楊純平, 王華川, 溫劍虎
(重慶醫(yī)科大學(xué)附屬第一醫(yī)院胸心外科,重慶 400016)
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DEC1基因過表達(dá)對人食管癌ECA109細(xì)胞增殖及侵襲能力的影響
楊純平, 王華川, 溫劍虎△
(重慶醫(yī)科大學(xué)附屬第一醫(yī)院胸心外科,重慶 400016)
目的: 探討DEC1基因過表達(dá)對人食管癌ECA109細(xì)胞增殖和侵襲能力的影響及可能機(jī)制。方法:將質(zhì)粒pcDNA3.1(-)/DEC1(DEC1組)和pcDNA3.1(-)(vector組)利用脂質(zhì)體分別轉(zhuǎn)染至人食管癌ECA109細(xì)胞中,通過real-time PCR檢測轉(zhuǎn)染48 h后的細(xì)胞內(nèi)DEC1 mRNA表達(dá),Western blot分別檢測轉(zhuǎn)染72 h后細(xì)胞內(nèi)DEC1、基質(zhì)金屬蛋白酶9(MMP9)及細(xì)胞周期蛋白cyclin D1的蛋白表達(dá);采用CCK-8實(shí)驗(yàn)、平板集落實(shí)驗(yàn)及Transwell實(shí)驗(yàn)分別檢測DEC1過表達(dá)對細(xì)胞的增殖和侵襲能力的影響。結(jié)果: 與vector組相比,DEC1組中DEC1的表達(dá)明顯增高(P<0.01);cyclin D1和MMP9的表達(dá)明顯降低(P<0.05);細(xì)胞增殖與侵襲能力明顯受到抑制(P<0.01)。結(jié)論:過表達(dá)DEC1可明顯抑制ECA109細(xì)胞的增殖和侵襲能力,DEC1可能通過影響MMP9和cyclin D1參與其中。
DEC1基因; 食管癌; ECA109細(xì)胞; 細(xì)胞增殖; 細(xì)胞侵襲
食管癌是嚴(yán)重危害人類健康的惡性腫瘤,資料顯示食管癌可能由多種因素致病[1-2],與患者的年齡、性別、職業(yè)、生活環(huán)境密切相關(guān),由于缺乏有效的早期診斷及綜合治療,而導(dǎo)致目前食管癌患者總體病死率仍居高不下[3-4]。因此,尋找和開發(fā)新的治療食管癌的方法和分子靶點(diǎn)是當(dāng)前研究的重要課題。
分化型胚胎軟骨發(fā)育基因1(differentiated embryo-chondrocyte expressed gene 1,DEC1)屬于生長發(fā)育基因,參與軟骨形成,神經(jīng)發(fā)生,細(xì)胞的分化等生理過程,同時還參與了腫瘤的發(fā)生與發(fā)展,在腫瘤細(xì)胞的增殖、凋亡和分化起著重要作用[5-6]。近年來研究發(fā)現(xiàn),DEC1在肺腺癌、乳腺癌和食管癌等多種腫瘤中高表達(dá)[7-8],且與缺氧誘導(dǎo)因子1α(hypoxia-inducible factor 1 alpha,HIF-1α)的表達(dá)顯著相關(guān)[9],可能是腫瘤缺氧的直接標(biāo)志[10]。本研究在此基礎(chǔ)上,將已構(gòu)建好的DEC1真核過表達(dá)質(zhì)粒轉(zhuǎn)染至食管癌ECA109細(xì)胞中,觀察其對ECA109細(xì)胞的增殖和侵襲能力的影響,并探討其可能機(jī)制。
1 主要試劑
兔抗人MMP9單克隆抗體購自北京博奧森公司;兔抗人cyclin D1單克隆抗體購自CST;兔抗人DEC1單克隆抗體購自Abcam;actin抗體購自Santa Cruz;HRP標(biāo)記的羊抗兔IgG購自北京鼎國生物技術(shù)有限公司;Total RNA提取試劑盒購自O(shè)mega;脂質(zhì)體轉(zhuǎn)染試劑LipofectamineTM2000購自Invitrogen。
2 細(xì)胞培養(yǎng)
人食管癌ECA109 細(xì)胞株由我院腫瘤研究中心提供,由含10%胎牛血清、1×105U/L青霉素和100 mg/L鏈霉素的RPMI-1640培養(yǎng)液,37 ℃、5% CO2培養(yǎng)箱中培養(yǎng),胰酶消化細(xì)胞,2~3 d傳代1次,取對數(shù)生長期細(xì)胞實(shí)驗(yàn)。
3 實(shí)驗(yàn)方法
3.1 過表達(dá)DEC1質(zhì)粒的構(gòu)建與轉(zhuǎn)染 根據(jù)DEC1基因在GenBank中cDNA序列(NM_003670.2)設(shè)計引物如下:P1:5’-TTTAAGCTTGCCACCATGGAGCG-GATCCCCAG-3’(含HindIII酶切位點(diǎn));P2:5’-CCGGTCTAGAGTCTTTGGTTTCTAAGT-3’(含XbaI酶切位點(diǎn)),引物由上海生工生物工程技術(shù)公司合成。從ECA109細(xì)胞提取總RNA,反轉(zhuǎn)錄獲得cDNA,以其為模板進(jìn)行PCR擴(kuò)增,切膠回收后,按照Invitrogen公司pcDNA3.1(-)說明書構(gòu)建重組質(zhì)粒,轉(zhuǎn)化DH5α感受態(tài)大腸桿菌,挑選轉(zhuǎn)化子,提取質(zhì)粒,經(jīng)酶切和測序鑒定pcDNA3.1(-)/DEC1構(gòu)建成功。按照脂質(zhì)體LipofectamineTM2000說明書操作,將pcDNA3.1(-)及pcDNA3.1(-)/DEC1,按質(zhì)粒與脂質(zhì)體質(zhì)量比為1:2轉(zhuǎn)染入ECA109細(xì)胞中,實(shí)驗(yàn)分組為DEC1組[pcDNA3.1(-)/DEC1]和vector組[pcDNA3.1(-)]。
3.2 Real-time PCR檢測轉(zhuǎn)染細(xì)胞中DEC1、cyclin D1和MMP9的mRNA表達(dá)水平 將轉(zhuǎn)染48 h后的vector組和DEC1組細(xì)胞提取總RNA,按TaKaRa逆轉(zhuǎn)錄反應(yīng)說明書合成cDNA,稀釋10倍后進(jìn)行real-time PCR,用于擴(kuò)增DEC1的上游引物為5’-GGCGGGGAATAAAACGGAGCGA-3’,下游引物為5’-CCTCACGGGCACAAGTCTGGAA-3’;β-actin的上游引物為5’-AGCGAGCATCCCCCAAAGTT-3’,下游引物為5’-GGGCACGAAGGCTCATCATT-3’。反應(yīng)條件為95 ℃ 3 min;95 ℃ 5 s,60 ℃ 15 s,72 ℃ 15 s,共39個循環(huán)。同一實(shí)驗(yàn)重復(fù)3次,實(shí)驗(yàn)數(shù)據(jù)分析采用2-ΔΔCt法計算。
3.3 Western blot 檢測轉(zhuǎn)染細(xì)胞中DEC1、cyclin D1和MMP9蛋白表達(dá)水平 提取各組ECA109細(xì)胞總蛋白,BCA法測定蛋白濃度,加入5×SDS上樣緩沖液100 ℃ 5 min,每孔40 μg,80 V恒壓SDS-PAGE,250 mA恒流2 h冰浴電轉(zhuǎn)至PVDF膜,5%的脫脂牛奶室溫封閉2 h,與特異性 I抗(DEC1 1∶2 000,MMP9 1∶1 000,cyclin D1 1∶1 000)4 ℃過夜,II抗孵育2 h后ECL化學(xué)發(fā)光并顯影。
3.4 CCK-8實(shí)驗(yàn) 將轉(zhuǎn)染24 h的各組細(xì)胞消化收集,按每孔2 000個接種于96孔板中,待細(xì)胞貼壁后記為0 h,分別在0 h、24 h、48 h、72 h、96 h加入10 μL CCK-8液,37 ℃孵育2 h后,在450 nm處測定吸光度。
3.5 平板集落實(shí)驗(yàn) 將轉(zhuǎn)染24 h的各組細(xì)胞消化收集,按每孔1 000個接種于6孔板中37 ℃、5% CO2培養(yǎng),2~3 d換液,10 d后取出,固定并結(jié)晶紫染色。
3.6 Transwell實(shí)驗(yàn)檢測其遷移能力 將Matrigel膠4 ℃過夜后,無血清RPMI1640培養(yǎng)基按8∶1稀釋并混勻,每Transwell小室上室均勻鋪入60 μL混合液,37 ℃孵育2 h,將轉(zhuǎn)染24 h的各組細(xì)胞消化收集并計數(shù),加入含3% 胎牛血清培養(yǎng)基制成細(xì)胞懸液,以每孔2 000個(100 μL)加入上室中,下室加入含15% 胎牛血清的培養(yǎng)基,37 ℃、5% CO2培養(yǎng)箱中孵育24 h,棉簽擦掉上室細(xì)胞,PBS洗3次,甲醇固定30 min,結(jié)晶紫染色10 min,PBS洗凈,室溫風(fēng)干,光鏡下隨機(jī)選取3個視野計數(shù)。
4 統(tǒng)計學(xué)處理
采用SPSS 17.0統(tǒng)計學(xué)軟件,計量資料用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示,兩組均數(shù)比較采用t檢驗(yàn),以P<0.05為差異有統(tǒng)計學(xué)意義。
1 轉(zhuǎn)染細(xì)胞中過表達(dá)DEC1的鑒定
Real-time PCR和Western blot檢測結(jié)果顯示,DEC1組DEC1基因的表達(dá)水平明顯高于vector組,差異有統(tǒng)計學(xué)意義(P<0.01),表明DEC1組的DEC1過表達(dá),見圖1、2。
Figure 1.The mRNA expression of DEC1 in the ECA109 cells transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group) detected by real-time PCR. Mean±SD.n=3.**P<0.01vsvector group.
圖1 Real-time PCR 檢測DEC1 mRNA的表達(dá)
Figure 2.The protein expression of DEC1 in the ECA109 cells transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3. 1 (-) (vector group) detected by Western blot. Mean±SD.n=3.*P<0.05vsvector group.
圖2 Western blot實(shí)驗(yàn)檢測DEC1蛋白的表達(dá)水平
2 過表達(dá)DEC1對ECA109細(xì)胞生長能力的影響
CCK-8實(shí)驗(yàn)結(jié)果顯示過表達(dá)DEC1基因后,ECA109細(xì)胞的生長明顯受到抑制,差異具有統(tǒng)計學(xué)意義(P<0.01),說明過表達(dá)DEC1基因抑制了細(xì)胞的生長,見圖3。
Figure 3.The growth curve of ECA109 cells transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group) evaluated by CCK-8 assay. Mean±SD.n=3.*P<0.05,**P<0.01vsvector group.
圖3 CCK-8測定過表達(dá)DEC1基因?qū)κ彻馨〦CA109細(xì)胞生長的影響
3 過表達(dá)DEC1對ECA109細(xì)胞克隆形成能力的影響
平板集落實(shí)驗(yàn)結(jié)果顯示,與vector組相比,DEC1組細(xì)胞克隆團(tuán)數(shù)量明顯減少(P<0.01),說明過表達(dá)DEC1后,ECA109細(xì)胞的克隆形成能力明顯減弱,見圖4。
Figure 4.Colony-forming capacity of the ECA109 cells transfec-ted with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group) detecded by colony formation assay. Mean±SD.n=3.**P<0.01vsvector group.
圖4 平板集落實(shí)驗(yàn)檢測過表達(dá)DEC1對ECA109細(xì)胞克隆形成能力的影響
4 過表達(dá)DEC1基因?qū)CA109細(xì)胞侵襲能力的影響
Transwell實(shí)驗(yàn) 結(jié)果顯示,與vector組相比,DEC1組穿過基底膜的細(xì)胞數(shù)明顯減少(P<0.05),說明過表達(dá)DEC1后ECA109細(xì)胞的侵襲能力減弱,見圖5。
Figure 5.The invasion ability of the ECA109 cells transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group) determined by Transwell invasion assay (×200). Mean±SD.n=3.*P<0.05vsvector group.
圖5 過表達(dá)DEC1基因?qū)CA109細(xì)胞侵襲能力的影響
5 過表達(dá)DEC1基因?qū)CA109細(xì)胞cyclin D1和MMP9表達(dá)的影響
Western blot檢測結(jié)果顯示,DEC1組的cyclin D1和MMP9蛋白表達(dá)水平明顯低于vector組(圖6),說明過表達(dá)DEC1基因能抑制cyclin D1和MMP9的表達(dá)。
Figure 6.The protein expression of cyclin D1 and MMP9 in the ECA109 cells transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group) determined by Western blot. Mean±SD.n=3.*P<0.05vsvector group.
圖6 過表達(dá)DEC1對cyclin D1和MMP9蛋白表達(dá)的影響
DEC家族包括DEC1和DEC2, 都是堿性螺旋-環(huán)-螺旋(basic helix-loop-helix, bHLH)結(jié)構(gòu)的轉(zhuǎn)錄因子。DEC1基因定位于人類染色體3p25.3-26 上, 大約5.7 kb,包括5個外顯子4個內(nèi)含子,其啟動子區(qū)域包括多個GC盒,5’端區(qū)域有包括cAMP 應(yīng)答元件及多個E-box在內(nèi)的多個轉(zhuǎn)錄因子結(jié)合位點(diǎn)[11]。DEC1蛋白由412個氨基酸組成,定位于細(xì)胞核,廣泛表達(dá)于軟骨、肺、腸、脾等大多數(shù)正常組織,在腦、胰腺和腎中表達(dá)較少[12]。
轉(zhuǎn)錄因子DEC1在腫瘤中的表達(dá)模式存在組織特異性,其表達(dá)與腫瘤增殖的關(guān)系尚存爭議,一方面研究資料表明在肺癌中沉默DEC1表達(dá)可以通過調(diào)控cyclin D1的表達(dá)進(jìn)而抑制細(xì)胞增殖[8],而乳腺癌中高表達(dá)的DEC1通過下調(diào)claudin-1的表達(dá)促進(jìn)乳腺癌細(xì)胞的侵襲[7],因此為了明確DEC1在腫瘤細(xì)胞中的生物學(xué)作用,深入研究不同腫瘤細(xì)胞中DEC1的表達(dá)調(diào)控模式至關(guān)重要。本研究通過過表達(dá)DEC1基因后研究其對食管癌的增殖和侵襲能力的影響并探討其可能機(jī)制,通過CCK-8和平板集落實(shí)驗(yàn)表明,過表達(dá)DEC1基因后食管癌ECA109細(xì)胞的生長和克隆形成能力明顯下降,而利用Transwell小室模型在體外模擬腫瘤細(xì)胞降解細(xì)胞外基質(zhì)穿過基底膜的遷移過程,實(shí)驗(yàn)表明過表達(dá)DEC1使ECA109細(xì)胞的遷移能力明顯下降,顯示DEC1在食管癌中可能作為抑癌基因調(diào)控食管癌的發(fā)生發(fā)展。同時我們檢測了過表達(dá)DEC1后cyclin D1與MMP9的表達(dá),發(fā)現(xiàn)在其蛋白表達(dá)水平明顯降低,cyclin D1與MMP9是調(diào)控腫瘤細(xì)胞增殖與侵襲的重要因子[14],也是TGF-β信號通路下游的調(diào)控因子,研究發(fā)現(xiàn)在胰腺癌中DEC1通過影響TGF-β通路進(jìn)而調(diào)控上皮-間質(zhì)轉(zhuǎn)換的發(fā)生[13],提示我們DEC1可能通過活化TGF-β信號通路,進(jìn)而調(diào)節(jié)食管癌的發(fā)生與發(fā)展。Xu等[15]在241例食管癌患者研究發(fā)現(xiàn)DEC1表達(dá)水平與食管癌患者年齡、食管鱗癌的浸潤深度,淋巴結(jié)轉(zhuǎn)移情況及pTNMs密切相關(guān),且與食管癌患者的術(shù)后生存率顯著相關(guān),提示DEC1可能作為食管鱗癌的一個潛在預(yù)后指標(biāo),對其的研究更有助于食管癌的前期診斷與臨床治療。
綜上所述,針對DEC1作為bHLH 類轉(zhuǎn)錄因子,是否可作為一個腫瘤標(biāo)志物,能否作為關(guān)鍵靶點(diǎn)控制腫瘤發(fā)生發(fā)展,探尋其自身的表達(dá)調(diào)控及轉(zhuǎn)錄調(diào)節(jié)的機(jī)制研究作為今后的實(shí)驗(yàn)重點(diǎn),而本實(shí)驗(yàn)以DEC1基因?qū)κ彻馨┘?xì)胞的增殖、侵襲能力的影響作為主要切入點(diǎn)進(jìn)行研究,為明確DEC1基因功能及其是否可作為食管癌的治療新靶點(diǎn)提供實(shí)驗(yàn)依據(jù)。
[1] Jemal A, Bray F, Center MM, et al. Global cancer statistics[J].CA Cancer J Clin, 2011, 61(2):69-90.
[2] 郭艷麗,郭 煒,鄺 鋼,等. 食管鱗狀細(xì)胞癌中SFRP基因家族啟動子區(qū)甲基化狀態(tài)的檢測[J]. 中國病理生理雜志, 2011, 27(2):278-283.
[3] Lu P, Yang X, Huang Y , et al. Antitumor activity of a combination of rAd2p53 adenoviral gene therapy and radiotherapy in esophageal carcinoma[J]. Cell Biochem Biophys, 2011, 59(3):147-152.
[4] Shimada H, Ochiai T. Gene therapy for esophageal squamous cell carcinoma[J]. Front Biosci, 2008, 13:3364-3372.
[5] Peng Y, Liu W, Xiong J, et al. Down regulation of diffe-rentiated embryonic chondrocytes 1 (DEC1) is involved in 8-methoxypsoralen-induced apoptosis in HepG2 cells [J].Toxicology, 2012, 301(1-3):58-65.
[6] Bhawal UK, Sato F, Arakawa Y, et al. Basic helix-loop-helix transcription factor DEC1 negatively regulates cyclin D1[J]. J Pathol, 2011, 224(3):420-429.
[7] Liu Y, Miao Y, Wang J, et al. DEC1 is positively asso-ciated with the malignant phenotype of invasive breast cancers and negatively correlated with the expression of claudin-1[J]. Int J Mol Med, 2013, 31(4):855-860.
[8] Liu Y, Wang L, Lin XY, et al. The transcription factor DEC1 (BHLHE40/STRA13/SHARP-2) is negatively associated with TNM stage in non-small-cell lung cancer and inhibits the proliferation through cyclin D1 in A549 and BE1 cells [J]. Tumour Biol, 2013, 34(3):1641-1650.
[9] Ma W, Shi X, Lu S, et al. Hypoxia-induced overexpression of DEC1 is regulated by HIF-1α in hepatocellular carcinoma[J]. Oncol Rep, 2013, 30(6):2957-2962.
[10]Li Y, Bi Z, Yan B, et al. UVB radiation induces expression of HIF-1alpha and VEGF through the EGFR/PI3K/DEC1 pathway[J]. Int J Mol Med, 2006, 18(4):713-719.
[11]Nishiwaki T, Daigo Y, Kawasoe T, et al. Isolation and mutational analysis of a novel human cDNA, DEC1 (deleted in esophageal cancer 1), derived from the tumor suppressor locus in 9q32[J]. Genes Chromosomes Cancer, 2000, 27(2): 169-176.
[12]Wykoff CC, Pugh CW, Maxwell PH, et al. Identification of novel hypoxia dependent and independent target genes of the von Hippel-Lindau (VHL) tumour suppressor by mRNA differential expression profiling[J]. Oncogene, 2000, 19(54):6297-6305.
[13]Wu Y, Sato F, Yamada T, et al. The BHLH transcription factor DEC1 plays an important role in the epithelial-mesenchymal transition of pancreatic cancer[J]. Int J Oncol, 2012, 41(4):1337-1346.
[14]秦瑞英,夏永華,任艷芳,等. AEG-1表達(dá)下調(diào)對人宮頸癌細(xì)胞細(xì)胞周期和侵襲能力的影響及其機(jī)制[J]. 中國病理生理雜志, 2013, 29(6):1020-1024.
[15]Xu Q, Ma P, Hu C, et al. Overexpression of the DEC1 protein induces senescenceinvitroand is related to better survival in esophageal squamous cell carcinoma[J]. PLoS One,2012, 7(7):e41862.
Effect ofDEC1 gene over-expression on proliferation and invasion abilities of human esophageal cancer ECA109 cells
YANG Chun-ping, WANG Hua-chuan, WEN Jian-hu
(DepartmentofCardiothoracicSurgery,ChongqingMedicalUniversity,Chongqing400016,China.E-mail:tiger001@163.com)
AIM: To investigate the effect ofDEC1 gene over-expression on the proliferation and invasion abilities of human esophageal cancer ECA109 cells. METHODS: ECA109 cells were transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group). The mRNA and protein levels of DEC1, cyclin D1 and MMP-9 were evaluated by real-time PCR and Western blot, respectively. The effects ofDEC1 over-expression on the proliferation and invasion abilities of the ECA109 cells were evaluated by CCK-8 assay, colony formation assay and Transwell test respectively. RESULTS: The DEC1 expression level in ECA109 cells in DEC1 group was significantly higher than that in vector group (P<0.01), but the levels of MMP9 and cyclin D1 expression were opposite (P<0.01). However, both the proliferation and invasion abilities of ECA109 cells in DEC1 groups decreased significantly as compared with those in vector group (P<0.05). CONCLUSION: The over-expression of DEC1 significantly inhibits the proliferation and invasion of ECA109 cells, which may be involved in the expression of cyclin D1 and MMP9.
DEC1 gene; Esophageal cancer; ECA109 cells; Cell proliferation; Cell invasion
1000- 4718(2015)04- 0620- 05
2014- 11- 20
2015- 01- 20
R735.1
A
10.3969/j.issn.1000- 4718.2015.04.008
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