Survivin shRNA载体的构建及其对大肠癌细胞体内外作用的研究
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摘要
背景和目的
大肠癌是人类最常见的恶性肿瘤之一,且发病率有逐年增加的趋势,在美国是导致恶性肿瘤死亡的第二大原因,且每年的发病人数达到135,000人。随着我国人民生活水平的提高,膳食结构的改变,大肠癌的发病率也逐年提高,发病率已占恶性肿瘤的第三位,严重威胁人类的健康。
目前绝大多数的大肠癌仍采取手术为主的综合治疗,随着治疗水平的提高,大肠癌的术后复发、转移逐步下降,但综合治疗的费用较高,副作用多,局部复发和肝转移仍然是治疗失败的主要原因,因此预防和早期发现局部复发和肝转移,筛选高危患者,预测复发和转移,以便及时治疗,对提高患者的生存质量和生存率具有重要的意义。随着分子生物学和基因工程的发展,基因治疗正成为肿瘤防治研究的重点。目前,肿瘤的基因治疗方法主要有基因替代,反义核酸技术,细胞因子基因治疗以及近年来最为关注的RNA干扰技术,肿瘤基因治疗的关键是找到合适的靶基因以及应用最先进的基因治疗手段。
Survivin是新近发现的具有独特结构的凋亡抑制蛋白(IAP)家族的新成员,在许多恶性肿瘤中高表达,近年研究发现Survivin在正常大肠上皮细胞中不表达而在大肠癌中高表达,Survivin的表达上调与大肠癌的凋亡指数下降、总体存活率缩短、预后不良和复发率增加有关。鉴于它的独特的生物学特点,Survivin有望成为肿瘤基因治疗的理想靶点,许多研究显示降低它的表达影响肿瘤细胞的生物学活性,对肿瘤的治疗有利。RNA干扰是目前最有效的基因沉默技术,具有简单、高效、特异的优点,它主要通过核酸酶将双链RNA(dsRNA)切割成21-25nt的小干扰RNA,即siRNA,由siRNA按碱基配对的原则特异性的识别并切割同源性靶mRNA分子而实现。短发夹样RNA(short hairpin RNA,shRNA)在细胞内会自动被加工成siRNA,使基因沉默,且比siRNA作用更稳定,高效。本研究构建针对Survivin基因的特异性shRNA表达载体,探索针对Survivin基因的RNA干扰技术对大肠癌细胞系生物学功能的影响,构建裸鼠人大肠癌动物模型,观察其在体内对肿瘤的作用。
研究方法
1、针对Survivin基因的一段靶序列:GGCTGGCTTCATCCACTGC(86-104)采用GeneChem~(TM)siRNA的设计工具,参照siRNA的设计原则,利用含绿色荧光蛋白的真核表达载体pGCsi-H1/Neo/GFP,构建真核表达质粒。两条寡核苷酸经退火、限制性内切酶Hind和BamH1双酶切、连接、转化、PCR鉴定阳性克隆、构建pGCH1/Survivin shRNA表达质粒,测序验证。
2、培养大肠癌细胞系SW480(Survivin高表达),用非脂质体法将真核表达质粒转染入SW480,并应用硫酸盐G418进行筛选阳性克隆,转染后以流式细胞仪、荧光显微镜、透射电镜、Western-blot方法检测肿瘤细胞的凋亡、转染和细胞周期及Survivin蛋白的表达情况。
3、MTT法测定质粒转染后SW480细胞的增殖情况。
4、Transwell小室法测定对细胞迁移能力的影响。
5、将SW480细胞和转染pGCH1/Survivin shRNA的SW480细胞分别接种裸鼠皮下,建立裸鼠人大肠癌荷瘤模型,观察shRNA-Survivin对成瘤性的影响
6、采用双盲法定期测量皮下肿瘤结节的二维值,计算每个肿瘤的体积和各组平均体积变化的差值,计算肿瘤抑制率。
7、免疫组织化学法测定Survivin蛋白的表达情况。
8、TUNEL法和透射电镜检测肿瘤细胞凋亡情况。
9、显微镜下观察肝,肾,肺等组织变化并测定血常规,了解siRNA对裸鼠血液系统,肝,肾,肺等器官的影响。
结果
1、成功构建了重组质粒pGCsi-H1/Neo/GFP-Survivin,经测序显示重组质粒shRNA编码序列与我们设计的靶向Survivin的核苷酸序列完全一致。
2、绿色荧光蛋白载体构建的真核表达质粒瞬时转染SW480细胞,G418筛选获得了稳定转染的细胞株。荧光显微镜观察转染效率达90%以上,流式细胞仪分析和透射电镜显示转染后的细胞出现了细胞周期的变化(G2/M期增高)和凋亡的出现(细胞核内的染色质聚集,核内呈团块状,可见凋亡小体)。Western-blot分析转染后的细胞Survivin蛋白的表达受到抑制,转染后48小时和稳定转染后Survivin蛋白的表达分别被下调了52.1%、73.6%、45.8%、41.7%,抑制的时间较长。
3、MTT测定显示转染重组质粒pGCsi-H1/Neo/GFP-Survivin后细胞的增殖受到抑制。
4、Transwell小室法分析细胞的迁移能力显示转染重组质粒pGCsi-H1/Neo/GFP-Survivin后的SW480细胞迁移能力较未转染和转染阴性质粒的SW480细胞明显下降,抑制率为44.08%。
5、接种转染细胞的裸鼠成瘤能力较未转染的裸鼠成瘤能力降低,成瘤时间延长,瘤结节的体积小,抑瘤率为67.86%。
6、免疫组化测定转染质粒的裸鼠移植瘤Survivin的表达降低,TUNEL法和透射电镜显示凋亡增加,凋亡指数(17.25±3.30)%vs(5.48±1.38)%,(P＜0.05)。
7、转染质粒的安全性分析显示,转染后的裸鼠与未转染组重要脏器的结构和功能以及血液系统都未受到影响。
结论
1、成功构建了重组质粒pGCsi-H1/Neo/GFP-Survivin。
2、靶向Survivin的shRNA表达载体可以较长时间抑制Survivin蛋白的表达;抑制大肠癌细胞的增殖,诱导细胞的凋亡,改变了细胞周期。
3、转染重组质粒后的SW480细胞迁移能力明显下降,说明Survivin除了抗凋亡和调节细胞周期的作用外,还可能参与和影响了肿瘤的转移过程,降低Survivin的表达还可能抑制肿瘤的转移。
4、SW480细胞可成功在裸鼠身上建立大肠癌模型。
5、事先转染特异的shRNA-Survivin的大肠癌SW480细胞成瘤性差,重组质粒pGCsi-H1/Neo/GFP-Survivin。能抑制大肠癌裸鼠移植瘤的发生和生长,诱导其凋亡。
6、重组质粒没有明显毒副作用。
Background and Objective
Colorectal cancer (CRC), one of the most common human malignant tumors, is the second leading cause of cancer-related deaths in the United States. Currently, most CRC are treated by comprehensive therapy, such as surgical resection, chemotherapy and radiotherapy. However, the recurrence and liver metastasis are still major causes leading to therapy failure. Thus, it is important to predict and discover early recurrence and metastasis in order to treat promptly, which could elevate living quality and survi- val rate. With the development of molecular biology and gene engineering, gene therapy,including gene replacement, antisensenucleic acids, cytokine therapy and RNA interference,has been an important method of tumor prevention and cure. The key of gene therapy is to find suitable target gene and to make use of most advanced gene therapeutic tool.
Survivin is a new member of the inhibitor-apoptosis gene family,owing special structure,overexpressed in many malignant tumor tissues. Recent studies indicate that the expression of Survivin was localized in the carcinoma cells and was not expressed in normal colorectal mucosas. Overexpression of Survivin in colorectal tumor cells has been correlated with descend of apoptotic index, shorten of overall survival rate,poor prognosis and increased recurrence. Many reports have shown that abrogation of Survivin is useful for tumor therapy. RNA interference is the most effective way to silence gene transcription. The process of RNAi is mediated by double-stranded RNAs that are homologous to the gene being suppressed. These dsRNAs are cleaved by Dicer into duplex 21-25nt in length,named small interfering RNA(siRNA).These fragments recognize and cleave homologous mRNA and lead to degradation of the gene. Short hairpin RNA(shRNA) can be automatically processed into siRNA in cell and is more stable and effective than siRNA. The aim of this study was to construct a shRNA expression vector against Survivin,explore its impact on colorectal cancer cells and construct nude mice bearing human colorectal carcinoma to see the vivo effect.
Methods
1.Eukaryon expression vector pGCsi-H1/Neo/GFP containing green fluorescence protein was used to construct shRNA plasmid directed to Survivin.
2.Colorectal carcinoma cells SW480(high expression of Survivin) were cultured , Eukaryon expression plsmid shRNA-Survivin was transfected into SW480 by use of Fuge6.Transfection was monitored by fluorescence microscope;cell cycle and the apoptosis cells were examined using flow cytometry and transmission electron microscope; Survivin protein expression was examined by Western-blotting.
3.Cell proliferation after transfection were analyzed by MTT.
4.The change of cell migration was detected by transwell chamber.
5.Construct nude mice bearing human colorectal carcinoma and observe the effect of shRNA on tumorigenesis in nude mice.
6.The tumors were measured two-dimensionally in double blind ;Tumor volume and average volume change were calculated , so did tumor inhibitation rate.
7.Survivin protein expression in nude mice were examined by immunohistochemical method.
8.Tumor cell apoptosis were monitored by use of TUNEL and transmission electron microscope.
9.The histological changes of liver, kidney and lung were observed with light microscope;peripheral blood hematological and biochemical parameter were detected.
Results
1 .Recombinant plasmid pGCsi-H1/Neo/GFP-Survivin was constructed successfully, and was confirmed by sequencing that shRNA encoding sequence was in coincidence with target nucleotide sequence of Survivin we engineered.
2.eukaryotic expression plasmid containing green fluorescent protein(GFP) could be transiently transfected into SW480,and obtained stably transfected cells after G418 screening. Transfection efficiency was more than 90% observed with fluorescence microscope. FCM and transmission electron microscope could see cell cycle change and apoptosis. Survivin protein expression was downregulated after transfection and lasted quite a long tine.
3.MTT showed that transfected cell proliferation was inhibited.
4.Cell migration experiment (transwell chamber) showed cell migration capability decreased significantly after transfection.
5.The tumorigenesis capability in nude mice injected with transfected cells was lower than that in nude mice injected with nontransfected cells. The tumor volume in transfected nude mice was smaller than in nontransfected nude mice,the inhibition rate was 67.86%.
6.Survivin protein expression of xenograft tumor in transfected nude mice was inhibited by immunohistochemical method,and apoptosis was increased by TUNEL and transmission electron microscope.
7.Plasmid security analysis revealed that the function and structure of important organs and hematological system in transfected nude mice did not appear abnormity.
Conclusion
1. Recombinant plasmid pGCsi-H1/Neo/GFP-Survivin was constructed successfuly.
2.shRNA expression vector directed against Survivin could downregulate Survivin protein expression for a long time,inhibit SW480 cell proliferation,induce cell apoptosis and result in cell cycle changes.
3.Colorectal tumor model could established in nude mice with SW480 cells.
4.Cell migration capability after transfection by recombinant plasmid descended obviously,which indicates that Survivin might participate and influence tumorous transfer process besides inhibiting apoptosis and regulating cell cycle.Downregulation of Survivin protein might hamper tumor metastasis.
5. The tumorigenesis capability in SW480 cells transfected by shRNA-Survivin reduced,the tumor growth was inhibited and apoptosis was induced.
6. Recombinant plasmid had no significant toxicity and side effects.
大肠癌是人类最常见的恶性肿瘤之一,且发病率有逐年增加的趋势,在美国是导致恶性肿瘤死亡的第二大原因,且每年的发病人数达到135,000人。随着我国人民生活水平的提高,膳食结构的改变,大肠癌的发病率也逐年提高,发病率已占恶性肿瘤的第三位,严重威胁人类的健康。
目前绝大多数的大肠癌仍采取手术为主的综合治疗,随着治疗水平的提高,大肠癌的术后复发、转移逐步下降,但综合治疗的费用较高,副作用多,局部复发和肝转移仍然是治疗失败的主要原因,因此预防和早期发现局部复发和肝转移,筛选高危患者,预测复发和转移,以便及时治疗,对提高患者的生存质量和生存率具有重要的意义。随着分子生物学和基因工程的发展,基因治疗正成为肿瘤防治研究的重点。目前,肿瘤的基因治疗方法主要有基因替代,反义核酸技术,细胞因子基因治疗以及近年来最为关注的RNA干扰技术,肿瘤基因治疗的关键是找到合适的靶基因以及应用最先进的基因治疗手段。
Survivin是新近发现的具有独特结构的凋亡抑制蛋白(IAP)家族的新成员,在许多恶性肿瘤中高表达,近年研究发现Survivin在正常大肠上皮细胞中不表达而在大肠癌中高表达,Survivin的表达上调与大肠癌的凋亡指数下降、总体存活率缩短、预后不良和复发率增加有关。鉴于它的独特的生物学特点,Survivin有望成为肿瘤基因治疗的理想靶点,许多研究显示降低它的表达影响肿瘤细胞的生物学活性,对肿瘤的治疗有利。RNA干扰是目前最有效的基因沉默技术,具有简单、高效、特异的优点,它主要通过核酸酶将双链RNA(dsRNA)切割成21-25nt的小干扰RNA,即siRNA,由siRNA按碱基配对的原则特异性的识别并切割同源性靶mRNA分子而实现。短发夹样RNA(short hairpin RNA,shRNA)在细胞内会自动被加工成siRNA,使基因沉默,且比siRNA作用更稳定,高效。本研究构建针对Survivin基因的特异性shRNA表达载体,探索针对Survivin基因的RNA干扰技术对大肠癌细胞系生物学功能的影响,构建裸鼠人大肠癌动物模型,观察其在体内对肿瘤的作用。
研究方法
1、针对Survivin基因的一段靶序列:GGCTGGCTTCATCCACTGC(86-104)采用GeneChem~(TM)siRNA的设计工具,参照siRNA的设计原则,利用含绿色荧光蛋白的真核表达载体pGCsi-H1/Neo/GFP,构建真核表达质粒。两条寡核苷酸经退火、限制性内切酶Hind和BamH1双酶切、连接、转化、PCR鉴定阳性克隆、构建pGCH1/Survivin shRNA表达质粒,测序验证。
2、培养大肠癌细胞系SW480(Survivin高表达),用非脂质体法将真核表达质粒转染入SW480,并应用硫酸盐G418进行筛选阳性克隆,转染后以流式细胞仪、荧光显微镜、透射电镜、Western-blot方法检测肿瘤细胞的凋亡、转染和细胞周期及Survivin蛋白的表达情况。
3、MTT法测定质粒转染后SW480细胞的增殖情况。
4、Transwell小室法测定对细胞迁移能力的影响。
5、将SW480细胞和转染pGCH1/Survivin shRNA的SW480细胞分别接种裸鼠皮下,建立裸鼠人大肠癌荷瘤模型,观察shRNA-Survivin对成瘤性的影响
6、采用双盲法定期测量皮下肿瘤结节的二维值,计算每个肿瘤的体积和各组平均体积变化的差值,计算肿瘤抑制率。
7、免疫组织化学法测定Survivin蛋白的表达情况。
8、TUNEL法和透射电镜检测肿瘤细胞凋亡情况。
9、显微镜下观察肝,肾,肺等组织变化并测定血常规,了解siRNA对裸鼠血液系统,肝,肾,肺等器官的影响。
结果
1、成功构建了重组质粒pGCsi-H1/Neo/GFP-Survivin,经测序显示重组质粒shRNA编码序列与我们设计的靶向Survivin的核苷酸序列完全一致。
2、绿色荧光蛋白载体构建的真核表达质粒瞬时转染SW480细胞,G418筛选获得了稳定转染的细胞株。荧光显微镜观察转染效率达90%以上,流式细胞仪分析和透射电镜显示转染后的细胞出现了细胞周期的变化(G2/M期增高)和凋亡的出现(细胞核内的染色质聚集,核内呈团块状,可见凋亡小体)。Western-blot分析转染后的细胞Survivin蛋白的表达受到抑制,转染后48小时和稳定转染后Survivin蛋白的表达分别被下调了52.1%、73.6%、45.8%、41.7%,抑制的时间较长。
3、MTT测定显示转染重组质粒pGCsi-H1/Neo/GFP-Survivin后细胞的增殖受到抑制。
4、Transwell小室法分析细胞的迁移能力显示转染重组质粒pGCsi-H1/Neo/GFP-Survivin后的SW480细胞迁移能力较未转染和转染阴性质粒的SW480细胞明显下降,抑制率为44.08%。
5、接种转染细胞的裸鼠成瘤能力较未转染的裸鼠成瘤能力降低,成瘤时间延长,瘤结节的体积小,抑瘤率为67.86%。
6、免疫组化测定转染质粒的裸鼠移植瘤Survivin的表达降低,TUNEL法和透射电镜显示凋亡增加,凋亡指数(17.25±3.30)%vs(5.48±1.38)%,(P＜0.05)。
7、转染质粒的安全性分析显示,转染后的裸鼠与未转染组重要脏器的结构和功能以及血液系统都未受到影响。
结论
1、成功构建了重组质粒pGCsi-H1/Neo/GFP-Survivin。
2、靶向Survivin的shRNA表达载体可以较长时间抑制Survivin蛋白的表达;抑制大肠癌细胞的增殖,诱导细胞的凋亡,改变了细胞周期。
3、转染重组质粒后的SW480细胞迁移能力明显下降,说明Survivin除了抗凋亡和调节细胞周期的作用外,还可能参与和影响了肿瘤的转移过程,降低Survivin的表达还可能抑制肿瘤的转移。
4、SW480细胞可成功在裸鼠身上建立大肠癌模型。
5、事先转染特异的shRNA-Survivin的大肠癌SW480细胞成瘤性差,重组质粒pGCsi-H1/Neo/GFP-Survivin。能抑制大肠癌裸鼠移植瘤的发生和生长,诱导其凋亡。
6、重组质粒没有明显毒副作用。
Background and Objective
Colorectal cancer (CRC), one of the most common human malignant tumors, is the second leading cause of cancer-related deaths in the United States. Currently, most CRC are treated by comprehensive therapy, such as surgical resection, chemotherapy and radiotherapy. However, the recurrence and liver metastasis are still major causes leading to therapy failure. Thus, it is important to predict and discover early recurrence and metastasis in order to treat promptly, which could elevate living quality and survi- val rate. With the development of molecular biology and gene engineering, gene therapy,including gene replacement, antisensenucleic acids, cytokine therapy and RNA interference,has been an important method of tumor prevention and cure. The key of gene therapy is to find suitable target gene and to make use of most advanced gene therapeutic tool.
Survivin is a new member of the inhibitor-apoptosis gene family,owing special structure,overexpressed in many malignant tumor tissues. Recent studies indicate that the expression of Survivin was localized in the carcinoma cells and was not expressed in normal colorectal mucosas. Overexpression of Survivin in colorectal tumor cells has been correlated with descend of apoptotic index, shorten of overall survival rate,poor prognosis and increased recurrence. Many reports have shown that abrogation of Survivin is useful for tumor therapy. RNA interference is the most effective way to silence gene transcription. The process of RNAi is mediated by double-stranded RNAs that are homologous to the gene being suppressed. These dsRNAs are cleaved by Dicer into duplex 21-25nt in length,named small interfering RNA(siRNA).These fragments recognize and cleave homologous mRNA and lead to degradation of the gene. Short hairpin RNA(shRNA) can be automatically processed into siRNA in cell and is more stable and effective than siRNA. The aim of this study was to construct a shRNA expression vector against Survivin,explore its impact on colorectal cancer cells and construct nude mice bearing human colorectal carcinoma to see the vivo effect.
Methods
1.Eukaryon expression vector pGCsi-H1/Neo/GFP containing green fluorescence protein was used to construct shRNA plasmid directed to Survivin.
2.Colorectal carcinoma cells SW480(high expression of Survivin) were cultured , Eukaryon expression plsmid shRNA-Survivin was transfected into SW480 by use of Fuge6.Transfection was monitored by fluorescence microscope;cell cycle and the apoptosis cells were examined using flow cytometry and transmission electron microscope; Survivin protein expression was examined by Western-blotting.
3.Cell proliferation after transfection were analyzed by MTT.
4.The change of cell migration was detected by transwell chamber.
5.Construct nude mice bearing human colorectal carcinoma and observe the effect of shRNA on tumorigenesis in nude mice.
6.The tumors were measured two-dimensionally in double blind ;Tumor volume and average volume change were calculated , so did tumor inhibitation rate.
7.Survivin protein expression in nude mice were examined by immunohistochemical method.
8.Tumor cell apoptosis were monitored by use of TUNEL and transmission electron microscope.
9.The histological changes of liver, kidney and lung were observed with light microscope;peripheral blood hematological and biochemical parameter were detected.
Results
1 .Recombinant plasmid pGCsi-H1/Neo/GFP-Survivin was constructed successfully, and was confirmed by sequencing that shRNA encoding sequence was in coincidence with target nucleotide sequence of Survivin we engineered.
2.eukaryotic expression plasmid containing green fluorescent protein(GFP) could be transiently transfected into SW480,and obtained stably transfected cells after G418 screening. Transfection efficiency was more than 90% observed with fluorescence microscope. FCM and transmission electron microscope could see cell cycle change and apoptosis. Survivin protein expression was downregulated after transfection and lasted quite a long tine.
3.MTT showed that transfected cell proliferation was inhibited.
4.Cell migration experiment (transwell chamber) showed cell migration capability decreased significantly after transfection.
5.The tumorigenesis capability in nude mice injected with transfected cells was lower than that in nude mice injected with nontransfected cells. The tumor volume in transfected nude mice was smaller than in nontransfected nude mice,the inhibition rate was 67.86%.
6.Survivin protein expression of xenograft tumor in transfected nude mice was inhibited by immunohistochemical method,and apoptosis was increased by TUNEL and transmission electron microscope.
7.Plasmid security analysis revealed that the function and structure of important organs and hematological system in transfected nude mice did not appear abnormity.
Conclusion
1. Recombinant plasmid pGCsi-H1/Neo/GFP-Survivin was constructed successfuly.
2.shRNA expression vector directed against Survivin could downregulate Survivin protein expression for a long time,inhibit SW480 cell proliferation,induce cell apoptosis and result in cell cycle changes.
3.Colorectal tumor model could established in nude mice with SW480 cells.
4.Cell migration capability after transfection by recombinant plasmid descended obviously,which indicates that Survivin might participate and influence tumorous transfer process besides inhibiting apoptosis and regulating cell cycle.Downregulation of Survivin protein might hamper tumor metastasis.
5. The tumorigenesis capability in SW480 cells transfected by shRNA-Survivin reduced,the tumor growth was inhibited and apoptosis was induced.
6. Recombinant plasmid had no significant toxicity and side effects.
引文
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