Livin基因转染对人骨肉瘤U-2OS细胞生长及化疗敏感性的影响
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摘要
前言
Livin是近年来发现的人类凋亡抑制蛋白(inhibitor of apoptosis family of protein,IAP)家族的新成员,在多数肿瘤组织中高表达,如黑色素瘤、乳腺癌、宫颈癌、结肠癌、膀胱癌、胃癌、前列腺癌、白血病以及淋巴瘤等。我们的前期研究表明,Livin在人正常组织中不表达,在骨肉瘤组织中表达;有研究表明肺癌的患者并且经过化学治疗的患者Livin表达水平明显升高,表明在化疗药物诱导癌细胞凋亡的同时,癌细胞合成表达抗凋亡的因子(如Livin)引起凋亡耐受,这可能是临床上肿瘤发生耐药的机制之一。进一步的体外实验证明,在黑色素瘤、淋巴瘤细胞、肺癌等中Livin的过表达与化疗耐药有关。因此,Livin可能在肿瘤化疗耐药过程中发挥作用。
Livin基因在抗凋亡作用中的生物学功能及在抗骨肉瘤细胞凋亡作用中的具体机制,以及其是否参与骨肉瘤细胞化疗耐药等还不清楚。我们构建了Livin真核表达载体,基因转染并获得高表达Livin基因的骨肉瘤U-2OS细胞,以探讨Livin基因对U-2OS细胞生长及化疗敏感性及凋亡耐受性的影响。
骨肉瘤是最常见的恶性骨肿瘤,恶性度高,预后差。其发生发展的过程尚不清楚,临床上也缺乏特异性的病理诊断指标。Livin是近年来发现的人类凋亡抑制蛋白家族的新成员,已发现在多数肿瘤中表达,与肿瘤发生有密切关系,可能作为诱导肿瘤凋亡治疗的新靶点。Caspase-3被认为是哺乳动物细胞凋亡的关键蛋白酶。然而,尚未见有关骨肉瘤患者Livin表达情况以及其与Caspase-3表达的关系,与骨肉瘤发生、发展、生物学特性及临床诊断价值的报道。本研究采用SABC免疫组化方法检测Livin和Caspase-3在骨肉瘤组织中的表达,探讨两者与骨肉瘤发生的关系及其临床诊断价值。
骨肉瘤是人类常见的原发性间叶组织恶性骨肿瘤,常发生于青少年,传统截肢疗法预后差,5年生存率不到20%。目前虽在术前术后给予化疗后生存率有所提高,但对于化疗耐受和转移复发的病例,化疗或手术常难以奏效。因此,应当在肿瘤的生物治疗方面深入研究,以探索有效的辅助治疗方法,提高骨肉瘤的治愈率。RNA干扰(RNA interference,RNAi)技术是近年来发展起来的新技术,它主要利用双链RNA(double-stranded RNA)在细胞内经Dicer酶的识别、结合、酶切产生有活性的长度为21～23 nt小干扰RNA(small interference RNA,siRNA),与目的基因mRNA结合并使之降解,从而抑制目的基因的表达。RNAi的发现为基因治疗学提供了更为有效的方法。Livin基因作为细胞凋亡抑制蛋白(inhibitor of apoptosis protein,IAP)家族的新成员,由于其具有在正常组织中不表达,而在人类肿瘤中高表达的特性,有望成为肿瘤治疗的选择性靶点,近年来引起学者们的广泛关注。我们的前期研究表明,Livin在人正常组织中不表达,在骨肉瘤组织中高表达,提示其在骨肉瘤的发生、发展中起重要作用。本研究通过RNAi技术靶向抑制人骨肉瘤MG-63细胞Livin基因表达,探讨利用体外转录的siRNA的方法诱导骨肉瘤细胞凋亡,从而为骨肉瘤的基因治疗提供新策略。
目的
第一部分:探讨Livin和Caspase-3的表达在骨肉瘤发生中的可能作用及其临床意义。第二部分:研究转染Livin基因对人骨肉瘤U-2OS细胞生长及化疗敏感性的影响。第三部分:体外转录合成凋亡抑制蛋白Livin基因siRNA,通过RNAi技术靶向抑制人骨肉瘤MG-63细胞Livin基因表达,观察其在骨肉瘤细胞株MG-63中抑制Livin表达后细胞增殖及细胞凋亡情况,从而为骨肉瘤的基因治疗提供新策略。
方法
第一部分:用SABC免疫组化方法检测Livin和Caspase-3在45例骨肉瘤及30例骨软骨瘤中的表达,探讨骨肉瘤组织中Livin和Caspase-3表达的关系及与临床病理因素的联系。第二部分:基因转染获得稳定表达Livin的U-2OS细胞克隆,逆转录-聚合酶链反应、Western blot及免疫荧光化学法了解Livin在转染细胞中基因水平和蛋白水平的表达情况;用平板克隆形成实验研究细胞生长情况;甲基偶氮唑蓝法研究细胞对化疗的敏感性的影响。第三部分:体外转录合成Livin序列特异性双链RNA(dsRNA),转染MG-63细胞株中,用荧光显微镜观察转染前后细胞形态的改变,RT-PCR和Western blot法检测转染前后Livin基因的mRNA和蛋白的干涉效果,流式细胞仪检测siRNA转染效率和细胞凋亡,平板克隆形成实验计算克隆形成率,MTT法检测细胞增殖,吖啶橙荧光染色法检测细胞凋亡,观察MG-63细胞生物学特性的改变。
结果
第一部分:Livin在骨肉瘤中表达阳性率为62.2%,在骨软骨瘤中为3.3%,二者相比差异有显著意义(P=0.000)。Caspase-3在骨肉瘤中表达阳性率为35.6%,在骨软骨瘤中为83.3%,二者相比差异有显著意义(P=0.001)。骨肉瘤中Livin和Caspase-3的表达呈显著负相关(r=—0.666,P=0.000)。Livin的高表达与骨肉瘤组织分型无关,与Price病理分级有关,Caspase-3的低表达与分化程度有关。第二部分:与转染前比较,Livin基因转染后,表达Livin的人骨肉瘤U-2OS细胞克隆形成能力提高约30%、细胞生长速度增快、倍增时间缩短9.2 h(P＜0.05),对多种化疗药物,转染Livin基因的U-2OS细胞对化疗药物敏感性降低(P＜0.01)。第三部分:转染特异性siRNA的细胞其Livin mRNA及蛋白表达均下调,与其它两组相比显著降低,(P＜0.05);干涉后部分细胞变圆,折光性降低,死亡细胞增多,细胞的增殖受到抑制,细胞集落形成明显减少,细胞凋亡增多,Western blot检测Caspase-3蛋白表达增多与其它两组比较有显著性差异(P＜0.05)。
结论
第一部分:Livin在骨肉瘤组织中高表达,Livin和Caspase-3与骨肉瘤的发生有关,且具有相关性,联合检测Livin和Caspase-3的表达将对骨肉瘤的早期诊断,判定恶性程度及预后具有指导意义。以Livin与Caspase-3为靶点,所进行的诱导骨肉瘤细胞凋亡的治疗,可望成为人骨肉瘤靶向凋亡诱导治疗的良好策略,将作为目前常规化疗的重要补充,在临床康复治疗中发挥出巨大的抗肿瘤效果。第二部分:由于肿瘤细胞内Livin的过表达,导致了肿瘤细胞对化学药物耐受,这些是临床上采用细胞因子和化学药物治疗肿瘤时要面临的肿瘤耐药问题。Livin参与骨肉瘤的发生发展,是骨肉瘤细胞对化疗耐受的重要机制之一,如果能找到一种药物可以降低Livin的表达,就可以抑制肿瘤细胞的增殖。阻断Livin的表达可促进骨肉瘤细胞的凋亡及增强化疗药物的抗癌作用,这为我们治疗骨肉瘤开辟了一条新的途径。第三部分:体外转录合成特异性Livin siRNA能有效抑制骨肉瘤细胞株MG-63中Livin的mRNA和蛋白表达,抑制细胞的增殖,诱导细胞凋亡,RNA干扰技术为骨肉瘤的治疗提供了一种新策略,Livin能作为诱导骨肉瘤细胞凋亡的分子靶点,Livin基因沉默靶向诱导骨肉瘤细胞凋亡可能作为手术、化疗、放疗等传统治疗的辅助手段。
Introduction
Livin, a novel member of inhibitor of apoptosis protein (IAP) family, is highly expressed in most tumor tissues, including melanoma, colon cancer, mammary cancer, cancer of the cervix,leukemia and lymphoma, and carcinom as of bladder, stomach, and prostate. Our previous study has showed that Livin is expressed in osteosarcoma, but is not found in normal bone tissue. Sun's research found that those patients who had underwent radiotherapy and chemotherapy demonstrated an obvious increase of Livin expression level, indicating that Livin may be one possible mechanism of drug resistance to lung adenocarcinoma. So that Livin may play an important role in mechanism of drug resistance to osteosarcoma.
The mechanism and biological function of Livin in the process of anti-apoptosis and whether it participate in mechanism of drug resistance to osteosarcoma are still not clear. Under this background, our study constructed eukaryotic expression vectors of Livin, then transfected them into U-2OS cell line to research on their effect on U-2OS cell growth. We aim at providing a basis for further exploring anti-apoptosis functions of Livin , and also an experimental basis for research on mechanism of chemotherapy resistance to osteosarcoma cell.
Osteosarcoma is one of the most common malignant bone tumor. It has high malinant grade and poor prognosis,but the relationship with osteosarcoma and the the course of its carcinogesisis not clear. Livin, a new member of inhibitor of apoptosis protein (IAP ) family, is highly expressed in most tumor tissues and has close relationship with tumorigenesis . It may be a new target for the therapy of tumor by inducing the apoptosis of tumor cells. Caspase-3 is considered a key proteolytic ferment of apoptosis of mammalian cell. However the expression and relationship of Livin and Caspase-3 is not clear,the report of their function in the tumorigenesis and biological characteristic are still not found yet. In this study, immunohistochemistry method was used to examine the expression of Livin and Caspase-3 , so as to explore the relationship between them and osteosarcoma.
Osteosarcoma is one of the most common malignant bone tumor which is originated from primary mesenchymal tissue. It is frequently occur among in adolescent. Traditional amputation has poor prognosis. Only 20% of the patient can live for five years. With the development of newly adjunctive chemotherapy, the survival rate has raised much,but the patient who show increased resistance to chemotherapy and recur soon have poor effect after chemotherapy and operation. So more attention should be payed to the biotherapy of tumor,as to explore more efficacious adjunctive therapy and enhance the recovery rate of osteosarcoma. RNA interfering technology is a newly developed method, it mainly utilize the identification and combination of double-stranded RNA by dicer enzyme. Then it was incised in 21～23 nt small interference RNA, the small interference RNA combine the messenger RNA of objective gene, so the expression of objective gene is silenced. The disvovery of RNA interference provide more effective method for gene therapy . Livin, a novel member of inhibitor of apoptosis protein (IAP) family, for the characteristics that is highly expressed in most tumor ti-ssues,may become the selective target in tumor therapy. Our previous study has showed that Livin is expressed in osteosarcoma,but is not found in normal bone tissue.it reveal that Livin may play an important role in the tumorigenesis and development of osteosarcoma. To study the inhibition of the expression of livin and the proliferation and apoptosis by livin small interterence RNA( siRNA) synthesized in vitro in osteosarcoma cell line MG-63. It may provide a new strategy for gene therapy of osteosarcoma.
Objective
The first part: To study the expression of Livin and Caspase-3 in osteosarcoma and their clinical significance. The second part: To express Livin in U-2OS cells by using gene transfection, and to observe its effect on cell growth and cell sensitivity to chemotherapy drugs . The third part: To study the inhibition of the expression of livin and the proliferation and apoptosis by livin small interterence RNA (siRNA) synthesized in vitro in Osteosarcoma cell line MG-63.
Methods
The first part: Livin and Caspase-3 were detected by SABC immunohistochemical method in 45 cases of paraffin embedded section of Osteosarcoma and 30 of osteochondroma. In addition to this, we explore the relationship between them and Osteosarcoma. The second part: Eukaryotic expression vectors of Livin were transfected into U-2OS cells and cell clones with stable expression were obtained Livin expression levels in the transfected U-2OS cells were assessed at mRNA level and protein level, res pectively. Cell growth status was assessed by biological features. MTT was performed to test effects of Livin on sensitivity of the U-2OS cells to chemotherapy drugs. The third part: Livin siRNA was chemically synthesized in vitro and then transfected into cell line MG-63. The expressions of livin mRNA and protein both before and after transfection were detected by reverse transcription polymerase chain reaction (RT-PCR) and West era Blotting. The proliferation of cells was determined by MTT method. The apoptosis morphous of Osteosarcoma cells is detected by AO fluorescent staining. The results of the three groups were analyzed and compared.
Results
The first part: The positive rate of Livin in Osteosarcoma was 62.2% and 3.3% in osteochondroma (P = 0.000). The positive rate of Caspase-3 in Osteosarcoma was 35.6% and 83.3% in osteochondroma (P = 0.001). The expression of Livin was correlated with Caspase-3 (r = -0.666,P = 0.000). Livin was related with differentiation instead of histotype ; Caspase-3 was also related with differentiation. The second part: After transfection, positive cells,especially U-2OS cells expressing Livin, showed an increase of about 30% in colony-forming ability, a shorter doubling time (P < 0.05) and lower sensitivity to chemotherapy drugs ( P < 0.01). Livin play an important role in the carcinogenesis of Osteosarcoma and are correlated with Osteosarcoma, it is one of the most important mechanism in the process of drug resistance to chemotherapy. The third part: Sequence specific siRNA targeting livin down-regulated the expression of livin mRNA and protein. The proliferation of cells was inhibited after transfection. The cell colony formation decreased, the apoptosis cell increased , meanwhile Caspase-3 protein increased compared with the other two groups(P < 0. 05).
Conclusion
The first part: Livin and Caspase-3 may play an important role in the carcinogenesis of Osteosarcoma and are correlated with Osteosarcoma. The detection of them might be a guide for the early diagnosis and determinant of malignancy and prognosis in Osteosarcoma. If we set Livin and Caspase-3 as a target and operate the therapy of Osteosarcoma by inducing the apoptosis of Osteosarcoma cells. It may become a good strategy of inducing the apoptosis of Osteosarcoma cells and use as an important supplement of conventional chemotherapy. The second part: Livin are implicated in genesis and development of human Osteosarcoma, thus may be an important mechanism for drug resistance of human Osteosarcoma cells. If we could find a drug which can reduce the expression of Livin, the proli- feration of cells would be inhibited .The silence of Livin, may induce the apoptosis of Osteosarcoma cells and enhance the anti-tumous effect of chemotherapeutics. It may provide us a new pathway for the therapy of Osteosarcoma. The third part: Sequence specific siRNA targeting Livin can efficiently inhibit the Livin expression and cell proliferation in Osteosarcoma cell line MG-63. The successful application of Livin extends the list of available therapeutic modalities in the treatment of Osteosarcoma. Inducing the apoptosis of Osteosarcoma cells by the silence of Livin gene , may become an important adjunctive method of traditional therapy.
Livin是近年来发现的人类凋亡抑制蛋白(inhibitor of apoptosis family of protein,IAP)家族的新成员,在多数肿瘤组织中高表达,如黑色素瘤、乳腺癌、宫颈癌、结肠癌、膀胱癌、胃癌、前列腺癌、白血病以及淋巴瘤等。我们的前期研究表明,Livin在人正常组织中不表达,在骨肉瘤组织中表达;有研究表明肺癌的患者并且经过化学治疗的患者Livin表达水平明显升高,表明在化疗药物诱导癌细胞凋亡的同时,癌细胞合成表达抗凋亡的因子(如Livin)引起凋亡耐受,这可能是临床上肿瘤发生耐药的机制之一。进一步的体外实验证明,在黑色素瘤、淋巴瘤细胞、肺癌等中Livin的过表达与化疗耐药有关。因此,Livin可能在肿瘤化疗耐药过程中发挥作用。
Livin基因在抗凋亡作用中的生物学功能及在抗骨肉瘤细胞凋亡作用中的具体机制,以及其是否参与骨肉瘤细胞化疗耐药等还不清楚。我们构建了Livin真核表达载体,基因转染并获得高表达Livin基因的骨肉瘤U-2OS细胞,以探讨Livin基因对U-2OS细胞生长及化疗敏感性及凋亡耐受性的影响。
骨肉瘤是最常见的恶性骨肿瘤,恶性度高,预后差。其发生发展的过程尚不清楚,临床上也缺乏特异性的病理诊断指标。Livin是近年来发现的人类凋亡抑制蛋白家族的新成员,已发现在多数肿瘤中表达,与肿瘤发生有密切关系,可能作为诱导肿瘤凋亡治疗的新靶点。Caspase-3被认为是哺乳动物细胞凋亡的关键蛋白酶。然而,尚未见有关骨肉瘤患者Livin表达情况以及其与Caspase-3表达的关系,与骨肉瘤发生、发展、生物学特性及临床诊断价值的报道。本研究采用SABC免疫组化方法检测Livin和Caspase-3在骨肉瘤组织中的表达,探讨两者与骨肉瘤发生的关系及其临床诊断价值。
骨肉瘤是人类常见的原发性间叶组织恶性骨肿瘤,常发生于青少年,传统截肢疗法预后差,5年生存率不到20%。目前虽在术前术后给予化疗后生存率有所提高,但对于化疗耐受和转移复发的病例,化疗或手术常难以奏效。因此,应当在肿瘤的生物治疗方面深入研究,以探索有效的辅助治疗方法,提高骨肉瘤的治愈率。RNA干扰(RNA interference,RNAi)技术是近年来发展起来的新技术,它主要利用双链RNA(double-stranded RNA)在细胞内经Dicer酶的识别、结合、酶切产生有活性的长度为21～23 nt小干扰RNA(small interference RNA,siRNA),与目的基因mRNA结合并使之降解,从而抑制目的基因的表达。RNAi的发现为基因治疗学提供了更为有效的方法。Livin基因作为细胞凋亡抑制蛋白(inhibitor of apoptosis protein,IAP)家族的新成员,由于其具有在正常组织中不表达,而在人类肿瘤中高表达的特性,有望成为肿瘤治疗的选择性靶点,近年来引起学者们的广泛关注。我们的前期研究表明,Livin在人正常组织中不表达,在骨肉瘤组织中高表达,提示其在骨肉瘤的发生、发展中起重要作用。本研究通过RNAi技术靶向抑制人骨肉瘤MG-63细胞Livin基因表达,探讨利用体外转录的siRNA的方法诱导骨肉瘤细胞凋亡,从而为骨肉瘤的基因治疗提供新策略。
目的
第一部分:探讨Livin和Caspase-3的表达在骨肉瘤发生中的可能作用及其临床意义。第二部分:研究转染Livin基因对人骨肉瘤U-2OS细胞生长及化疗敏感性的影响。第三部分:体外转录合成凋亡抑制蛋白Livin基因siRNA,通过RNAi技术靶向抑制人骨肉瘤MG-63细胞Livin基因表达,观察其在骨肉瘤细胞株MG-63中抑制Livin表达后细胞增殖及细胞凋亡情况,从而为骨肉瘤的基因治疗提供新策略。
方法
第一部分:用SABC免疫组化方法检测Livin和Caspase-3在45例骨肉瘤及30例骨软骨瘤中的表达,探讨骨肉瘤组织中Livin和Caspase-3表达的关系及与临床病理因素的联系。第二部分:基因转染获得稳定表达Livin的U-2OS细胞克隆,逆转录-聚合酶链反应、Western blot及免疫荧光化学法了解Livin在转染细胞中基因水平和蛋白水平的表达情况;用平板克隆形成实验研究细胞生长情况;甲基偶氮唑蓝法研究细胞对化疗的敏感性的影响。第三部分:体外转录合成Livin序列特异性双链RNA(dsRNA),转染MG-63细胞株中,用荧光显微镜观察转染前后细胞形态的改变,RT-PCR和Western blot法检测转染前后Livin基因的mRNA和蛋白的干涉效果,流式细胞仪检测siRNA转染效率和细胞凋亡,平板克隆形成实验计算克隆形成率,MTT法检测细胞增殖,吖啶橙荧光染色法检测细胞凋亡,观察MG-63细胞生物学特性的改变。
结果
第一部分:Livin在骨肉瘤中表达阳性率为62.2%,在骨软骨瘤中为3.3%,二者相比差异有显著意义(P=0.000)。Caspase-3在骨肉瘤中表达阳性率为35.6%,在骨软骨瘤中为83.3%,二者相比差异有显著意义(P=0.001)。骨肉瘤中Livin和Caspase-3的表达呈显著负相关(r=—0.666,P=0.000)。Livin的高表达与骨肉瘤组织分型无关,与Price病理分级有关,Caspase-3的低表达与分化程度有关。第二部分:与转染前比较,Livin基因转染后,表达Livin的人骨肉瘤U-2OS细胞克隆形成能力提高约30%、细胞生长速度增快、倍增时间缩短9.2 h(P＜0.05),对多种化疗药物,转染Livin基因的U-2OS细胞对化疗药物敏感性降低(P＜0.01)。第三部分:转染特异性siRNA的细胞其Livin mRNA及蛋白表达均下调,与其它两组相比显著降低,(P＜0.05);干涉后部分细胞变圆,折光性降低,死亡细胞增多,细胞的增殖受到抑制,细胞集落形成明显减少,细胞凋亡增多,Western blot检测Caspase-3蛋白表达增多与其它两组比较有显著性差异(P＜0.05)。
结论
第一部分:Livin在骨肉瘤组织中高表达,Livin和Caspase-3与骨肉瘤的发生有关,且具有相关性,联合检测Livin和Caspase-3的表达将对骨肉瘤的早期诊断,判定恶性程度及预后具有指导意义。以Livin与Caspase-3为靶点,所进行的诱导骨肉瘤细胞凋亡的治疗,可望成为人骨肉瘤靶向凋亡诱导治疗的良好策略,将作为目前常规化疗的重要补充,在临床康复治疗中发挥出巨大的抗肿瘤效果。第二部分:由于肿瘤细胞内Livin的过表达,导致了肿瘤细胞对化学药物耐受,这些是临床上采用细胞因子和化学药物治疗肿瘤时要面临的肿瘤耐药问题。Livin参与骨肉瘤的发生发展,是骨肉瘤细胞对化疗耐受的重要机制之一,如果能找到一种药物可以降低Livin的表达,就可以抑制肿瘤细胞的增殖。阻断Livin的表达可促进骨肉瘤细胞的凋亡及增强化疗药物的抗癌作用,这为我们治疗骨肉瘤开辟了一条新的途径。第三部分:体外转录合成特异性Livin siRNA能有效抑制骨肉瘤细胞株MG-63中Livin的mRNA和蛋白表达,抑制细胞的增殖,诱导细胞凋亡,RNA干扰技术为骨肉瘤的治疗提供了一种新策略,Livin能作为诱导骨肉瘤细胞凋亡的分子靶点,Livin基因沉默靶向诱导骨肉瘤细胞凋亡可能作为手术、化疗、放疗等传统治疗的辅助手段。
Introduction
Livin, a novel member of inhibitor of apoptosis protein (IAP) family, is highly expressed in most tumor tissues, including melanoma, colon cancer, mammary cancer, cancer of the cervix,leukemia and lymphoma, and carcinom as of bladder, stomach, and prostate. Our previous study has showed that Livin is expressed in osteosarcoma, but is not found in normal bone tissue. Sun's research found that those patients who had underwent radiotherapy and chemotherapy demonstrated an obvious increase of Livin expression level, indicating that Livin may be one possible mechanism of drug resistance to lung adenocarcinoma. So that Livin may play an important role in mechanism of drug resistance to osteosarcoma.
The mechanism and biological function of Livin in the process of anti-apoptosis and whether it participate in mechanism of drug resistance to osteosarcoma are still not clear. Under this background, our study constructed eukaryotic expression vectors of Livin, then transfected them into U-2OS cell line to research on their effect on U-2OS cell growth. We aim at providing a basis for further exploring anti-apoptosis functions of Livin , and also an experimental basis for research on mechanism of chemotherapy resistance to osteosarcoma cell.
Osteosarcoma is one of the most common malignant bone tumor. It has high malinant grade and poor prognosis,but the relationship with osteosarcoma and the the course of its carcinogesisis not clear. Livin, a new member of inhibitor of apoptosis protein (IAP ) family, is highly expressed in most tumor tissues and has close relationship with tumorigenesis . It may be a new target for the therapy of tumor by inducing the apoptosis of tumor cells. Caspase-3 is considered a key proteolytic ferment of apoptosis of mammalian cell. However the expression and relationship of Livin and Caspase-3 is not clear,the report of their function in the tumorigenesis and biological characteristic are still not found yet. In this study, immunohistochemistry method was used to examine the expression of Livin and Caspase-3 , so as to explore the relationship between them and osteosarcoma.
Osteosarcoma is one of the most common malignant bone tumor which is originated from primary mesenchymal tissue. It is frequently occur among in adolescent. Traditional amputation has poor prognosis. Only 20% of the patient can live for five years. With the development of newly adjunctive chemotherapy, the survival rate has raised much,but the patient who show increased resistance to chemotherapy and recur soon have poor effect after chemotherapy and operation. So more attention should be payed to the biotherapy of tumor,as to explore more efficacious adjunctive therapy and enhance the recovery rate of osteosarcoma. RNA interfering technology is a newly developed method, it mainly utilize the identification and combination of double-stranded RNA by dicer enzyme. Then it was incised in 21～23 nt small interference RNA, the small interference RNA combine the messenger RNA of objective gene, so the expression of objective gene is silenced. The disvovery of RNA interference provide more effective method for gene therapy . Livin, a novel member of inhibitor of apoptosis protein (IAP) family, for the characteristics that is highly expressed in most tumor ti-ssues,may become the selective target in tumor therapy. Our previous study has showed that Livin is expressed in osteosarcoma,but is not found in normal bone tissue.it reveal that Livin may play an important role in the tumorigenesis and development of osteosarcoma. To study the inhibition of the expression of livin and the proliferation and apoptosis by livin small interterence RNA( siRNA) synthesized in vitro in osteosarcoma cell line MG-63. It may provide a new strategy for gene therapy of osteosarcoma.
Objective
The first part: To study the expression of Livin and Caspase-3 in osteosarcoma and their clinical significance. The second part: To express Livin in U-2OS cells by using gene transfection, and to observe its effect on cell growth and cell sensitivity to chemotherapy drugs . The third part: To study the inhibition of the expression of livin and the proliferation and apoptosis by livin small interterence RNA (siRNA) synthesized in vitro in Osteosarcoma cell line MG-63.
Methods
The first part: Livin and Caspase-3 were detected by SABC immunohistochemical method in 45 cases of paraffin embedded section of Osteosarcoma and 30 of osteochondroma. In addition to this, we explore the relationship between them and Osteosarcoma. The second part: Eukaryotic expression vectors of Livin were transfected into U-2OS cells and cell clones with stable expression were obtained Livin expression levels in the transfected U-2OS cells were assessed at mRNA level and protein level, res pectively. Cell growth status was assessed by biological features. MTT was performed to test effects of Livin on sensitivity of the U-2OS cells to chemotherapy drugs. The third part: Livin siRNA was chemically synthesized in vitro and then transfected into cell line MG-63. The expressions of livin mRNA and protein both before and after transfection were detected by reverse transcription polymerase chain reaction (RT-PCR) and West era Blotting. The proliferation of cells was determined by MTT method. The apoptosis morphous of Osteosarcoma cells is detected by AO fluorescent staining. The results of the three groups were analyzed and compared.
Results
The first part: The positive rate of Livin in Osteosarcoma was 62.2% and 3.3% in osteochondroma (P = 0.000). The positive rate of Caspase-3 in Osteosarcoma was 35.6% and 83.3% in osteochondroma (P = 0.001). The expression of Livin was correlated with Caspase-3 (r = -0.666,P = 0.000). Livin was related with differentiation instead of histotype ; Caspase-3 was also related with differentiation. The second part: After transfection, positive cells,especially U-2OS cells expressing Livin, showed an increase of about 30% in colony-forming ability, a shorter doubling time (P < 0.05) and lower sensitivity to chemotherapy drugs ( P < 0.01). Livin play an important role in the carcinogenesis of Osteosarcoma and are correlated with Osteosarcoma, it is one of the most important mechanism in the process of drug resistance to chemotherapy. The third part: Sequence specific siRNA targeting livin down-regulated the expression of livin mRNA and protein. The proliferation of cells was inhibited after transfection. The cell colony formation decreased, the apoptosis cell increased , meanwhile Caspase-3 protein increased compared with the other two groups(P < 0. 05).
Conclusion
The first part: Livin and Caspase-3 may play an important role in the carcinogenesis of Osteosarcoma and are correlated with Osteosarcoma. The detection of them might be a guide for the early diagnosis and determinant of malignancy and prognosis in Osteosarcoma. If we set Livin and Caspase-3 as a target and operate the therapy of Osteosarcoma by inducing the apoptosis of Osteosarcoma cells. It may become a good strategy of inducing the apoptosis of Osteosarcoma cells and use as an important supplement of conventional chemotherapy. The second part: Livin are implicated in genesis and development of human Osteosarcoma, thus may be an important mechanism for drug resistance of human Osteosarcoma cells. If we could find a drug which can reduce the expression of Livin, the proli- feration of cells would be inhibited .The silence of Livin, may induce the apoptosis of Osteosarcoma cells and enhance the anti-tumous effect of chemotherapeutics. It may provide us a new pathway for the therapy of Osteosarcoma. The third part: Sequence specific siRNA targeting Livin can efficiently inhibit the Livin expression and cell proliferation in Osteosarcoma cell line MG-63. The successful application of Livin extends the list of available therapeutic modalities in the treatment of Osteosarcoma. Inducing the apoptosis of Osteosarcoma cells by the silence of Livin gene , may become an important adjunctive method of traditional therapy.
引文
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3 吕建,陈智超.Livin与肿瘤研究新进展.中华肿瘤防治杂志,2006:13(17):1347-1350.
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1 Lin J H,Deng G,Huang Q,et al.KIAP,a novel member of the inhibitor of apoptosis protein family.Biochem Biophys Res Commun.2000;279(3):820-831.
2 Kasof GM,Gomes BC.Livin,a novel inhibitor of apoptosis protein family member J Biol Chem.2001;276(5):3238-3246.
3 Vucic D,Stennicke HR,Pisabarro MT,et al.ML-IAP,a novel inhibitor of apoptosis that is preferentially expressed in human melanomas.Curr Biol.2000;10(11):1359-1366.
4 Ashhab Y,Allan A,Polliack A,et al.Two splicing variant s of a new inhibitor of apoptosis gene with different biological properties and tissue distribution pattern.FEBS Lett.2001;495(4):56-60.
5 Boaz N,Yaqoub A,Vered B,et al.Caspase-mediated cleavage converts livin from an antiapoptotic to a proapoptotic factor:implications for drug-resistant melanoma.Cancer Res.2003;63(10):6340-6343.
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8 Sanna MG,da Silva Correia J,Ducrey O,et al.IAP suppression of apoptosis involves distinct mechanisms:The TAK1/ JNK1 signaling cascade and caspase inhibition.Mol Cell Biol.2002;22(6):1754-1766.
9 Vucic D,Deshayes K,Ackerly H,et al.SMAC negatively regulates the anti-apoptotic activity of melanoma inhibitor of apoptosis(ML-IAP).J Biol Chem.2002;277:12275-12279.
10 Nachmias B,Ashhab Y,Bucholtz V,et al.caspase-mediated cleavage converts Livin from an antiapoptotic to a proapoptotic factor:implications for drug-resistant melanoma.Cancer Res.2003;63(10):6340-6349.
11 Atsuhito Yagihashi,Koichi Asanuma,Naoki Tsuji,et al.Detection of anti-livin antibody in gastrointestinal cancer patients.Clin Chem.2003;49(7):1026-1030.
12 孙建国,廖荣霞,陈正堂,等.一种新的凋亡抑制蛋白Livin在非小细胞肺癌组织中的表达.重庆医学.2004:33(7):982-984.
13 Yagihashi A , Asanuma K,Tsuji N , et al . Detection of anti-Livin antibody in gastrointestinal cancer patients . Clin Chem. 2003; 49 (7): 1206-1208.
14 Yagihashi A , Asanuma K, Nakamura M, et al . Detection of anti-Survivin antibody in gastrointestinal cancer patients . Clin Chem. 2001; 47 (7): 1729-1731.
15 Tanabe H , Yagihashi A , Tsuji N , et al. Expression of Survivin mRNA and Livin mRNA in non- small-cell lung cancer . Lung Cancer .2004; 46(3): 299 - 304.
16 Gazzaniga P , Gradilone A , Giuliani L , et al. Expression and prognostic significance of LIVIN , SURVIVIN and other apoptosis-related genes in the progression of superficial bladder cancer . Ann Oncol . 2003; 14 (1): 85-90.
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20 Kleinberg L, Lie AK, Florenes VA, et al. Expression of inhibitor-of-apoptosis protein family members in malignant mesothelioma. Human Pathology. 2007 Jul; 38(7): 986-994.
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22 Crnkovic-Mertens I, Semzow J, Hoppe-Seyler F, et al. Isoform-specific silencing of the Livin gene by RNA interference defines Livin beta as key mediator of apoptosis inhibition in HeLa cells. J Mol Med. 2006 Mar; 84(3): 232-240.
23 Liu B, Han M, Wen JK, et al. Livin/ML-IAP as a new target for cancer treatment . Cancer Letters, 2007 Jun 8; 250(2): 168-176.
24 Crnkovic-Mertens , Felix Hoppe-Seyler , Karin Butz. Induction of apoptosis in tumor cells by siRNA-mediated silencing of the livin/ ML-IAP/ KIAP gene . Oncogene , 2003; 53 (11): 8330-8336
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28 Qiuping Z, JieX, Youxin J, et al. Selectively frequent expression of CXCR5 enhances resistance to apoptosis in CD8 (+)CD34 (+) T cells from patients with T-cell-lineage acute lymphocytic leukemia. Oncogene, 2005; 24 (4): 573-584.
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36 Chang H, Schimmer AD. Livin/melanoma inhibitor of apoptosis protein as a potential therapeutic target for the treatment of malignancy. Molecular Cancer Therapeutics, 2007 Jan; 6(1): 24-30.
2 Lopes RB,Gangeswaran R,Mcneish IA,et al.Expression of the IAP protein family is dysregulated in pancreatic cancer cells and is important for resistance to chemotherapy.International Journal of Cancer.2007 Jun 1;120(11):2344 - 2352.
3 吕建,陈智超.Livin与肿瘤研究新进展.中华肿瘤防治杂志,2006:13(17):1347-1350.
4 Kleinberg L,Lie AK,Florenes VA,et al.Expression of inhibitor-of -apoptosis protein family members in malignant mesothelioma.Human Pathology.2007 Jul;38(7):986-994.
5 Yagihashi A,Asanuma K,Tsuji N,et al.Detection of anti-Livin antibody in gastrointestinaL cancer patients.Clin Chem.2003 Jul;49(7):1206-1208.
6 Gazzaniga P,Gradilone A,Giuliani L,et al.Expression and prognostic significance of LIVIN,SURVIVIN and other apoptosis-related genes in the progression of superficial bladder cancer.Ann Oncol.2003 Jan;14(1):85-90.
7 Crnkovic-Mertens I,Hoppe-Seyler F,Butz K.Induction of apoptosis in tumor cells by siRNA-mediated silencing of the Livin /ML-IAP /KIAP gene.Oncogene.2003 Nov 13;22(51):8330-8336.
8 Nakopoulou L,Alexandrou P,Stefanaki K,et al.Immunohistochemical expression of caspase-3 as an adverse indicator of the clinical outcome in human breast cancer.Pathobiology.2001;69(5):266-273.
9 Lin JH,Deng G,Huang Q,et al.KIAP,a novel member of the inhibitor of apoptosis protein family.Biochem Biophys Res Commun.2000;279:820-831.
10 Kasof GM,Gomes BC.Livin,a novel inhibitor of apoptosis protein Family member.J Biol Chem.2001;276:3238-3246.
11 Ashhab Y,Alian A,Polliack A,et al.Two splicing variants of a new inhibitor of apoptosis gene with different biological properties and tissue distribution pattern.FEBS Lett.2001;495:56-60.
12 Vucic D,Stennicke HR,Pisabarro MT,et al.ML-IAP,a novel inhibitor of apoptosis that is preferentially expressed in human melanomas.Curr Biol.2000,10:1359-1366.
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