組織中普遍低表達(dá)[15]。本課題組研究也顯示,Kin17在乳腺正常上皮細(xì)胞Hs-578Bst與MCF-10A中表達(dá)較低,該蛋白在MCF-10A細(xì)胞受血清刺激增殖后表達(dá)升高;而且通過(guò)轉(zhuǎn)染攜帶Kin17基因的重組質(zhì)粒來(lái)上調(diào)Kin17表達(dá)水平,能明顯促進(jìn)MCF-10A細(xì)胞的增殖速度[16]。因此,Kin17與乳腺上皮細(xì)胞的生長(zhǎng)增殖密切相關(guān)。我們還發(fā)現(xiàn),該分子在常見(jiàn)乳腺癌細(xì)胞系(MDA-MB-231、BT474、MCF-7、SKBr-3等)及臨床乳腺癌標(biāo)本組織中表達(dá)明顯升高,提示其很可能在乳腺癌細(xì)胞的快速增殖及癌癥轉(zhuǎn)化中發(fā)揮重要作用[12]。在本研究中,通過(guò)感染攜帶特異性siRNA的病毒重組載體,穩(wěn)定、明顯敲減了MDA-MB-231細(xì)胞中的Kin17基因與蛋白表達(dá)水平,而且顯著抑制了該乳腺癌細(xì)胞的生長(zhǎng)增殖速度。因此,沉默Kin17很可能是一種潛在的乳腺癌治療新策略。特別是對(duì)于ER-PR-HER-2-表型的患者,Kin17也許是一種潛在的有效靶點(diǎn)。
目前,Kin17在乳腺癌細(xì)胞增殖中的調(diào)控機(jī)制尚有待闡明[8]。我們已證實(shí),上調(diào)Kin17表達(dá)能促進(jìn)MCF-10A細(xì)胞的DNA復(fù)制活性[16];化療藥物阿霉素能誘發(fā)MCF-10A細(xì)胞產(chǎn)生較多的DNA損傷,同時(shí)也引起Kin17蛋白反應(yīng)性升高[12]。因此,Kin17很可能在乳腺上皮細(xì)胞加速增殖中支持其DNA復(fù)制及損傷修復(fù)功能。本前期研究還發(fā)現(xiàn),上調(diào)Kin17能促進(jìn)MCF-10A細(xì)胞中的ERK1/2的磷酸化活性,提示該蛋白也許參與EGFR癌癥信號(hào)途徑[16]。本課題組還發(fā)現(xiàn),上調(diào)Kin17表達(dá)能促進(jìn)MCF-10A細(xì)胞中的cyclin D1的表達(dá)水平[16]。另有學(xué)者也證實(shí),上調(diào)Kin17能上調(diào)肝癌細(xì)胞的cyclin D1水平[8]。這充分表明了該蛋白對(duì)細(xì)胞周期中G1/S轉(zhuǎn)換的調(diào)控作用。對(duì)Kin17在乳腺癌細(xì)胞中功能及其分子調(diào)控機(jī)制的深入探究,將有助于揭示乳腺癌細(xì)胞快速增殖及癌癥轉(zhuǎn)化的潛在機(jī)制,為乳腺癌治療新方案的摸索提供有力的科學(xué)依據(jù)。
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Silencing of Kin17 inhibits proliferation of breast cancer MDA-MB-231 cells
SUN Zhenya1,LIANG Lin2,HE Jieting2,WANG Lingling2,ZENG Tao3,4★
(1.Department of Laboratory Medicine,Central Hospital of Longhua New District,Shenzhen,Guangdong,China,518110;2.The First Clinical Medicine College,Southern Medical University,Guangzhou,Guangdong,China,510515;3.Laboratory Medicine Center,Nanfang Hospital,Southern Medical University,Guangzhou,Guangdong,China,510515;4.School of Laboratory Medicine,Guangdong Medical University,Dongguan,Guangdong,China,523808)
Objective To explore the effectof Kin17 silencing on the proliferation of breast cancer cells,and assess the potential of Kin17 asanew drug target.Methods MDA-MB-231 cells were infected with the lentiviral recombinant vectors carrying Kin17 siRNA or normal controlled siRNA,and were screened with puromycin.The rates of the positive cells were observed under a fluorescence microscope.Real-time PCR and Western blot assay were performed to detect them RNA level and protein level of Kin17 in MDA-MB-231 cells transfected with the lentiviral recombinant vectors.And the CCK-8 assay was used to test the cell proliferation. Results The infection of the lentiviral recombinant vector carrying Kin17 siRNA stably knocked down the mRNA level and protein level of Kin17,and slowed down the cell grow th significantly in MDA-MB-231 cells. Conclusion Our findings indicate that the silence of Kin17 expression could inhibit the cell proliferation of triple-negative breast cancer cells,andwould bea potential therapeutic target for breast cancer.
Kin17;Gene silence;Breast cancer cell;Cell proliferation;Triple-negative
廣東省醫(yī)學(xué)科學(xué)技術(shù)研究基金項(xiàng)目(A2013622);廣東省大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目(201512121118);東莞市醫(yī)療衛(wèi)生科技計(jì)劃一般項(xiàng)目(20131051010005);南方醫(yī)科大學(xué)大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目(201512121245)
1.深圳市龍華新區(qū)中心醫(yī)院檢驗(yàn)科,廣東,深圳518110 2.南方醫(yī)科大學(xué)第一臨床醫(yī)學(xué)院,廣東,廣州510515 3.南方醫(yī)科大學(xué)南方醫(yī)院檢驗(yàn)醫(yī)學(xué)科,廣東,廣州510515 4.廣東醫(yī)科大學(xué)醫(yī)學(xué)檢驗(yàn)學(xué)院,廣東,東莞523808
★通訊作者:曾濤,E-mail:zengt@smu.edu.cn