表达Cdc42-shRNA重组慢病毒载体的构建及其对膀胱癌细胞影响的实验研究
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摘要
研究背景膀胱癌在西方国家其发病率居泌尿系统肿瘤的第二位，在中国居第一位，而且有逐渐增加的趋势。然而，由于缺乏有效的治疗手段，晚期膀胱癌患者的长期生存率很低。为了寻找更有效的治疗手段，一个关键的问题是找到那些易被检测并与肿瘤的发展和恶性程度一致的分子标记。细胞分裂周期蛋白42(Cdc42)属于Rho GTP酶家族，作为一个GTP结合蛋白开关调节多条信号传导途径，控制一些关键的细胞活动，如细胞增殖、存活、细胞形态、粘附、迁移和转录调节等。有些资料表明，Cdc42分子在膀胱癌组织比非肿瘤的膀胱组织表达增多，而且Cdc42的过表达与不少肿瘤的发生和发展相关。作为一种特异而有效的基因沉默技术，RNAi(RNA干扰)已广泛用于分析与肿瘤发生发展相关基因的功能，而且RNAi技术在许多肿瘤已展示其巨大的治疗潜力。
目的构建表达Cdc42-shRNA的慢病毒载体质粒，评估其转染EJ和T24两种膀胱癌细胞后对Cdc42 mRNA和蛋白质表达、对STAT3 mRNA和蛋白质表达以及对细胞形态学变化、细胞生长、细胞凋亡和细胞粘附的影响。
方法化学合成的寡核苷酸由两个19 nt方向相反的干扰序列及其在两序列之间插入7 nt的间隔序列和3′末端的5个脱氧胸苷酸组成。模板寡核苷酸退火后与线性化的pSIH-H1载体连接产生重组慢病毒载体质粒pSIH-H1-Cdc42-shRNA，此载体再经酶切、PCR和测序确认。然后分别用pSIH-H1-Cdc42-shRNA和pSIH-H1-Luciferase-shRNA(对照)转染EJ和T24细胞。利用普通倒置显微镜和荧光倒置显微镜观察细胞形态和转染效率，借助流式细胞仪检测细胞周期，利用MTT分析检测膀胱癌细胞的生长。细胞粘附实验用于检测肿瘤细胞对细胞外基质的粘附性，而Hoechst 33258染色和DNA梯状电泳用于细胞凋亡分析。此外，利用半定量RT-PCR评估Cdc42和STAT3 mRNA表达水平，而Western blotting用于检测Cdc42和STAT3蛋白表达。
结果(1)成功构建了重组慢病毒载体质粒pSIH-H1-Cdc42-shRNA。(2)重组载体转染EJ和T24细胞72小时后，其转染效率均50％以上。Cdc42的下调导致肿瘤细胞形态学改变，主要表现为细胞变圆。(3)用pSIH-H1-Cdc42-shRNA转染后，Cdc42 mRNA和蛋白质的表达逐渐减少。转染72小时后，其mRNA和蛋白质的表达在EJ细胞分别下降89％和81％，在T24细胞分别下降90％和85％，而pSIH-H1-Luciferase-shRNA转染的两种细胞Cdc42基因表达没有明显改变。(4)Cdc42沉默能显著减少两种膀胱癌细胞磷酸化STAT3蛋白水平，但STAT3 mRNA和总STAT3蛋白质水平与对照组相比没有改变。(5)与对照组相比，Cdc42沉默后，T24和EJ细胞的增殖能力显著下降。而且Cdc42下调也能显著减少S期细胞的百分率(EJ细胞减少71.2％，T24细胞减少48.0％)，G_1期细胞百分率相应增加。(6)Hoechst33258染色EJ细胞后，发现Cdc42沉默能使细胞核碎片增加并出现凋亡小体，而DNA片段的琼脂糖凝胶电泳中亦发现典型的DNA梯状现象。此外，Cdc42沉默能显著减少肿瘤细胞与胞外基质粘附的能力(EJ细胞减少65.3％，T24细胞减少74.5％)。
结论构建的靶向Cdc42的慢病毒载体质粒在两种膀胱癌细胞中能有效地下调Cdc42 mRNA和蛋白质的表达、导致细胞形态学改变、抑制细胞生长、诱导细胞凋亡、减少细胞粘附以及下调STAT3的磷酸化。这些资料表明RNAi介导的Cdc42沉默可能是膀胱肿瘤基因治疗的一种新途径。
Background Bladder cancer is the second most common urological malignancy inthe Western society, but it is the most common urological malignancy in China with arising incidence. However, the bladder cancer at late-stage has poor long-termsurvival rates due to the absence of effective treatment methods. In order to developeffective anti-cancer therapies, one of the key issues is to identify molecular markersthat can effectively detect and distinguish the progression and malignancy of bladdertumors. Cdc42 (Cell division cycle 42), a member of Rho GTPases, acts as aGTP-binding protein switch to regulate multiple signal transduction pathways thatcontrol key cellular processes such as cell proliferation, survival, cytoarchitecture,adhesion, migration, and transcriptional regulation. Some data suggest that RhoGTPases including Cdc42 are more abundant in bladder tumor tissues than innon-tumor bladder tissues. Furthermore, the overexpression of Cdc42 is associatedwith carcinogenesis and progression of many tumors. RNAi (RNA interference), aspecific and efficient method for gene silencing, is widely used to analyze thefunctional roles of genes associated with cancer initiation and progression. And thetherapeutic potential of the RNAi technique has been demonstrated in many cancercells.
Objectives To construct a lentivirus-vector plasmid expressing shRNAs againstCdc42 and evaluate its effects on the expression of Cdc42 mRNA and protein,morphological changes, cell growth, cell apoptosis, cell adhesion and STAT3expression after transfected into two human bladder cancer cell lines, EJ and T24cells.
Methods Chemically synthesized oligonucleotides were composed of two 19 ntsequences placed in opposition to each other with the insertion of a 7 nt spacer sequence and the attachment of five Ts at the 3'-end of the sequence. The templateoligonucleotides were annealed and ligated into the linearized pSIH-H1 vector togenerate the recombinant lentiviral vector plasmid pSIH-H1-Cdc42-shRNA. Thisvector was evaluated by using enzyme digestion, PCR and DNA sequencing. EJ andT24 cells were transfected with pSIH-H1-Cdc42-shRNA and pSIH-H1-Luciferase-shRNA, respectively. The morphological changes were observed by phase-contrastmicroscopy and fluorescence microscope. Moreover, both the proliferation and thecell cycle of bladder cancer cells were detected by MTT assay and flow cytometry,respectively. Cell adhesion to the extracellular matrices was measured by an adhesionassay. Hoechst 33258 staining and DNA ladder electrophoresis were used as cellapoptosis analysis. In addition, the levels of Cdc42 mRNA and STAT3 mRNAexpression were evaluated by semi-quantitative RT-PCR assay, whereas theexpression of Cdc42 and STAT3 proteins was determined by Western blotting.
Results①The recombinant lentiviral vector plasmid pSIH-H1-Cdc42-shRNA wassuccessfully constructed.②Its transfection efficiency in EJ and T24 cells was morethan 50% 72 h after transfection. The ablation of Cdc42 led to the morphologicalchanges of tumor cells, which are mainly to become round shape.③Cdc42expression was gradually decreased at its mRNA and protein levels after transfectionwith pSIH-H1-Cdc42-shRNA, and at 72 h after transfection the levels of its mRNAand protein were decreased by 89% and 81% in EJ cells, and by 90% and 85% in T24cells, respectively, whereas no changes were observed in EJ and T24 cells transfectedwith pSIH-H1-Luciferase-shRNA.④Cdc42 silencing can significantly decrease thelevels of p-STAT3 protein in two bladder cancer cells. However, no difference wasobserved at the levels of STAT3 mRNA and protein compared with two controlgroups.⑤After Cdc42 silencing, the proliferation ability of T24 or EJ cells wasmarkedly decreased compared with that of two control groups. Furthermore, thesilencing of Cdc42 exhibited a significantly decreased percentage of S-phase cells(decreased by 71.2% in EJ cells and by 48.0% in T24 cells) and a compensatoryincrease in the population of G_1-phase cells.⑥After Cdc42 deletion, an increasedlevel of nuclear fragmentation and apoptotic bodies was detected by Hoechst 33258 staining, and ladders of DNA fragmentation were observed by agarose-gelelectrophoresis of DNA in EJ cells. In addition, Cdc42 silencing can markedly reducethe adhesive ability of tumor cells to the extracellular matrices (by 65.3% in EJ cellsand by 74.5% in T24 cells).
Conclusion The constructed lentiviral vector plasmid targeting Cdc42 caneffectively knock-down the expression of Cdc42 mRNA and protein, result in themorphological changes, inhibit cell proliferation, induce cell apoptosis, decrease celladhesion and down-regulate the levels of p-STAT3 in human bladder cancer EJ andT24 cells. These data suggest that RNAi-mediated Cdc42 silencing may be a novelapproach for gene therapy of bladder cancer.
目的构建表达Cdc42-shRNA的慢病毒载体质粒，评估其转染EJ和T24两种膀胱癌细胞后对Cdc42 mRNA和蛋白质表达、对STAT3 mRNA和蛋白质表达以及对细胞形态学变化、细胞生长、细胞凋亡和细胞粘附的影响。
方法化学合成的寡核苷酸由两个19 nt方向相反的干扰序列及其在两序列之间插入7 nt的间隔序列和3′末端的5个脱氧胸苷酸组成。模板寡核苷酸退火后与线性化的pSIH-H1载体连接产生重组慢病毒载体质粒pSIH-H1-Cdc42-shRNA，此载体再经酶切、PCR和测序确认。然后分别用pSIH-H1-Cdc42-shRNA和pSIH-H1-Luciferase-shRNA(对照)转染EJ和T24细胞。利用普通倒置显微镜和荧光倒置显微镜观察细胞形态和转染效率，借助流式细胞仪检测细胞周期，利用MTT分析检测膀胱癌细胞的生长。细胞粘附实验用于检测肿瘤细胞对细胞外基质的粘附性，而Hoechst 33258染色和DNA梯状电泳用于细胞凋亡分析。此外，利用半定量RT-PCR评估Cdc42和STAT3 mRNA表达水平，而Western blotting用于检测Cdc42和STAT3蛋白表达。
结果(1)成功构建了重组慢病毒载体质粒pSIH-H1-Cdc42-shRNA。(2)重组载体转染EJ和T24细胞72小时后，其转染效率均50％以上。Cdc42的下调导致肿瘤细胞形态学改变，主要表现为细胞变圆。(3)用pSIH-H1-Cdc42-shRNA转染后，Cdc42 mRNA和蛋白质的表达逐渐减少。转染72小时后，其mRNA和蛋白质的表达在EJ细胞分别下降89％和81％，在T24细胞分别下降90％和85％，而pSIH-H1-Luciferase-shRNA转染的两种细胞Cdc42基因表达没有明显改变。(4)Cdc42沉默能显著减少两种膀胱癌细胞磷酸化STAT3蛋白水平，但STAT3 mRNA和总STAT3蛋白质水平与对照组相比没有改变。(5)与对照组相比，Cdc42沉默后，T24和EJ细胞的增殖能力显著下降。而且Cdc42下调也能显著减少S期细胞的百分率(EJ细胞减少71.2％，T24细胞减少48.0％)，G_1期细胞百分率相应增加。(6)Hoechst33258染色EJ细胞后，发现Cdc42沉默能使细胞核碎片增加并出现凋亡小体，而DNA片段的琼脂糖凝胶电泳中亦发现典型的DNA梯状现象。此外，Cdc42沉默能显著减少肿瘤细胞与胞外基质粘附的能力(EJ细胞减少65.3％，T24细胞减少74.5％)。
结论构建的靶向Cdc42的慢病毒载体质粒在两种膀胱癌细胞中能有效地下调Cdc42 mRNA和蛋白质的表达、导致细胞形态学改变、抑制细胞生长、诱导细胞凋亡、减少细胞粘附以及下调STAT3的磷酸化。这些资料表明RNAi介导的Cdc42沉默可能是膀胱肿瘤基因治疗的一种新途径。
Background Bladder cancer is the second most common urological malignancy inthe Western society, but it is the most common urological malignancy in China with arising incidence. However, the bladder cancer at late-stage has poor long-termsurvival rates due to the absence of effective treatment methods. In order to developeffective anti-cancer therapies, one of the key issues is to identify molecular markersthat can effectively detect and distinguish the progression and malignancy of bladdertumors. Cdc42 (Cell division cycle 42), a member of Rho GTPases, acts as aGTP-binding protein switch to regulate multiple signal transduction pathways thatcontrol key cellular processes such as cell proliferation, survival, cytoarchitecture,adhesion, migration, and transcriptional regulation. Some data suggest that RhoGTPases including Cdc42 are more abundant in bladder tumor tissues than innon-tumor bladder tissues. Furthermore, the overexpression of Cdc42 is associatedwith carcinogenesis and progression of many tumors. RNAi (RNA interference), aspecific and efficient method for gene silencing, is widely used to analyze thefunctional roles of genes associated with cancer initiation and progression. And thetherapeutic potential of the RNAi technique has been demonstrated in many cancercells.
Objectives To construct a lentivirus-vector plasmid expressing shRNAs againstCdc42 and evaluate its effects on the expression of Cdc42 mRNA and protein,morphological changes, cell growth, cell apoptosis, cell adhesion and STAT3expression after transfected into two human bladder cancer cell lines, EJ and T24cells.
Methods Chemically synthesized oligonucleotides were composed of two 19 ntsequences placed in opposition to each other with the insertion of a 7 nt spacer sequence and the attachment of five Ts at the 3'-end of the sequence. The templateoligonucleotides were annealed and ligated into the linearized pSIH-H1 vector togenerate the recombinant lentiviral vector plasmid pSIH-H1-Cdc42-shRNA. Thisvector was evaluated by using enzyme digestion, PCR and DNA sequencing. EJ andT24 cells were transfected with pSIH-H1-Cdc42-shRNA and pSIH-H1-Luciferase-shRNA, respectively. The morphological changes were observed by phase-contrastmicroscopy and fluorescence microscope. Moreover, both the proliferation and thecell cycle of bladder cancer cells were detected by MTT assay and flow cytometry,respectively. Cell adhesion to the extracellular matrices was measured by an adhesionassay. Hoechst 33258 staining and DNA ladder electrophoresis were used as cellapoptosis analysis. In addition, the levels of Cdc42 mRNA and STAT3 mRNAexpression were evaluated by semi-quantitative RT-PCR assay, whereas theexpression of Cdc42 and STAT3 proteins was determined by Western blotting.
Results①The recombinant lentiviral vector plasmid pSIH-H1-Cdc42-shRNA wassuccessfully constructed.②Its transfection efficiency in EJ and T24 cells was morethan 50% 72 h after transfection. The ablation of Cdc42 led to the morphologicalchanges of tumor cells, which are mainly to become round shape.③Cdc42expression was gradually decreased at its mRNA and protein levels after transfectionwith pSIH-H1-Cdc42-shRNA, and at 72 h after transfection the levels of its mRNAand protein were decreased by 89% and 81% in EJ cells, and by 90% and 85% in T24cells, respectively, whereas no changes were observed in EJ and T24 cells transfectedwith pSIH-H1-Luciferase-shRNA.④Cdc42 silencing can significantly decrease thelevels of p-STAT3 protein in two bladder cancer cells. However, no difference wasobserved at the levels of STAT3 mRNA and protein compared with two controlgroups.⑤After Cdc42 silencing, the proliferation ability of T24 or EJ cells wasmarkedly decreased compared with that of two control groups. Furthermore, thesilencing of Cdc42 exhibited a significantly decreased percentage of S-phase cells(decreased by 71.2% in EJ cells and by 48.0% in T24 cells) and a compensatoryincrease in the population of G_1-phase cells.⑥After Cdc42 deletion, an increasedlevel of nuclear fragmentation and apoptotic bodies was detected by Hoechst 33258 staining, and ladders of DNA fragmentation were observed by agarose-gelelectrophoresis of DNA in EJ cells. In addition, Cdc42 silencing can markedly reducethe adhesive ability of tumor cells to the extracellular matrices (by 65.3% in EJ cellsand by 74.5% in T24 cells).
Conclusion The constructed lentiviral vector plasmid targeting Cdc42 caneffectively knock-down the expression of Cdc42 mRNA and protein, result in themorphological changes, inhibit cell proliferation, induce cell apoptosis, decrease celladhesion and down-regulate the levels of p-STAT3 in human bladder cancer EJ andT24 cells. These data suggest that RNAi-mediated Cdc42 silencing may be a novelapproach for gene therapy of bladder cancer.
引文
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