PTEN甲基化在骨肉瘤发生中的作用及5-Aza-CdR去甲基化对其MG-63细胞生物学行为的影响
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摘要
骨肉瘤(Osteosarcoma)是起源于间胚叶组织的最常见的原发性恶性骨肿瘤,其生物学性状极为复杂,有关其发病机理仍未完全清楚,诊断和治疗的方法依然有限。现代医学认为,肿瘤的发生是一个多阶段、多因素和多基因综合作用的过程,其生物学本质是细胞内遗传调控和表观遗传调控的紊乱与失调。近年来以不基于基因序列改变为特征的表观遗传调控在肿瘤发生中的作用备受关注,其主要表现形式是DNA甲基化,它可以直接导致相关基因的表观遗传转录沉默。基因DNA甲基化有助于肿瘤发生的早期预测及预后评估。癌基因的低甲基化、抑癌基因的高甲基化和整体基因组的低甲基化是DNA甲基化失衡状态的三种经典现象,其中尤以抑癌基因的高甲基化与肿瘤的关系最为密切,它是抑癌基因转录失活的主要途径,甚至可能成为调控基因转录的唯一的机制。
PTEN是迄今为止发现的第一个具有双特异性磷酸酶活性的抑癌基因,是抑癌基因家族中继P53发现后在人类肿瘤中突变及缺失率最高的又一明星分子。PTEN具有抑制肿瘤生长、促进细胞凋亡及抑制血管生成等作用,能负性调控PI3K/AKt及抑制MAPK的信号转导通路对肿瘤产生影响。已有研究表明在很多人类恶性肿瘤中都存在PTEN的异常甲基化,但关于其与骨肉瘤的关系却鲜见报道。了解骨肉瘤中PTEN的甲基化状态,对寻找骨肉瘤早期诊断可能的分子标志物具有重要的临床意义。
由于DNA甲基化不涉及DNA序列改变,因此,它是一个可逆的表观遗传学修饰过程。任何一个基因的甲基化状态是一个甲基化和去甲基化的动态平衡过程,处于甲基化异常的抑癌基因对阻断其DNA甲基化的药物非常敏感,临床上可利用这种敏感性进行基因治疗。5-Aza-CdR是已被美国FDA批准应用于骨髓增殖异常综合征及急性粒细胞白血病临床治疗的去甲基化药物,它能在体内外激发DNA的去甲基化过程,导致被甲基化沉默的抑癌基因持续激活。目前已有5-Aza-CdR作用于乳腺癌、非小细胞肺癌及前列腺癌等肿瘤的相关研究,但其与骨肉瘤的关系的研究却还是空白。因此,探讨5-Aza-CdR对肿瘤的去甲基化作用机制对防治骨肉瘤亦具有重要的临床意义。
本研究分成三个部分。第一部分,通过MSP技术检测骨肉瘤标本中PTEN的甲基化状态并将其与患者的临床病理参数结合进行分析,以期探讨PTEN甲基化在骨肉瘤发生中的作用及其相关预后影响因素,为探寻骨肉瘤早期诊断可能的分子靶标提供依据；第二部分,通过MSP技术对三株人骨肉瘤细胞MG-63、SAOS-2和U2-OS进行筛选,选取其中PTEN完全甲基化的一株细胞进行后续细胞学功能实验,以5-Aza-CdR对其进行去甲基化干预,探讨5-Aza-CdR作用的最佳浓度与时间、细胞的生物学行为及甲基化状态,为阐明5-Aza-CdR的体外去甲基化作用机制提供实验依据；第三部分,建立人骨肉瘤MG-63细胞荷瘤裸鼠模型,以5-Aza-CdR经尾静脉注射去甲基化干预4周,观测移植瘤体积生长的动态变化及检测移植瘤的病理形态学、PTEN蛋白表达及其甲基化情况,明确5-Aza-CdR的体内去甲基化作用机制。
第一部分骨肉瘤中PTEN的甲基化情况及其与相关临床病理参数的关系
目的明确骨肉瘤中抑癌基因PTEN的甲基化情况,寻找早期诊断可能的分子标志物。分析PTEN甲基化和骨肉瘤临床病理参数的关系,探讨PTEN甲基化对其相关预后影响因素的影响。
方法采集于2001年4月～2011年4月间在湖南省肿瘤医院、中南大学湘雅二医院及湘雅医院进行治疗并确诊的46例骨肉瘤患者的病理标本,其中新鲜组织21例(同时采集癌旁组织21例)、石蜡标本25例,采用HE染色、免疫组化(S-P)法检测肿瘤组织的病理形态学及PTEN表达水平,然后采用甲基化特异性PCR (MSP)技术检测骨肉瘤组织中PTEN启动子区甲基化的状态；整合所纳入骨肉瘤患者的临床资料,采用x2检验或Fisher's确切概率法分析PTEN启动子区甲基化与临床病理参数之间的关系。
结果HE染色及免疫组化结果显示,骨肉瘤组织中病理形态学呈明显肉瘤特征,而PTEN表达较癌旁组织明显减少,两组比较有统计学差异(P<0.05)。 MSP结果显示,骨肉瘤组织中存在PTEN高甲基化状态,癌旁组织中呈低甲基化或未甲基化状态,两组比较差异有统计学意义(P<0.05)。 PTEN高甲基化与骨肉瘤的Enneking肿瘤外科分期(P=0.023)。 Price组织学分级(P=0.001)及肿瘤发生的部位(P=0.030)表现出显著的正相关(P<0.05),而与年龄、性别、病程及化疗前AKP水平等一般临床资料无明显相关(P>0.05)。PTEN高甲基化患者术后5年生存率明显低于PTEN低甲基化患者,两组比较差异有统计学意义(P<0.05)。
结论1、PTEN在骨肉瘤中表达下调,其DNA启动子区存在高甲基化状态,PTEN高甲基化是骨肉瘤早期发生的分子特征之一。2、PTEN高甲基化状态与骨肉瘤的病理分期及分级呈显著正相关,是其独立的预后影响因素之一。
第二部分5-Aza-CdR去甲基化对人骨肉瘤MG-63细胞生物学行为及PTEN甲基化的影响
目的筛选PTEN完全甲基化的骨肉瘤细胞株,明确5-Aza-CdR对骨肉瘤细胞的体外去甲基化作用及对PTEN甲基化的影响,证实PTEN负性调控骨肉瘤中PI3K/AKt信号转导通路的作用机制。
方法选取三株经典的人成骨肉瘤细胞株MG-63、SAOS-2和U2-OS,用MSP法检测其中PTEN甲基化的情况,筛选出PTEN完全甲基化的一株细胞进行后续实验,以5-Aza-CdR对其进行去甲基化干预,同时筛选出PTEN甲基化状态被完全去除时5-Aza-CdR作用的最佳浓度及时间点,并检测该细胞的生物学行为及其PTEN甲基化的情况,采用Western-Blotting法检测经去甲基化干预后该细胞株中PI3K和AKt信号分子的蛋白表达水平。
结果在三株人骨肉瘤MG-63、SAOS-2和U2-OS中,MG-63细胞中PTEN完全甲基化,其被5-Aza-CdR完全去甲基化的最佳浓度和时间点分别为10μmol/L和24h。PTEN被完全去甲基化后的MG-63细胞在细胞凋亡、细胞周期、细胞侵袭迁移能力等细胞生物学行为方面与处理前比较差异有统计学意义(P<0.05)。 Western-Blotting结果显示,经5-Aza-CdR干预的MG-63细胞中PI3K和AKt分子蛋白的表达水平较对照组明显降低,两者表现出统计学差异(P<0.05)。
结论1、PTEN在人骨肉瘤MG-63细胞中完全甲基化；2、MG-63细胞中PTEN甲基化被5-Aza-CdR完全去除时具有时间和浓度的依赖性；3、5-Aza-CdR能对MG-63细胞的生物学行为产生影响,能增加肿瘤细胞的凋亡率、抑制细胞生长周期及降低细胞的侵袭转移能力；4、5-Aza-CdR能降低MG-63细胞中PI3K和AKt蛋白的表达水平,证实了PTEN负性调控MG-63细胞中PI3K/AKt信号转导通路的作用机制。
第三部分5-Aza-CdR去甲基化对裸鼠模型移植瘤PTEN表达及其甲基化的影响
目的构建人骨肉瘤MG-63细胞荷瘤裸鼠模型；探讨5-Aza-CdR对荷瘤裸鼠模型移植瘤的影响及其在动物体内的去甲基化作用机制。
方法采用细胞悬液异种移植皮下接种法,将人骨肉瘤MG-63细胞单悬液以1×107/200μl的活细胞浓度接种于4～6周龄裸鼠左腋肩部皮下,共接种18只动物,构建荷瘤裸鼠模型。成功建模后,将实验动物分成实验组和对照组,每组9只动物；实验组以10μmol/L浓度、1μ/g剂量的5-Aza-CdR进行尾静脉注射行去甲基化干预,而对照组则注射等量的PBS溶液,4周后处死动物,解剖移植瘤,观测并动态比较两组移植瘤体积大小和病理形态学的变化。采用IHC免疫组化法检测两组移植瘤瘤体组织中PTEN的表达情况,采取MSP技术检测两组瘤体组织中PTEN的甲基化状态,以Western-Blotting法检测并比较两组瘤体组织中PTEN蛋白表达的情况。
结果细胞接种4周后裸鼠移植瘤的大小为5mm3,表明成功地建立了人骨肉瘤MG-63细胞荷瘤裸鼠模型。两组裸鼠模型移植瘤体积大小动态比较,在5-Aza-CdR干预至第4周时两组瘤体体积大小比较差异有统计学意义(P<0.05),实验组优于对照组。IHC结果显示,实验组移植瘤中PTEN表达明显上调,较对照组有统计学差异(P<0.05)。MSP结果显示,实验组移植瘤中PTEN呈低甲基化状态,与对照组比较有统计学差异(P<0.05)。Western-Blotting结果显示,实验组移植瘤中PTEN蛋白表达量明显增加,较对照组比较差异有统计学意义(P<0.05)。
结论1、采用1×107/200μ1浓度的细胞悬液行皮下接种法能成功建立人骨肉瘤MG-63细胞荷瘤裸鼠动物模型；2、5-Aza-CdR能抑制人骨肉瘤MG-63细胞裸鼠模型移植瘤的生长,上调移植瘤中PTEN的表达,抑制其中PTEN的高甲基化状态,在体内发挥了其去甲基化作用。
BACKGROUND Osteosarcoma is one of primary neoplasms of the anterior mediastinum with highly variable histopathological characteristics. This heterogeneity has long hindered the development of a standardized protocol for diagnostic and prognostic evaluation of Osteosarcoma. The modified Enneking staging system, is currently the most commonly employed scoring method, but its value is limited by the fact that it assigns scores only for specific macroscopic surgical, but not histological, characteristics, making it difficult to reliably assess tumor severity in patients. Previous studies showed that multiple genetic alterations involved in many kinds of carcinomas, but the molecular mechanisms of Osteosarcoma is poorly understood. Recent data suggests that carcinomas appear to be a process that is caused by genetic alterations and by epigenetic mechanism. It mainly involves DNA methylation and histone modification. DNA methylation is one of the major mechanisms of epigenetics. Aberrant methylation of promoter regions resulting in inactivation of human tumor suppressor gene expression has been proposed to be an important mechanism in cancer. Cancer cell DNA is frequently characterized by localized gene-specific hypermethylation in TSG, the expression and status of DNA methylation of gene PTEN have already been reported in many kinds of tumor, such as lung cancer, colon cancer and oral carcinoma ea tl. Although multiple genetic and epigenetic changes have been detected in many kinds of cancer, the precise molecular mechanisms in the development and/or progression of Osteosarcoma still remain unknown. PTEN is the first TSG which has both Lipid phosphatase and Dual specificphosphatase action. The DNA mythlation can be altered by demethylation. The drug5-Aza-CdR, the DNA methyltransferase inhibitor, is the most mainly demethylation drug which has been applied for clinical treatment widely and be authorized by FDA,USA. To clarify the demethylation mechanism of the5-Aza-CdR is relatively important to the treatment of Osteosarcoma.
There are3parts content in our research works. In part1, the DNA metyhlation status of PTEN in tissues of the Osteosarcoma was detected by MSP method, and the methylation results and the clinical data were analysed together. In part2, the cell behavior and the DNA methylation status of the MG-63cell were detected by the cytology test and the MSP method. In part3, To establish nude mouse animal models and intervene demethylatedly it through5-Aza-CdR injection, and the expression of PTEN and the DNA methylation of PTEN of the transplantation tumor were detected by WB and MSP methods.
Part I PTEN Abnormal DNA Methylation and Its Clinical Significance in Osteosarcoma
Objective:To investigate the expression and the promoter hypermethylation of PTEN and potential clinical significance in patients with Osteosarcoma.
Methods:Methylation-specific PCR (MSP) method was performed for evaluating the expression and promoter region methylation patterns of PTEN in Osteosarcoma. The correlation between TSGs mRNA, TSGs methylation and clinicopathological data were analyzed.46patients with Osteosarcoma were surgically treated in the HN province tumor hospital, the2nd xiangya hospital and the xiangya hospital, and from April,2001to April,2011. The samples of Osteosarcoma were be detected by HE dye, immunohistochemistry and MSP, and the methylation status of the samples and the clinical data of the Osteosarcoma patients were analysed together for discussing the relationship between the above two.
Results:There has been significant defference between the tumor tissue group and the non-tumor tissue group about the results of the expression of PTEN and the DNA methylation status of PTEN (P<0.05). PTEN hyermethylation is correlative with the tumor grades and stages of Osteosarcoma. Enneking surgery stages of tumor (P=0.002)、Price surgery classification (P=0.003)、position of tumor occurring (P= 0.030).
Conclusions:1、The down-regulation of PTEN in osteosarcoma and the status of DNA methylation of promote region is hymethylation.2、PTEN hymethylation is relatively correlative with the tumor grades and stages of Osteosarcoma.
Part II Influence of Human Osteosarcoma Cell MG-63was Demethylated by5-Aza-CdR on Cytology behavior and PTEN Methylation
Objective:To screen completed methylation cell in the3Osteosarcoma cell lines, and to explicit the effect of demethylation of5-Aza-CdR in vitro, and to confirm the mechanism of action of the PI3K/AKt pathway can be negatively regulated by PTEN in Osteosarcoma cell line.
Methods:The3Osteosarcoma cell lines MG-63,SAOS-2and U2-OS were detected by MSP, and the PTEN completely methylation cell line was screened, and this cell line was intervened by5-Aza-CdR in terms of difference drug density and active time. The protein of the PI3K and the AKt were detected by WB method in this cell line.
Results:The MG-63cell line display the completely methylation in3cell lines, and the best drug density and effect time are separately10μmol/L and24h after5-Aza-CdR intervention. There has been significant difference between the intervening group and the control group (P<0.05), meanwhile, same significant difference between2groups on protein expression of the PI3K and AKt (P<0.05).
Conclusions:1、The human Osteosarcoma MG-63cell line display the completely mythlation on PTEN.2、The MG-63cell line demonstrates the dependency of drug density and effect time after5-Aza-CdR intervention.3、5-Aza-CdR can exert an influence on cell behavior of MG-63.4、The protein expression of the PI3K and the AKt were down-regulated by5-Aza-CdR in Osteosarcoma cell line MG-63, and the mechanism of action of PTEN negatively regulates the PI3K/AKt pathway in Osteosarcoma was confirmed.
Part Ⅲ Influence of Expression and DNA Mythlation of PTEN in Osteosarcoma Nude Mice Model Loaded Transplantation Tumor Intervened Demethylation by5-Aza-CdR
Objective:To establish nude mice model loaded Osteosarcoma cell line MG-63transplantation tumor and to clear the demethylation effect mechanism of5-Aza-CdR in vivo.
Methods:18nude mices which4～6weeks were used subcutaneous vaccination by1×10/200μl cell line MG-63suspension for establishing transplantation tumor loaded nude mice model. After modeling success, the animal models were divided into the group experimental(A) and control(B) separately9animals. The nude mice models of group A were intervened by5-Aza-CdR in terms of drug density10μmol/L and dose1μl/g. After4weeks, the animals were executed and the transplantation tumor were dissected. The volume of the transplantation tumor were compared between group A and B. The expression of PTEN of the transplantation tumor were detected separately by IHC method and Western-Blotting method, meanwhile, the status of DNA methylation in PTEN of it by MSP technique.
Results:The human Osteosarcoma cell line MG-63transplantation tumor loaded nude mice models can be established successfully. There has been significant difference between the group A and B on the volume of transplantation tumor (P<0.05), and similar difference between the groups2on IHC results (P<0.05). The MSP results demonstrates that the PTEN hypomethylation status can be detected in transplantation tumor of group A and there has significant difference between the group A and B (P<0.05).Western-Blotting results demonstrates that the protein expression of the group A were up-regulation, and the same difference between the group A and B (P<0.05)
Conclusions:1、The human Osteosarcoma cell line MG-63nude mice model loaded transplantation tumor can be established successfully through using subcutaneous vaccination method.2、The grow of the transplantation tumor of nude mice model can be inhibited by5-Aza-CdR, and the expression of PTEN of the tissue can be up-regulated and the status of PTEN hypermethylation can be inhibited by5-Aza-CdR in vivo.
PTEN是迄今为止发现的第一个具有双特异性磷酸酶活性的抑癌基因,是抑癌基因家族中继P53发现后在人类肿瘤中突变及缺失率最高的又一明星分子。PTEN具有抑制肿瘤生长、促进细胞凋亡及抑制血管生成等作用,能负性调控PI3K/AKt及抑制MAPK的信号转导通路对肿瘤产生影响。已有研究表明在很多人类恶性肿瘤中都存在PTEN的异常甲基化,但关于其与骨肉瘤的关系却鲜见报道。了解骨肉瘤中PTEN的甲基化状态,对寻找骨肉瘤早期诊断可能的分子标志物具有重要的临床意义。
由于DNA甲基化不涉及DNA序列改变,因此,它是一个可逆的表观遗传学修饰过程。任何一个基因的甲基化状态是一个甲基化和去甲基化的动态平衡过程,处于甲基化异常的抑癌基因对阻断其DNA甲基化的药物非常敏感,临床上可利用这种敏感性进行基因治疗。5-Aza-CdR是已被美国FDA批准应用于骨髓增殖异常综合征及急性粒细胞白血病临床治疗的去甲基化药物,它能在体内外激发DNA的去甲基化过程,导致被甲基化沉默的抑癌基因持续激活。目前已有5-Aza-CdR作用于乳腺癌、非小细胞肺癌及前列腺癌等肿瘤的相关研究,但其与骨肉瘤的关系的研究却还是空白。因此,探讨5-Aza-CdR对肿瘤的去甲基化作用机制对防治骨肉瘤亦具有重要的临床意义。
本研究分成三个部分。第一部分,通过MSP技术检测骨肉瘤标本中PTEN的甲基化状态并将其与患者的临床病理参数结合进行分析,以期探讨PTEN甲基化在骨肉瘤发生中的作用及其相关预后影响因素,为探寻骨肉瘤早期诊断可能的分子靶标提供依据；第二部分,通过MSP技术对三株人骨肉瘤细胞MG-63、SAOS-2和U2-OS进行筛选,选取其中PTEN完全甲基化的一株细胞进行后续细胞学功能实验,以5-Aza-CdR对其进行去甲基化干预,探讨5-Aza-CdR作用的最佳浓度与时间、细胞的生物学行为及甲基化状态,为阐明5-Aza-CdR的体外去甲基化作用机制提供实验依据；第三部分,建立人骨肉瘤MG-63细胞荷瘤裸鼠模型,以5-Aza-CdR经尾静脉注射去甲基化干预4周,观测移植瘤体积生长的动态变化及检测移植瘤的病理形态学、PTEN蛋白表达及其甲基化情况,明确5-Aza-CdR的体内去甲基化作用机制。
第一部分骨肉瘤中PTEN的甲基化情况及其与相关临床病理参数的关系
目的明确骨肉瘤中抑癌基因PTEN的甲基化情况,寻找早期诊断可能的分子标志物。分析PTEN甲基化和骨肉瘤临床病理参数的关系,探讨PTEN甲基化对其相关预后影响因素的影响。
方法采集于2001年4月～2011年4月间在湖南省肿瘤医院、中南大学湘雅二医院及湘雅医院进行治疗并确诊的46例骨肉瘤患者的病理标本,其中新鲜组织21例(同时采集癌旁组织21例)、石蜡标本25例,采用HE染色、免疫组化(S-P)法检测肿瘤组织的病理形态学及PTEN表达水平,然后采用甲基化特异性PCR (MSP)技术检测骨肉瘤组织中PTEN启动子区甲基化的状态；整合所纳入骨肉瘤患者的临床资料,采用x2检验或Fisher's确切概率法分析PTEN启动子区甲基化与临床病理参数之间的关系。
结果HE染色及免疫组化结果显示,骨肉瘤组织中病理形态学呈明显肉瘤特征,而PTEN表达较癌旁组织明显减少,两组比较有统计学差异(P<0.05)。 MSP结果显示,骨肉瘤组织中存在PTEN高甲基化状态,癌旁组织中呈低甲基化或未甲基化状态,两组比较差异有统计学意义(P<0.05)。 PTEN高甲基化与骨肉瘤的Enneking肿瘤外科分期(P=0.023)。 Price组织学分级(P=0.001)及肿瘤发生的部位(P=0.030)表现出显著的正相关(P<0.05),而与年龄、性别、病程及化疗前AKP水平等一般临床资料无明显相关(P>0.05)。PTEN高甲基化患者术后5年生存率明显低于PTEN低甲基化患者,两组比较差异有统计学意义(P<0.05)。
结论1、PTEN在骨肉瘤中表达下调,其DNA启动子区存在高甲基化状态,PTEN高甲基化是骨肉瘤早期发生的分子特征之一。2、PTEN高甲基化状态与骨肉瘤的病理分期及分级呈显著正相关,是其独立的预后影响因素之一。
第二部分5-Aza-CdR去甲基化对人骨肉瘤MG-63细胞生物学行为及PTEN甲基化的影响
目的筛选PTEN完全甲基化的骨肉瘤细胞株,明确5-Aza-CdR对骨肉瘤细胞的体外去甲基化作用及对PTEN甲基化的影响,证实PTEN负性调控骨肉瘤中PI3K/AKt信号转导通路的作用机制。
方法选取三株经典的人成骨肉瘤细胞株MG-63、SAOS-2和U2-OS,用MSP法检测其中PTEN甲基化的情况,筛选出PTEN完全甲基化的一株细胞进行后续实验,以5-Aza-CdR对其进行去甲基化干预,同时筛选出PTEN甲基化状态被完全去除时5-Aza-CdR作用的最佳浓度及时间点,并检测该细胞的生物学行为及其PTEN甲基化的情况,采用Western-Blotting法检测经去甲基化干预后该细胞株中PI3K和AKt信号分子的蛋白表达水平。
结果在三株人骨肉瘤MG-63、SAOS-2和U2-OS中,MG-63细胞中PTEN完全甲基化,其被5-Aza-CdR完全去甲基化的最佳浓度和时间点分别为10μmol/L和24h。PTEN被完全去甲基化后的MG-63细胞在细胞凋亡、细胞周期、细胞侵袭迁移能力等细胞生物学行为方面与处理前比较差异有统计学意义(P<0.05)。 Western-Blotting结果显示,经5-Aza-CdR干预的MG-63细胞中PI3K和AKt分子蛋白的表达水平较对照组明显降低,两者表现出统计学差异(P<0.05)。
结论1、PTEN在人骨肉瘤MG-63细胞中完全甲基化；2、MG-63细胞中PTEN甲基化被5-Aza-CdR完全去除时具有时间和浓度的依赖性；3、5-Aza-CdR能对MG-63细胞的生物学行为产生影响,能增加肿瘤细胞的凋亡率、抑制细胞生长周期及降低细胞的侵袭转移能力；4、5-Aza-CdR能降低MG-63细胞中PI3K和AKt蛋白的表达水平,证实了PTEN负性调控MG-63细胞中PI3K/AKt信号转导通路的作用机制。
第三部分5-Aza-CdR去甲基化对裸鼠模型移植瘤PTEN表达及其甲基化的影响
目的构建人骨肉瘤MG-63细胞荷瘤裸鼠模型；探讨5-Aza-CdR对荷瘤裸鼠模型移植瘤的影响及其在动物体内的去甲基化作用机制。
方法采用细胞悬液异种移植皮下接种法,将人骨肉瘤MG-63细胞单悬液以1×107/200μl的活细胞浓度接种于4～6周龄裸鼠左腋肩部皮下,共接种18只动物,构建荷瘤裸鼠模型。成功建模后,将实验动物分成实验组和对照组,每组9只动物；实验组以10μmol/L浓度、1μ/g剂量的5-Aza-CdR进行尾静脉注射行去甲基化干预,而对照组则注射等量的PBS溶液,4周后处死动物,解剖移植瘤,观测并动态比较两组移植瘤体积大小和病理形态学的变化。采用IHC免疫组化法检测两组移植瘤瘤体组织中PTEN的表达情况,采取MSP技术检测两组瘤体组织中PTEN的甲基化状态,以Western-Blotting法检测并比较两组瘤体组织中PTEN蛋白表达的情况。
结果细胞接种4周后裸鼠移植瘤的大小为5mm3,表明成功地建立了人骨肉瘤MG-63细胞荷瘤裸鼠模型。两组裸鼠模型移植瘤体积大小动态比较,在5-Aza-CdR干预至第4周时两组瘤体体积大小比较差异有统计学意义(P<0.05),实验组优于对照组。IHC结果显示,实验组移植瘤中PTEN表达明显上调,较对照组有统计学差异(P<0.05)。MSP结果显示,实验组移植瘤中PTEN呈低甲基化状态,与对照组比较有统计学差异(P<0.05)。Western-Blotting结果显示,实验组移植瘤中PTEN蛋白表达量明显增加,较对照组比较差异有统计学意义(P<0.05)。
结论1、采用1×107/200μ1浓度的细胞悬液行皮下接种法能成功建立人骨肉瘤MG-63细胞荷瘤裸鼠动物模型；2、5-Aza-CdR能抑制人骨肉瘤MG-63细胞裸鼠模型移植瘤的生长,上调移植瘤中PTEN的表达,抑制其中PTEN的高甲基化状态,在体内发挥了其去甲基化作用。
BACKGROUND Osteosarcoma is one of primary neoplasms of the anterior mediastinum with highly variable histopathological characteristics. This heterogeneity has long hindered the development of a standardized protocol for diagnostic and prognostic evaluation of Osteosarcoma. The modified Enneking staging system, is currently the most commonly employed scoring method, but its value is limited by the fact that it assigns scores only for specific macroscopic surgical, but not histological, characteristics, making it difficult to reliably assess tumor severity in patients. Previous studies showed that multiple genetic alterations involved in many kinds of carcinomas, but the molecular mechanisms of Osteosarcoma is poorly understood. Recent data suggests that carcinomas appear to be a process that is caused by genetic alterations and by epigenetic mechanism. It mainly involves DNA methylation and histone modification. DNA methylation is one of the major mechanisms of epigenetics. Aberrant methylation of promoter regions resulting in inactivation of human tumor suppressor gene expression has been proposed to be an important mechanism in cancer. Cancer cell DNA is frequently characterized by localized gene-specific hypermethylation in TSG, the expression and status of DNA methylation of gene PTEN have already been reported in many kinds of tumor, such as lung cancer, colon cancer and oral carcinoma ea tl. Although multiple genetic and epigenetic changes have been detected in many kinds of cancer, the precise molecular mechanisms in the development and/or progression of Osteosarcoma still remain unknown. PTEN is the first TSG which has both Lipid phosphatase and Dual specificphosphatase action. The DNA mythlation can be altered by demethylation. The drug5-Aza-CdR, the DNA methyltransferase inhibitor, is the most mainly demethylation drug which has been applied for clinical treatment widely and be authorized by FDA,USA. To clarify the demethylation mechanism of the5-Aza-CdR is relatively important to the treatment of Osteosarcoma.
There are3parts content in our research works. In part1, the DNA metyhlation status of PTEN in tissues of the Osteosarcoma was detected by MSP method, and the methylation results and the clinical data were analysed together. In part2, the cell behavior and the DNA methylation status of the MG-63cell were detected by the cytology test and the MSP method. In part3, To establish nude mouse animal models and intervene demethylatedly it through5-Aza-CdR injection, and the expression of PTEN and the DNA methylation of PTEN of the transplantation tumor were detected by WB and MSP methods.
Part I PTEN Abnormal DNA Methylation and Its Clinical Significance in Osteosarcoma
Objective:To investigate the expression and the promoter hypermethylation of PTEN and potential clinical significance in patients with Osteosarcoma.
Methods:Methylation-specific PCR (MSP) method was performed for evaluating the expression and promoter region methylation patterns of PTEN in Osteosarcoma. The correlation between TSGs mRNA, TSGs methylation and clinicopathological data were analyzed.46patients with Osteosarcoma were surgically treated in the HN province tumor hospital, the2nd xiangya hospital and the xiangya hospital, and from April,2001to April,2011. The samples of Osteosarcoma were be detected by HE dye, immunohistochemistry and MSP, and the methylation status of the samples and the clinical data of the Osteosarcoma patients were analysed together for discussing the relationship between the above two.
Results:There has been significant defference between the tumor tissue group and the non-tumor tissue group about the results of the expression of PTEN and the DNA methylation status of PTEN (P<0.05). PTEN hyermethylation is correlative with the tumor grades and stages of Osteosarcoma. Enneking surgery stages of tumor (P=0.002)、Price surgery classification (P=0.003)、position of tumor occurring (P= 0.030).
Conclusions:1、The down-regulation of PTEN in osteosarcoma and the status of DNA methylation of promote region is hymethylation.2、PTEN hymethylation is relatively correlative with the tumor grades and stages of Osteosarcoma.
Part II Influence of Human Osteosarcoma Cell MG-63was Demethylated by5-Aza-CdR on Cytology behavior and PTEN Methylation
Objective:To screen completed methylation cell in the3Osteosarcoma cell lines, and to explicit the effect of demethylation of5-Aza-CdR in vitro, and to confirm the mechanism of action of the PI3K/AKt pathway can be negatively regulated by PTEN in Osteosarcoma cell line.
Methods:The3Osteosarcoma cell lines MG-63,SAOS-2and U2-OS were detected by MSP, and the PTEN completely methylation cell line was screened, and this cell line was intervened by5-Aza-CdR in terms of difference drug density and active time. The protein of the PI3K and the AKt were detected by WB method in this cell line.
Results:The MG-63cell line display the completely methylation in3cell lines, and the best drug density and effect time are separately10μmol/L and24h after5-Aza-CdR intervention. There has been significant difference between the intervening group and the control group (P<0.05), meanwhile, same significant difference between2groups on protein expression of the PI3K and AKt (P<0.05).
Conclusions:1、The human Osteosarcoma MG-63cell line display the completely mythlation on PTEN.2、The MG-63cell line demonstrates the dependency of drug density and effect time after5-Aza-CdR intervention.3、5-Aza-CdR can exert an influence on cell behavior of MG-63.4、The protein expression of the PI3K and the AKt were down-regulated by5-Aza-CdR in Osteosarcoma cell line MG-63, and the mechanism of action of PTEN negatively regulates the PI3K/AKt pathway in Osteosarcoma was confirmed.
Part Ⅲ Influence of Expression and DNA Mythlation of PTEN in Osteosarcoma Nude Mice Model Loaded Transplantation Tumor Intervened Demethylation by5-Aza-CdR
Objective:To establish nude mice model loaded Osteosarcoma cell line MG-63transplantation tumor and to clear the demethylation effect mechanism of5-Aza-CdR in vivo.
Methods:18nude mices which4～6weeks were used subcutaneous vaccination by1×10/200μl cell line MG-63suspension for establishing transplantation tumor loaded nude mice model. After modeling success, the animal models were divided into the group experimental(A) and control(B) separately9animals. The nude mice models of group A were intervened by5-Aza-CdR in terms of drug density10μmol/L and dose1μl/g. After4weeks, the animals were executed and the transplantation tumor were dissected. The volume of the transplantation tumor were compared between group A and B. The expression of PTEN of the transplantation tumor were detected separately by IHC method and Western-Blotting method, meanwhile, the status of DNA methylation in PTEN of it by MSP technique.
Results:The human Osteosarcoma cell line MG-63transplantation tumor loaded nude mice models can be established successfully. There has been significant difference between the group A and B on the volume of transplantation tumor (P<0.05), and similar difference between the groups2on IHC results (P<0.05). The MSP results demonstrates that the PTEN hypomethylation status can be detected in transplantation tumor of group A and there has significant difference between the group A and B (P<0.05).Western-Blotting results demonstrates that the protein expression of the group A were up-regulation, and the same difference between the group A and B (P<0.05)
Conclusions:1、The human Osteosarcoma cell line MG-63nude mice model loaded transplantation tumor can be established successfully through using subcutaneous vaccination method.2、The grow of the transplantation tumor of nude mice model can be inhibited by5-Aza-CdR, and the expression of PTEN of the tissue can be up-regulated and the status of PTEN hypermethylation can be inhibited by5-Aza-CdR in vivo.
引文
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