丁克云 徐娟 滿昌峰 范鈺

RNA干擾下調(diào)PLK1基因?qū)盒院谒亓黾?xì)胞侵襲和失巢凋亡的影響

丁克云 徐娟 滿昌峰 范鈺

目的探討RNA干擾技術(shù)下調(diào)PLK1基因?qū)盒院谒亓黾?xì)胞侵襲的影響和可能機(jī)制。方法用PLK1小干擾核糖核酸分子(siRNA)轉(zhuǎn)染人惡性黑素瘤A375細(xì)胞后,分別用實(shí)時(shí)定量PCR和Western印跡法檢測(cè)PLK1mRNA和蛋白表達(dá),Transwell方法檢測(cè)細(xì)胞侵襲能力,以平板集落形成方法了解癌細(xì)胞集落形成情況,分別用瓊脂糖凝膠電泳和TUNEL方法檢測(cè)癌細(xì)胞失巢凋亡情況。結(jié)果惡性黑素瘤A375細(xì)胞經(jīng)PLK1 siRNA轉(zhuǎn)染后,PLK1 mRNA和蛋白表達(dá)明顯下調(diào);siRNA轉(zhuǎn)染組癌細(xì)胞生長(zhǎng)明顯受到抑制。與空白對(duì)照組和脂質(zhì)體對(duì)照組比較,siRNA細(xì)胞轉(zhuǎn)染組侵襲性明顯下降。失巢凋亡檢測(cè)發(fā)現(xiàn),瓊脂糖凝膠電泳出現(xiàn)明顯的梯度圖譜。TUNEL結(jié)果顯示,PLK1 siRNA可誘導(dǎo)惡性黑素瘤A375細(xì)胞凋亡,siRNA細(xì)胞轉(zhuǎn)染組較空白對(duì)照組和脂質(zhì)體對(duì)照組凋亡指數(shù)明顯升高[3.86%±0.35%(3.125 nmol/L siRNA組),7.35%±0.36%(6.250 nmol/L siRNA組),17.56%±0.38%(12.500 nmol/L siRNA組)比1.15%±0.25%和1.18% ±0.22%),均P＜0.01]。結(jié)論P(yáng)LK1 siRNA可抑制惡性黑素瘤細(xì)胞的侵襲,其機(jī)制可能與誘導(dǎo)失巢凋亡有關(guān)。

黑色素瘤;RNA干擾;基因,PLK1;腫瘤浸潤(rùn)

作者單位:212002江蘇鎮(zhèn)江,江蘇大學(xué)附屬人民醫(yī)院腫瘤研究所

PLK1是哺乳類Polo激酶家族的重要成員之一。研究發(fā)現(xiàn),PLK1在有絲分裂指數(shù)增高的細(xì)胞和組織中如惡性腫瘤中表達(dá)增高[1-4]。有學(xué)者發(fā)現(xiàn),PLK1在黑素瘤組織和細(xì)胞中表達(dá)明顯增高[5-6],提示PLK1基因在惡性黑素瘤發(fā)生發(fā)展中具有重要的作用,但尚不清楚PLK1基因在惡性黑素瘤增殖和侵襲中的內(nèi)在機(jī)制。本研究用RNA干擾(iRNA)技術(shù),針對(duì)PLK1設(shè)計(jì)合成小干擾核糖核酸分子(siRNA)轉(zhuǎn)染處理人惡性黑素瘤A375細(xì)胞,觀察對(duì)細(xì)胞侵襲和失巢凋亡的影響。

材料與方法

一、材料

人惡性黑素瘤A375細(xì)胞產(chǎn)自南京凱基生物科技有限公司,本院組織細(xì)胞生物標(biāo)本庫(kù)凍存。PLK1 siRNA序列:正向:5'-GAGGAGGCUGAGGAUCCUG TT-3',反向:5'-CAGGAUCCUCAGCCUCCUCTT-3',目標(biāo)位點(diǎn)1253-1273,與已知人類基因組序列無(wú)重復(fù),由美國(guó)羅氏公司合成高純度的21nt寡核苷酸siRNA。PLK1抗體產(chǎn)自美國(guó) Santa Cruz公司。TRIzol、RNase抑制劑、反轉(zhuǎn)錄酶 SSRTⅡ、Taq 酶、轉(zhuǎn)染試劑Oligofectamine均產(chǎn)自美國(guó)Invitrogen公司。DNA抽提試劑盒產(chǎn)自上海申能博彩公司。

二、方法

1.細(xì)胞培養(yǎng)及轉(zhuǎn)染處理:惡性黑素瘤A375細(xì)胞在含10%胎牛血清的RPMI 1640培養(yǎng)液,37℃、5%CO2、飽和濕度環(huán)境的條件下連續(xù)培養(yǎng)。轉(zhuǎn)染前1天,將1.0×105對(duì)數(shù)期生長(zhǎng)的細(xì)胞接種于24孔培養(yǎng)板,1 ml/孔,培養(yǎng)過(guò)夜。次日進(jìn)行轉(zhuǎn)染。操作按說(shuō)明書進(jìn)行。分為空白對(duì)照組、脂質(zhì)體對(duì)照組、siRNA組(10%胎牛血清的RPMI 1640中含已用脂質(zhì)體包埋PLK1基因siRNA)3個(gè)組。

2.PLK1基因檢測(cè):①mRNA水平檢測(cè):用實(shí)時(shí)定量PCR檢測(cè)。用TRIzol抽提細(xì)胞總RNA,取總RNA 1 μg,以oligo dT為引物逆轉(zhuǎn)錄合成cDNA第一鏈,以此cDNA鏈2 μl為模板進(jìn)行PCR擴(kuò)增,步驟參照說(shuō)明書。引物序列:上游:5'-CAAGCTCATC TTGTGCCCACT-3';下游:5'-GAGAAAGGGCTGCC AGGC-3'。FAM 探針:5'-CCGCTTCTCGTCGATGTA GGTCACG-3'-TAMRA。以GAPDH為內(nèi)參。PCR反應(yīng)條件為:95℃,預(yù)變性3min,95℃、30 s;52℃、45 s;72℃、45 s;35個(gè)循環(huán)后72℃再延伸7min;②蛋白水平檢測(cè):轉(zhuǎn)染48 h后,提取各實(shí)驗(yàn)組細(xì)胞總蛋白,蛋白定量后,以β肌動(dòng)蛋白為內(nèi)參照,進(jìn)行Western印跡法檢測(cè)。

3.集落形成試驗(yàn):用集落形成方法檢測(cè)。主要步驟如下:①細(xì)胞消化、計(jì)數(shù)、配制成濃度為1×103個(gè)/ml的細(xì)胞懸液,6孔細(xì)胞培養(yǎng)板中每孔加入1 ml細(xì)胞懸液(每孔1×103個(gè)細(xì)胞);②6孔細(xì)胞培養(yǎng)板置于37℃、5%CO2培養(yǎng)箱中培養(yǎng)14 d;③棄去培養(yǎng)基,PBS清洗細(xì)胞2次后,加入甲醇1 ml/孔,固定15min后,棄去固定液,以流水緩慢沖洗干凈,每孔加入l ml的姬姆薩液染色30min,以流水緩慢洗去染色液,通風(fēng)櫥中干燥,顯微鏡下觀察計(jì)數(shù)。

4.體外侵襲試驗(yàn):根據(jù)文獻(xiàn)[7]方法,用Transwell方法檢測(cè)。細(xì)胞計(jì)數(shù):用棉簽擦去基質(zhì)膠和上室內(nèi)的細(xì)胞,移去Transwells,倒置,風(fēng)干,24孔板中加入500 μl含0.1%結(jié)晶紫,將小室置于其中,使膜浸沒(méi)在培養(yǎng)基中,37℃、30min后取出,PBS清洗,直徑上取4個(gè)視野,照相,計(jì)數(shù)。

5.細(xì)胞失巢凋亡檢測(cè):①失巢凋亡模型制備:以鋪有多聚-HEMA的培養(yǎng)皿作為失巢凋亡模型。按照文獻(xiàn)[8]制備該模型。簡(jiǎn)述如下,將多聚HEMA溶于無(wú)水乙醇,制備成10mg/ml的多聚-HEMA溶液。將培養(yǎng)皿用PBS洗3遍,然后加上2 ml多聚-HEMA溶液包被;②細(xì)胞處理:收集轉(zhuǎn)染48 h的細(xì)胞,吹打均勻,制成5×105細(xì)胞/ml的單細(xì)胞懸液。將2 ml細(xì)胞接種于多聚HEMA的培養(yǎng)皿中,放入培養(yǎng)箱中繼續(xù)培養(yǎng)。12 h后,收集細(xì)胞,分別用瓊脂糖凝膠電泳和TUNEL檢測(cè)細(xì)胞失巢凋亡;③瓊脂糖凝膠電泳;用DNA抽提試劑盒按照說(shuō)明書操作提取DNA,無(wú)水乙醇沉淀。25℃瓊脂糖凝膠電泳,電壓60 V,1.5 h。用MPIAS-1000多媒體病理圖文分析系統(tǒng)進(jìn)行吸光度測(cè)定;④TUNEL檢測(cè):蓋玻片上細(xì)胞用多聚甲醛固定,室溫1 h。用PBS洗滌蓋玻片,然后在破膜固定液(permeabilization solution)冰上孵育2min。蓋玻片上加50 μl TUNEL反應(yīng)液,37℃孵育1 h。最后,光鏡下觀察。TUNEL陽(yáng)性(凋亡)細(xì)胞呈亮綠色。凋亡指數(shù)(%)=凋亡細(xì)胞/細(xì)胞總數(shù)×100%。

結(jié) 果

一、siRNA轉(zhuǎn)染對(duì)PLK1 mRNA和蛋白水平影響

用PLK1 siRNA轉(zhuǎn)染惡性黑素瘤A375細(xì)胞后,分別采用定量PCR和Western印跡檢測(cè)PLK1基因mRNA和蛋白水平。結(jié)果發(fā)現(xiàn),與對(duì)照組細(xì)胞比較,siRNA組PLK1基因mRNA水平明顯下調(diào),差異有統(tǒng)計(jì)學(xué)意義(P＜0.01),見表1。Western印跡結(jié)果顯示,與空白對(duì)照組比較,PLK1蛋白水平明顯下調(diào),見圖1。

二、PLK-1 siRNA轉(zhuǎn)染對(duì)癌細(xì)胞集落的影響

下調(diào)PLK-1表達(dá)后,對(duì)錨著不依賴性增殖和癌細(xì)胞穿越基質(zhì)能力的影響。結(jié)果顯示,A375細(xì)胞在體外半固體培養(yǎng)體系中可以自發(fā)地形成集落。而經(jīng)siRNA轉(zhuǎn)染的細(xì)胞集落生長(zhǎng)呈劑量依賴性減少。當(dāng)siRNA 濃度為 3.125、6.250、12.500 nmol/L 時(shí)集落數(shù)分別為52±5、36±6、17±5、與空白對(duì)照組對(duì)照組集落數(shù)78±6比較,siRNA組軟瓊脂集落形成數(shù)明顯降低(P＜0.05)。

表1 siRNA對(duì)惡性黑素瘤A375細(xì)胞PLK1 mRNA水平的影響(±s,%)

表1 siRNA對(duì)惡性黑素瘤A375細(xì)胞PLK1 mRNA水平的影響(±s,%)

注:n=3

組別 24 h 48 h 72 h空白對(duì)照組 100±1.5 100±1.3 100±1.2脂質(zhì)體對(duì)照組 100±1.3 100±1.2 100±1.1 siRNA組3.125 nmol/L 61.8±2.1 49.5±1.9 40.8±1.6 6.250 nmol/L 45.6±1.8 35.8±1.5 26.5±1.7 12.500 nmol/L 29.6±1.6 18.1±1.3 8.9±1.2

三、PLK-1 siRNA轉(zhuǎn)染對(duì)癌細(xì)胞侵襲的影響

收集轉(zhuǎn)染48 h的細(xì)胞,用Transwell方法檢測(cè)癌細(xì)胞侵襲情況。結(jié)果發(fā)現(xiàn),當(dāng)siRNA濃度為3.125、6.250、12.500 nmol/L時(shí)穿膜細(xì)胞數(shù)分別為39±5、19±5、9±3,與空白對(duì)照組組56±5比較,siRNA組穿過(guò)濾膜的細(xì)胞數(shù)明顯下降(P＜0.01)。

圖1 siRNA對(duì)惡性黑素瘤A375細(xì)胞PLK1蛋白的抑制(48 h)

圖2 PLK1 siRNA對(duì)惡性黑素瘤細(xì)胞DNA梯形條帶的影響(48 h) M:標(biāo)準(zhǔn)參照物;1:空白對(duì)照組;2:脂質(zhì)體對(duì)照組;3:siRNA 6.25 nmol/L

表2 PLK1 siRNA對(duì)惡性黑素瘤A375細(xì)胞凋亡指數(shù)的影響(±s)

表2 PLK1 siRNA對(duì)惡性黑素瘤A375細(xì)胞凋亡指數(shù)的影響(±s)

注:n=3

組別 凋亡指數(shù)(%)空白對(duì)照組 1.15±0.25脂質(zhì)體對(duì)照組 1.18±0.22 siRNA轉(zhuǎn)染組3.125 nmol/L 3.86±0.35 6.250 nmol/L 7.35±0.36 12.500 nmol/L 17.56±0.38

四、siRNA對(duì)癌細(xì)胞抗失巢凋亡的影響

A375細(xì)胞無(wú)論是貼壁培養(yǎng)時(shí)還是在鋪在多聚HEMA的培養(yǎng)皿上培養(yǎng)均未見明顯的凋亡現(xiàn)象,說(shuō)明A375細(xì)胞具有一定的抗失巢凋亡的特性。將轉(zhuǎn)染后的癌細(xì)胞轉(zhuǎn)到鋪有多聚HEMA的培養(yǎng)皿上,9 h后收集細(xì)胞,分別用瓊脂糖凝膠電泳和TUNEL檢測(cè)。瓊脂糖凝膠電泳檢測(cè)結(jié)果顯示,與空白對(duì)照組、脂質(zhì)體對(duì)照組比較,siRNA轉(zhuǎn)染組明顯增強(qiáng),空白對(duì)照組、脂質(zhì)體對(duì)照組和siRNA組吸光度值分別為(18 620±1 120、17 880±1 250和118 116±2 520。見圖2。為了解PLK1 siRNA對(duì)惡性黑素瘤細(xì)胞凋亡的影響,我們用TUNEL方法檢測(cè)凋亡指數(shù),結(jié)果發(fā)現(xiàn),與空白對(duì)照組比較,siRNA組凋亡指數(shù)明顯增高。見表2。

討 論

正常真核細(xì)胞,除成熟血細(xì)胞外,大多需黏附于特定的細(xì)胞外基質(zhì)上才能抑制凋亡而存活,稱為錨著依賴性。腫瘤細(xì)胞可以錨著不依賴性增殖。軟瓊脂形成集落的多少可反映腫瘤細(xì)胞錨著不依賴性的特性,且與其惡性程度呈正相關(guān)。癌細(xì)胞侵襲能力強(qiáng),則在軟瓊脂上形成的集落數(shù)目多。本研究發(fā)現(xiàn),經(jīng)PLK1 siRNA處理惡性黑素瘤細(xì)胞軟瓊脂集落數(shù)目明顯減少;Transwell方法檢測(cè)發(fā)現(xiàn),經(jīng)siRNA處理后的惡性黑素瘤細(xì)胞穿膜細(xì)胞數(shù)減少。說(shuō)明PLK1 siRNA可抑制惡性黑素瘤細(xì)胞的錨著不依賴性增殖和侵襲能力。

癌細(xì)胞侵襲是一個(gè)復(fù)雜的過(guò)程,涉及到很多機(jī)制。近年來(lái),失巢凋亡抵抗引起人們重視。細(xì)胞與細(xì)胞外基質(zhì)脫離黏附接觸或者是接觸不完全后出現(xiàn)的凋亡現(xiàn)象,被稱為失巢凋亡(anoikis)[8-9]。正常上皮細(xì)胞或內(nèi)皮細(xì)胞脫離于細(xì)胞外基質(zhì)的聯(lián)系時(shí)會(huì)發(fā)生失巢凋亡,而大多數(shù)來(lái)源于上皮或內(nèi)皮的腫瘤細(xì)胞則喪失了這種特性,尤其是容易發(fā)生轉(zhuǎn)移的惡性腫瘤細(xì)胞,可以遷徙到其他部位再次生長(zhǎng)。這種存在于某些腫瘤細(xì)胞的抗失巢凋亡現(xiàn)象,是腫瘤獲得轉(zhuǎn)移增殖功能特性的重要原因之一[10]。同樣地,惡性黑素瘤細(xì)胞在體內(nèi)增殖轉(zhuǎn)移可能是失巢凋亡抵抗的結(jié)果[11]。有學(xué)者研究發(fā)現(xiàn),某些癌基因參與了一些惡性腫瘤抗失巢凋亡的調(diào)控[9-11]。但目前尚不了解PLK1基因與惡性黑素瘤細(xì)胞抗失巢凋亡的關(guān)系。

研究失巢凋亡現(xiàn)象一般在包被有多聚HEMA的petri-dish培養(yǎng)皿上進(jìn)行。多聚HEMA是一種陰離子聚合物,具有均勻的非離子特性,鋪在容器的底部可阻止細(xì)胞接觸和ECM的沉淀,可阻止細(xì)胞貼壁,細(xì)胞呈懸浮狀態(tài),從而誘發(fā)失巢凋亡。本研究發(fā)現(xiàn),A375細(xì)胞無(wú)論是貼壁培養(yǎng)時(shí)還是在鋪有多聚HEMA的培養(yǎng)皿上培養(yǎng)均未見明顯的凋亡現(xiàn)象,即表現(xiàn)為抗失巢凋亡。本研究將不同濃度的PLK-1 siRNA轉(zhuǎn)染處理惡性黑素瘤細(xì)胞后,轉(zhuǎn)到包被有多聚HEMA的petri-dish培養(yǎng)皿中培養(yǎng)。于不同時(shí)間收集細(xì)胞,分別采用瓊脂糖凝膠電泳和TUNEL檢測(cè)。瓊脂糖凝膠電泳顯示,基因組DNA凝膠電泳顯現(xiàn)明顯的梯狀圖譜。凋亡指數(shù)結(jié)果顯示,轉(zhuǎn)染組細(xì)胞凋亡指數(shù)明顯增高,且呈濃度依賴性。提示PLK-1 siRNA增強(qiáng)了癌細(xì)胞對(duì)失巢凋亡的敏感性。

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2013-07-01)

(本文編輯:吳曉初)

Impacts of RNA interference targeting the polo-like kinase-1 gene on the invasion of and anoikis in human malignant melanoma cells

Ding Keyun,Xu Juan,Man Changfeng,Fan Yu.Cancer Institute,Affiliated People's Hospital of Jiangsu University,Zhenjiang 212002,Jiangsu,China

Fan Yu,Email:yuf36@sina.com

ObjectiveTo investigate the effects of down-regulation of polo-like kinase-1(PLK1)gene by RNA interference(RNAi)on the invasion of a human malignant melanoma cell line A375 and their possible mechanisms.MethodsCultured A375 cells were classified into several groups:blank control group receiving no treatment,liposome group transfected with lipofectamine only,and three siRNA groups transfected with three concentrations of a small interference RNA(siRNA)targeting PLK1 respectively.After additional culture,real time quantitative PCR and Western blot analysis were performed to quantify the expressions of PLK1 mRNA and protein in A375 cells respectively,Transwell invasion assay to evaluate the invasive capacity of A375 cells,agarose gel electrophoresis and terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labelling(TUNEL)to detect anoikis in A375 cells.The colony-forming capacity was also evaluated for A375 cells.Statistical analysis was carried out by one-factor analysis of variance.ResultsThere was a significant decrease in PLK1 mRNA and protein expressions as well as in colony-forming units in the siRNA groups compared with the blank control group(allP＜0.05).The invasive capacity of A375 cells was significantly inhibited in the siRNA groups with the number of migrating cells in Transwell assay being 39±5,19±5 and 9±3 in A375 cells transfected with 3.125,6.250 and 12.500 nmol/L siRNAs respectively,compared to 56±5 in the blank control group(allP＜0.05).A characteristic DNA ladder was observed on agarose gel electrophoresis in the siRNA(6.250 nmol/L)group.Compared with the blank control group and liposome group,the three siRNA groups showed increased apoptotic index(3.86%±0.35%(3.125 nmol/L siRNA),7.35%±0.36%(6.250 nmol/L siRNA)and 17.56%±0.38%(12.500 nmol/L siRNA)vs.1.15%±0.25%(blank control group)and 1.18%±0.22%(liposome group),allP＜0.05).ConclusionsPLK1 siRNA can inhibit the invasion of malignant melanoma cells,likely by inducing anoikis in these cells.

Melanoma;RNA interference;Genes,PLK1;Neoplasm invasiveness

10.3760/cma.j.issn.0412-4030.2014.06.012

江蘇省鎮(zhèn)江市社會(huì)發(fā)展基金(SH2013048)

范鈺,Email:yuf36@sina.com