【作者】 李颖；

【导师】 陈建斌；

【作者基本信息】 重庆医科大学 ， 内科学， 2012， 硕士

【摘要】 多发性骨髓瘤（multiple myeloma, MM）是浆细胞克隆增殖性恶性肿瘤，在血液系统恶性肿瘤中所占的比例超过10％，现有治疗手段除骨髓移植外难以有效治愈。研究表明多发性骨髓瘤的发生发展与多种癌基因过表达及抑癌基因失活密切相关，但目前对其发生发展的精确分子机制尚不完全清楚。DNA甲基化是肿瘤中最常见的表观遗传改变之一，启动子CpG岛异常甲基化是导致抑癌基因沉默的重要机制之一。近年来50多个基因启动子区域异常甲基化先后被报道，作为一个转录调节器它在多种人类肿瘤发生发展中具有不可小觑的作用。研究发现基因启动子区异常甲基化与多发性骨髓瘤有关，其中大部分为抑癌基因。表观遗传机制调节的PCDH10沉默发生在多种人类癌症。位于PCDH10启动子5′-侧462-bp片段的区域具有高密度的CpG岛，而CpG岛正是DNA甲基化发生的区域。PCDH10基因的沉默可能减弱了机体对癌细胞迁移的抑制作用，从而有助于癌症的发展。研究发现PCDH10基因在人类胃癌中有潜在的抑癌作用，已有报道该基因的甲基化在胃癌早期是一个独立的预后评价因素，动态监测PCDH10基因甲基化对胃癌癌前病变的患者具有重要意义。本课题拟探讨PCDH10基因在MM发病中的作用及相关机制。实验方法及结果1测定PCDH10基因在两株MM细胞系及患者样本中表达状态、甲基化状态及两者相关性1.1PCDH10表达水平的测定：MM细胞株及随机9例MM患者、3例正常志愿者骨髓样本（对照）提取RNA，RT-PCR显示MM细胞株及MM患者骨髓标本中PCDH10基因下调或缺失，正常志愿者骨髓中PCDH10基因均表达。1.2PCDH10甲基化测定：甲基化特异性PCR（MSP）测定两株MM细胞株及44例MM患者骨髓标本中PCDH10甲基化水平，以9例正常志愿者骨髓标本为对照组。收集MM患者的临床信息，统计学分析其与DNA甲基化相关性。MSP示34/44（77.27%）MM患者样本存在PCDH10基因甲基化，而9例对照组样本中均未检测出PCDH10基因甲基化。临床统计分析表明PCDH10基因甲基化状态与患者年龄、性别、ISS分期、血肌酐Cr、血清钙等未见明显统计学差异（P＞0.05）。1.3探讨DNA甲基化修饰是否PCDH10基因下调或缺失的原因：应用5-氮杂-2′-脱氧胞苷(5-aza-2’-deoxycytidine，Aza)和曲古抑菌素A（trichostatin A，TSA)处理PCDH10基因沉默的细胞株，MSP及RT-PCR分别测定DNA甲基化状态变化及基因表达状态。在KM3、RPMI-8226骨髓瘤细胞系经过A+T（Aza+TSA）处理后甲基化特异性扩增产物条带减弱，经A+T处理后均可扩增出非甲基化产物。RT-PCR显示A+T处理后PCDH10基因恢复表达。2PCDH10基因对多发性骨髓瘤的影响构建PCDH10表达载体pcDNA3.1-PCDH10，以脂质体2000为载体转染多发性骨髓瘤细胞系RPMI-8226细胞，以最小致死浓度G418筛选表达PCDH10基因的阳性克隆，Western blot鉴定转染后PCDH10蛋白表达。探讨PCDH10基因表达对多发性骨髓瘤细胞增殖及血管新生的影响。2.1增殖：克隆形成实验显示PCDH10基因阳性表达后MM细胞增殖缓慢，克隆细胞数减少、克隆形成率减低，有统计学差异；流式细胞分析PCDH10基因阳性表达后进入G1期的细胞数有所减少，细胞阻滞于S期，有统计学差异；2.2血管新生：鸡胚尿囊膜实验显示PCDH10基因阳性表达后，新生血管产生畸形，血管节点数下降。结论1PCDH10基因在MM中表达下调或缺失；2DNA甲基化是MM的PCDH10基因沉默的重要机制之一；3PCDH10基因在MM细胞系中具有抑制细胞增殖、抑制血管新生的作用。更多还原

【Abstract】 Multiple myeloma (multiple myeloma, MM) is a plasma cell tumor,which takes up more than10%in haematological malignancies. Beside thebone marrow transplantation，MM is difficult to cure by the existingtreatment. Studies have shown that multiple myeloma closely related withmultiple oncogene overexpression, and inactivation of tumor suppressorgenes. But at present the development of the precise molecular mechanismis not fully understood.DNA methylation in human tumors is one of the most commonphenomenon. Aberrant promoter methylation in CpG island has beenrecognized as a tumor suppressor gene silencing mechanism. In recent years,more than50gene promoter region methylation anomalies has been reported.Clearly shown, as a transcriptional regulator DNA methylation plays a keyrole in human cancers. Have found that the gene promoter hypermethylationclosely associated with MM, these susceptibility genes almost involved in allknown cell information pathways. Most of these gene is tumor suppressor,hypermethylation inactivation leads to gene expression induced by loss of function, make the individual predisposition to MM.Epigenetic mechanisms regulating PCDH10silencing occurs in avariety of human cancers. In the PCDH10promoter,5’-side462-bpfragment areas with high density of CpG Island, is DNA methylation occursregion. The PCDH10gene silencing may weaken the inhibition of cancercell migration, which contributes to the cancer development. Studies showthat PCDH10can be used as a gastric cancer suppressor gene, methylationcan be used as an independent prognostic factors, and as a tumor marker fordynamic monitoring of precancerous lesions, which has importantapplication value in the early stage of gastric cancer prevention andtreatment.Our study do some degree of interpretation the inactivation of PCDH10gene in MM.Methods and Results：1. Determinated the expression state of PCDH10in MM1.1PCDH10expression level determination: Determination theexpression level of PCDH10of MM cell lines and9random MM patientsbone marrow by RT-PCR, with3normal volunteers as controls. RT-PCRshows PCDH10in MM cell lines and MM patients’ bone marrow specimensexpress very low or silenced, while normal bone marrow PCDH10highexpressed in normal volunteers.1.2methylation assay: Determination the methylation level of PCDH10 in MM cell lines and in44MM patients bone marrow by methylationspecific PCR (MSP), with9normal volunteers as controls. And thecollection clinical information of MM patients, analysis its correlation withDNA methylation.MSP shows34/44(77.27%) patients with MM samples appearedmethylation, and in9cases of control group PCDH10were not detectedmethylation. Clinical statistical analysis showed that the PCDH10genemethylation status has no obvious statistical difference with patients’ age,gender, ISS staging, serum creatinine, serum calcium, Cr (P>0.05).1.3Explored whether PCDH10inactivation cause of DNAmethylation:A+T(5-aza-2’-deoxycytidine (Aza) and trichostatin A (TSA))application in the PCDH10gene silencing in cell lines. RT-PCR showedA+T treatment may amplified PCDH10unmethylation specific product strip,proofed that after treatment with A+T, KM3、RPMI-8226cell recoveryPCDH10’s expression.2. PCDH10tumor suppressor function in multiple myeloma:Building a PCDH10expression vector pcDNA3.PCDH10, withliposomes2000vector transfection of human multiple myeloma cell lineRPMI-8226cells, G418screening the clone which PCDH10geneexpression. Western blot identificated PCDH10protein expression. Studythe effect of PCDH10gene expression multiple myeloma cells on apoptosis and angiogenic:2.1Proliferation: Colony formation assays show that after PCDH10expression in MM cells, clone number decreased, colony formation rate isreduced; flow cytometric analysis show G1phase of the cell numberdecreased by PCDH10expression, also cells arrest in S phase;2.2Angiogenesis: Chick chorioallantoic membrane (CAM) assaysshow that if PCDH10expressed in MM, angiogenesis will generatemalformations, with vascular node number droped.Conclusion：1PCDH10inactivation in MM;2DNA methylation play a role in the silencement of PCDH10in MM;3PCDH10is involved in angiogenesis and proliferation correlated withmultiple myeloma.更多还原