胆管癌表遗传学研究
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摘要
胆管癌是一种恶性程度很高的肿瘤性疾病，早期无特异性诊断标准，诊断明确时多已是晚期，已失去了手术时机；且目前临床常用化疗药物对胆管癌均疗效较差。因此，寻找新型的对胆管癌敏感的治疗药物具有非常重要的意义。
表遗传学主要研究没有DNA序列改变的可遗传表达改变，即提供何时、何地和如何应用遗传学信息的指令，以确保基因适当的表达或关闭。当人类进入后基因组时代，尤其是人类表观基因组计划的提出和实施后，表遗传学引起了越来越多的关注，并逐渐成为阐明基因组功能的关键研究领域，也为研究肿瘤的治疗提出一个全新的方向。表遗传的改变主要是对基因序列的修饰，而不改变基因序列本身，因此它是可逆的，这为利用表遗传学方法治疗肿瘤提供了理论依据。
现阶段，许多肿瘤亚型缺少染色体或者微卫星不稳定性的事实挑战了基因组不稳定在启动和维持肿瘤发生、发展上起主要作用的假说。肿瘤特异性的以表遗传学改变为基础的通过异常DNA甲基化、异常组蛋白修饰以及异常的核小体定位而导致的基因表达的改变被认可为肿瘤发生的驱动因素，其中肿瘤抑制基因的灭活和原癌基因的活化是癌变的中心事件。因此，通过表遗传药物逆转肿瘤细胞异常表遗传改变，恢复沉默抑癌基因表达，进而达到治疗肿瘤的目的，成为了我们课题组的研究方向。
在邹声泉教授的带领下，本课题组研究证实了DNA甲基化酶抑制剂5-Aza-CdR具有在体外抑制胆管癌细胞增殖、促进细胞调亡的作用；并证实联合下调DNA甲基转移酶DNMT1和DNMT3b能使RASSF1A基因启动子区域去甲基化并诱导其重新表达，表明抑制DNA甲基转移酶可使抑癌基因启动子区去甲基化，恢复表达。近年来，研究者逐步发现，抑癌基因沉默的根本原因是染色质结构变得致密，影响了转录起始因子与DNA上相应转录起始部位的结合，而正是抑癌基因启动子区的超甲基化和组蛋白的脱乙酰化决定了染色质的变构。根据这一发现，和以前研究基础，本试验研究了DNA甲基化酶抑制剂(肼屈嗪)与组蛋白脱乙酰化酶抑制剂(丙戊酸)联用对胆管癌细胞QBC_(939)增殖、细胞侵袭转移力以及细胞中抑癌基因表达的影响。
另外，近两年发现第三类组蛋白脱乙酰化酶Sir2家族中SIRT1蛋白在导致抑癌基因沉默以至肿瘤的发生中有非常重要的作用，为此我们专门探讨了DNA甲基化酶抑制剂、组蛋白脱乙酰化酶抑制剂与SIRT1之间的关联性。在研究中我们发现对于组蛋白脱乙酰化酶抑制剂不敏感的SIRT1在组蛋白脱乙酰化酶抑制剂与DNA甲基化酶抑制剂联合作用下表达减少。进一步研究后明确了恢复HIC1基因表达，抑制SIRT1表达是组蛋白脱乙酰化酶抑制剂与DNA甲基化酶抑制剂联合作用胆管癌细胞中抑癌基因表达的机制之一。本研究为进一步明确组蛋白脱乙酰化酶抑制剂与DNA甲基化酶抑制剂联合作用机制、明确表遗传学变化间的关系奠定了基础，并为利用表遗传学方法治疗胆管癌提供了实验依据。
第一部分：DNMTi与HDACi对胆管癌细胞的干预效应
论文1．DNMTi与HDACi对胆管癌细胞中抑癌基因表达的影响
目的研究肼屈嗪与丙戊酸联合作用对于体外胆管癌细胞QBC_(939)中P16、RASSF1A、E-Cadherin等抑癌基因mRNA及蛋白表达，以及启动子区甲基化状态的影响。
方法将胆管细胞QBC_(939)分为空白组、单用丙戊酸组、单用肼屈嗪组与两药合用组，相应给予丙戊酸、肼屈嗪以及两药联用，空白组给予等量RPIM-1640培养液，干预48h后，RT-PCR、Western-blot法检测各组细胞中P16、RASSF1A、E-Cadherin基因mRNA及其蛋白表达；MSP法检测各组细胞中P16、RASSF1A、E-Cadherin基因启动子区甲基化状态。
结果1．空白组胆管癌细胞QBC_(939)中P16基因表达量极低，RASSF1A与E-Cadherin基因均未见表达；单用丙戊酸组细胞中P16、RASSF1A及E-Cadherin基因表达量与其空白组一致；单用肼屈嗪组细胞中P16、RASSF1A及E-Cadherin基因表达量较空白组及单用丙戊酸组增多(P＜0．01)；两药联用组细胞中P16、RASSF1A及E-Cadherin基因表达量较其它三组明显增高(P＜0．01)。2．空白组中胆管癌细胞QBC_(939)P16蛋白表达量极低，未见RASSF1A与E-Cadherin蛋白表达；单用丙戊酸组细胞三种蛋白表达量与其空白组一致；单用肼屈嗪组细胞中三种蛋白表达量较空白组和单用丙戊酸组增多(P＜0．01)；两药联用组细胞中P16、RASSF1A及E-Cadherin蛋白表达量较其余三组明显增高(P＜0．01)。3．空白组胆管癌细胞QBC_(939)中，P16基因启动子区呈部分去甲基化；RASSF1A、E-Cadherin基因启动子区超甲基化；单用丙戊酸组细胞RASSF1A、E-Cadherin基因启动子区仍呈超甲基化状态，P16基因启动子区仍呈部分去甲基化状态；单用肼屈嗪组细胞中三种基因启动子区均呈部分去甲基化状态；两药联组细胞中三种抑癌基因启动子区呈完全去甲基化状态。
结论肼屈嗪与丙戊酸两药合用可诱导胆管癌细胞QBC_(939)中P16、RASSF1A、E-Cadherin三种抑癌基因启动子区完全去甲基化，并可促进三种基因mRNA及其蛋白大量恢复表达；而单用一种药物效果明显弱于两药合用。
论文2．DNMTi与HDACi对胆管癌细胞生长与侵袭转移力的影响
目的探讨肼屈嗪与丙戊酸联合作用对于体外胆管癌细胞QBC_(939)生长与侵袭转移力的影响。
方法将胆管细胞QBC_(939)分为空白组、单用丙戊酸组、单用肼屈嗪组与两药合用组，相应给予丙戊酸、肼屈嗪以及两药联用，空白组给予等量RPIM-1640培养液，48h后1．流式细胞术Annexin V／PI双标法检测各组细胞的凋亡情况；2．Transwell法检测各组细胞侵袭转移力；
结果1．空白组细胞凋亡率为(2．855±0．936)％，单用丙戊酸组细胞凋亡率为(1 1．56±1．590)％，单用肼屈嗪组细胞凋亡率为(24．65±2．330)％；两药合用组细胞凋亡率为(47．93±3．973)％。两药合用组凋亡细胞较单用药组调亡细胞数量明显增多(P＜0．01)。2．计数移至微孔下层的细胞，结果显示空白组细胞侵袭转移力最强(P＜0．01)；肼屈嗪与丙戊酸联合作用组细胞侵袭转移力最弱(P＜0．01)；肼屈嗪组细胞侵袭转移力较丙戊酸组弱(P＜0．01)。
结论单用肼屈嗪与单用丙戊酸均可引起体外胆管癌细胞QBC_(939)不同程度凋亡；但两药合用致使细胞凋亡数量远多于单用一种药物；单用肼屈嗪与单用丙戊酸均可引起体外胆管癌细胞QBC_(939)侵袭转移力不同程度减弱，但两药合用效果明显强于单用一种药物。表明两药合用在致细胞凋亡及降低胆管癌细胞QBC939侵袭转移力方面有增效作用。
第二部分：DNMTi与HDACi对胆管癌细胞中SIRT1基因表达的调节
论文3．DNMTi与HDACi通过恢复胆管癌细胞中HIC1基因表达抑制SIRT1基因表达
目的探讨肼屈嗪与丙戊酸对胆管癌细胞QBC_(939)中SIRT1基因表达的影响及调节机制。
方法将正常胆管上皮细胞HIBEC-HR和胆管癌细胞QBC_(939)均分别分为空白组、单用丙戊酸组、单用肼屈嗪组及两药合用组，相应给予丙戊酸、肼屈嗪以及两药联用，空白组给予等量RPIM-1640培养液，干预48h后，RT-PCR、Westen blot法检测各组胆管癌细胞QBC_(939)中SIRT1、HIC1基因mRNA及其蛋白表达；RT-PCR法检测各组正常胆管上皮细胞HIBEC-HR中SIRT1基因mRNA及其蛋白表达；构建含HIC1基因干扰片段质粒，将胆管癌细胞QBC_(939)分为两药合用组、两药合用+阴性对照组、两药合用+干扰质粒组，两药合用+阴性对照组与两药合用+干扰质粒组内分别转染阴性质粒和干扰质粒，两药合用组转染液中不含质粒，转染完成后以两药联用干预各组细胞，48h后，RT-PCR、Westenblot法检测干扰HIC1基因后两药联用对SIRT1基因mRNA及其蛋白表达的影响
结果1．空白组、单用肼屈嗪组与单用丙戊酸组胆管癌细胞QBC_(939)间SIRT1基因及其蛋白表达无统计学差异(P＞0．05)，两药联用组胆管癌细胞QBC_(939)内SIRT1基因及蛋白的表达较其余三组明显降低(P＜0．01)；两药联用组内HIC1基因及其蛋白表达较单用药组以及空白组明显增高(P＜0．01)，空白组与单用丙戊酸组内无HIC1基因及其蛋白表达，单用肼屈嗪组中可见HIC1基因及蛋白的少量表达。2．干预后各组间正常胆管上皮细胞HIBEC-HR内SIRT1基因及蛋白表达无统计学差异(P＞0．05)。3．抑制HIC1基因表达后，两药合用+干扰质粒组内细胞SIRT1基因及其蛋白表达较两药合用组和两药合用+阴性质粒组明显增强(P＜0．01)，而后两组间SIRT1表达无统计学差异(P＞0．05)。
结论肼屈嗪与丙戊酸联合作用通过上调HIC1基因表达降低胆管癌细胞QBC_(939)内SIRT1基因表达。
第三部分：DNMTi与HDACi联合作用抑制胆管癌细胞中SIRT1表达恢复RASSF1A基因表达
论文4．DNMTi与HDACi联合作用抑制胆管癌细胞中SIRT1表达恢复RASSF1A基因表达
目的探讨SIRT1在肼屈嗪与丙戊酸联合恢复胆管癌细胞QBC_(939)RASSF1A基因表达中的作用。
方法成功构建含HIC1基因干扰片段质粒，将培养的胆管癌细胞QBC939分为：两药合用组(H+V组)；两药合用+阴性对照组(H+V+pHK组)；两药合用+干扰质粒组(H+V+pSihHIC1-2组)，两药合用+阴性对照组与两药合用+干扰质粒组内分别转染阴性质粒和干扰质粒，两药合用组转染液中不含质粒，转染完成后以两药联用干预各组细胞，48h后，RT-PCR、Western blot法检测各组细胞中RASSF1A基因及其蛋白表达；另取胆管癌细胞分为两组，分别为：两药合用+干扰质粒组(H+V+pSihHIC1-2组)和两药合用+干扰质粒+烟酰胺组(H+V+pSihHIC1-2+NAM组)，转染含HIC1基因干扰片段质粒成功后给予相应药物干预48h后，RT-PCR、Western blot法检测各组细胞中RASSF1A基因mRNA及其蛋白表达；MSP法检测各组细胞中RASSF1A基因启动子区甲基化状态；
结果1．RT-PCR结果显示，两药合用组与两药合用+阴性对照组中RASSF1A基因mRNA表达较两药合用+干扰质粒组强(P＜0．01)；Western blot结果与RT-PCR一致，两药合用组与两药合用+阴性对照组中RASSF1A蛋白表达较两药合用+干扰质粒组高(P＜0．01)，MSP结果显示，三组细胞RASSF1A基因启动子区甲基化状态一致，都呈完全去甲基化状态。2．RT-PCR结果显示，两药合用+干扰质粒组中RASSF1A基因mRNA表达较两药合用+干扰质粒+烟酰胺组明显减弱(P＜0．01)；Western blot结果显示两药合用+干扰质粒组中RASSF1A蛋白表达较两药合用+干扰质粒+烟酰胺组明显减弱(P＜0．01)。3．各组细胞中RASSF1A基因启动子区均呈去甲基化状态
结论SIRT1参与了肼屈嗪与丙戊酸恢复胆管癌细胞QBC_(939)中抑癌基因RASSF1A表达的过程；恢复HIC1表达继而抑制SIRT1表达，是肼屈嗪与丙戊酸恢复胆管癌细胞QBC_(939)中抑癌基因RASSF1A表达的机制之一。SIRT1表达改变对抑癌基因启动子区甲基化状态无影响。
小结
本课题从利用DNA甲基转移酶抑制剂(肼屈嗪)与组蛋白脱乙酰化酶抑制剂(丙戊酸)对体外胆管癌细胞的干预效果入手，研究了两药对于体外胆管癌细胞QBC_(939)的干预效应；并通过RNA干扰技术抑制HIC1基因表达研究了肼屈嗪与丙戊酸对于SIRT1表达的调节作用，继而研究了SIRT1基因在肼屈嗪与丙戊酸联合作用恢复体外胆管癌细胞QBC939细胞抑癌基因表达中发挥的作用。根据以上研究，得出以下结果和创新点：
1．本课题首次研究了甲基化酶抑制剂(肼屈嗪)与组蛋白脱乙酰化酶抑制剂(丙戊酸)对于体外胆管细胞QBC_(939)的干预效应，发现两药在恢复QBC_(939)细胞中抑癌基因表达、诱导细胞调亡以及降低细胞侵袭转移力上具有增效作用，即1+1＞2的作用。显示出两药合用对于胆管癌的治疗有较好的前景。
2．同时用肼屈嗪与丙戊酸干预胆管癌细胞QBC_(939)与正常胆管上皮细胞HIBEC-HR，发现QBC_(939)细胞中SIRT1表达降低而HIBEC-HR细胞中SIRT1表达不变，表明QBC_(939)细胞中SIRT1发生了异常表达。
3．通过构建并转染含HIC1基因干扰片段质粒到胆管癌细胞QBC_(939)中，再给予两药同时干预转染后细胞，检测SIRT1表达发现，甲基化酶抑制剂与组蛋白脱乙酰化酶抑制剂联合作用可通过恢复HIC1基因表达降低第三类组蛋白脱乙酰化酶抑制剂(Sir2)成员SIRT1的表达。
4．通过转染含HIC1基因干扰片段质粒进QBC_(939)细胞内，同时给予肼屈嗪丙戊酸和烟酰胺干预转染后细胞，观察QBC_(939)细胞中RASSF1A基因表达变化，发现SIRT1参与了肼屈嗪与丙戊酸恢复胆管癌细胞QBC_(939)中抑癌基因RASSF1A表达的过程。这一结果表明，恢复HIC1表达继而抑制SIRT1表达，是肼屈嗪与丙戊酸联会作用恢复胆管癌细胞QBC_(939)中抑癌基因RASSF1A表达的机制之一。
5．本试验揭示了DNA甲基转移酶、组蛋白脱乙酰化酶对SIRT1异常表达的调节作用，阐明了SIRT1在DNA甲基转移酶与组蛋白脱乙酰化酶恢复胆管癌细胞QBC939抑癌基因RASSF1A表达中的作用。同时，发现了SIRT1这一DNA甲基转移酶与组蛋白脱乙酰化酶联合作用的效应因子，为今后继续发现其他效应因子及针对效应因子研究肿瘤的治疗奠定了基础。
Bile duct carcinoma is a malignancy originating from the biliary epithelium.To datethere is no imaging techniques or molecular markers that can aid in the accurate diagnosisand evaluation of bile duct carcinoma in the early stage of the disease.So many patientspresented with unresectable diseases when the diagnosis was definite.And also Bile ductcarcinoma is an aggressive malignancy typified by and unresponsiveness to the existingchemotherapy and radiotherapy regimes in the vast majority of cases.So,it is importantto explore new drugs for the treatment of bile duct carcinoma.
Epigenetics is a new science that studies the change of gene expression caused bynon-gene sequence alteration.It decides gene transcription and the way of gene expression.Since human being enters the post-genome era,epigeneitcs has attracted more and morescholars' attention and is becoming one of the major study fields for the function ofgenome.Epigenetics provides a brand-new research direction for the study of the tumortherapy.Epigenetics refers to alternate phenotypic states that are not based on differencesin genotype,and are potentially reversible,which provides the theoretical basis for the useof epigenetie therapy for tumors.
Nowadays,the fact that subsets of many cancers lack chromosomal or microsatelliteinstability argues against the hypothesis that genomic instability plays an essential role inthe initiation and maintenance of oncogenesis.Cancer-specific,epigenetically-basedchanges in gene expression caused by abnormalities in DNA methylation,histonemodifications,and nucleosome positioning are gaining recognition as driving events intumorigenesis,in which the epigenetic gene silencing is considered to be a key event.Soour research focuses on reversing the abnormal epigenetic pattern of tumor cells,especiallyreactivating the silent tumor suppressor gene by epigenetic drugs.
In our previous studies,we observed the cell cycle alteration of human biliary tractcarcinoma cell line QBC_(939) after treatment with DNMTs inhibitor 5-Aza-CdR and foundthat 5-Aza-CdR could suppress the growth and proliferation of QBC_(939) in vivo and in vitro and induce apoptosis,we also found that using antisense RNA technology to down-regulateDNMT1 and DNMT3b expression levels in human biliary tract carcinoma cell line QBC_(939)could make the promoter region of the RASSF1A gene to be demethylated and couldreactivate it.These experimental results suggested that DNA methyltransferases might playan important role in re-expressing tumor suppressor gene of QBC_(939) cells.In recent years,advances in research of chromatin structure,histone modification,transcriptional activityand DNA methylation have resulted in an increasingly integrated view of epigenetics.It isconfirmed that aberrant gene transcription resulting from epigenetic changes are frequentevents in the molecular pathogenesis of malignant transformation.DNA hypermethylationand histone deacetylation are critical for determining a closed chromatin structureresponsible for or related with aberrant gene transcription in malignancies.Based on suchobservations,in this research we focus on the effect of DNA methylationinhibitors(hydralazine) and histone deacetylase inhibitors(valproate) on the proliferationand invasiveness of the QBC_(939) cells and the expression of tumor suppressor gene of thecells.
In addition,it is reported that SIRT1,a member of silentin formation regulator 2family,plays an important role in the remodeling of chromatin and tumorigenesis.And inour research,we also found that using hydralazine and valproate in combination coulddown-regulate the SIRT1 expression of QBC939 cells,which was insensitive to histonedeacetylase inhibitors.So we further explored the mechanism of the effect of hydralazineand valproate on SIRT1 gene,and the role of SIRT1 gene in re-expressing RASSF1A byusing hydralazine and valproate in combination.This research may lay a foundation for theepigenetic therapy of bile duct carcinoma and clarify the mechanism of the effect of DNAmethylation inhibitors and histone deacetylase inhibitors on tumor cells.
PartⅠIntervention effect of DNMTi and HDACi on cells of humanbile duct carcinoma in vitro
Paper 1 The effect of DNMTi and HDACi on the expression of tumorsuppressor genes of QBC_(939) cells in vitro
Objective To investigate the effects of hydralazine and valproate on P16,RASSIF1A andE-cardherin gene of QBC_(939) cells in vitro.
Methods The QBC_(939) cells were divided into four groups.The experimental groups weretreated separately with hydralazine and valproate either alone or combined(the finalconcentration was 10mmol/L),and the control group was added with RPIM-1640 culturemedium.After 48hours,the expression of the mRNA and the protein of the three genes wasevaluated by reverse transcription-polymerase chain reaction and Western blot assay,andthe methylation status of promoter region of the three genes was detected byMSP(mehtylation specific PCR).
Results It was found that the promoter regions of P16,RASSIF 1A and E-cardherin gene ofQBC_(939) cells were hypermethylated;valproate alone could not contribute to demethyaltionof the three genes,while hydralazine could make them to be partially demethylated.However,the methylation status of the three genes could be thoroughly reversed by usingvalproate and hydralazine in combination.It was comfirmed that the three genes of QBC_(939)cells could not be transcriptionally reactivated by valproate alone,while hydralazine alonecould induce minimal re-expression of the three genes.However,using valproate andhydralazine in combination ccould result in robust re-expression of the threegenes(P＜0.01).
Conclusion The two drugs can synergistically reactivate the P16,RASSF1A andE-cardherin gene of QBC_(939) cells and make the promoter region of the three genes to bedemethylated.
Paper 2 The effect of DNMTi and HDACi on the growth and invasion ofQBC_(939) cells in vitro
Objective To investigate the effect of hydralazine and valproate on the growth and invasionof QBC_(939) cells in vitro
Methods The QBC_(939) cells were divided into four groups.The experimental groups weretreated separately with hydralazine and valproate either alone or combined(the finalconcentration was 10mmol/L),and the control group was added with RPIM-1640 culturemedium.After 48hours,the apoptosis of treated cells was detected by flow cytometry withAnnexin V/PI staining,and the in vitro invassiveness of the treated cells was detected byTranswell permeable assays.
Results 1.The flow cytometry assays showed that the treated cells were induced toapoptosis in different degree,but the number of apoptotic cells in both drugs group wasmuch higher than that of the other two groups(P＜0.01).It also showed that usinghydralazine alone could induce more cells to apoptosis than valproate could(P＜0.01).
2.The Transwell assays demonstrated that the invasiveness of treated cells wasreduced in different extent,but a combination of two drugs worked better than either drugalone(P＜0.01).It was also found that hydralazine worked better than valproate on reducingthe invasiveness of QBC_(939) cells.Conclusion The two drugs have synergy effect on reducing the growth and invasiveness ofthe QBC_(939) cells.
PartⅡMechanism of the effect of hydralazine and valproate onSIRT1 gene of QBC_(939) cells
Paper3 DNMTi and HDACi decrease the expression of SIRT1 viare-expressing the HIC 1 gene
Objective To investigate the mechanism of the effect of hydralazine and valproate onSIRT1 gene of QBC_(939).
Methods 1.The QBC_(939) cells were divided into four groups,and HIBEC-HR cells weretreated identically.Both of them were treated separately with hydralazine and valproateeither alone or combined(the final concentration was 10mmol/L),and the control groupwas added with RPIM-1640 culture medium.After 48hours,the expression of SIRT1 andHIC1 gene of the treated cells and cells in the contral group were detected with RT-PCRand Western blot.
2.One pair of HIC1 target sequence-specific small interfering RNA (siRNA) was designedand synthesized,then siRNA/liposome complex was used to transfect QBC cells.Aftertreating the transfected cells with hydralazine and valproate inhibitors for 48 hours,theexpression of SIRT1 and HIC1 gene of these cells was detected with RT-PCR and Westernblot.
Results The expression of SIRT1 gene was detected to be decreased only when the cellswere treated with hydralazine and valproate in combination(P＜0.01);the inhibition ofhydralazine and valproate on SIRT1 gene was weakened when HIC1 gene wasslieneed(P＜0.01).
Conclusion Hydralazine and valproate decrease the expression of SIRT1 via re-expressingthe HIC1 gene.
PartⅢUsing hydralazine and valproate in combination toreactivate the RASSF1A gene by repressing the SIRT1 gene ofQBC_(939) cells
Paper4 Using hydralazine and valproate in combination to reactivate theRASSF1A gene by repressing the SIRT1 gene of QBC_(939) cells
Objective To investigate the role of SIRT1 gene in re-expressing RASSF1A of QBC_(939)cells by using hydralazine and valproate in combination.
Methods a batch of QBC_(939) cells were divided into three groups,then siRNA/liposomecomplex was used to transfect one group of QBC_(939) cells,the other two groups of cellswere transfected with pHK or none.After treating the transfected cells with hydralazine andvalproate for 48 hours,the expression of RASSF1A was evaluated by reversetranscription-PCR and western blot.Another batch of QBC_(939) cells were divided into twogroups,After siRNA/liposome complex was used to transfect these cells,hydralazine andvalproate were added into the cells.Then one group of cells were added with nicotinamide,the other group were added with RPIM-1640.48h later,the expression of RASSF1A wasevaluated by reverse transcription-PCR and western blot.Finally the promoter regionmethylation status of RASSF1A of the five groups of cells was detected withMSP(mehtylation specific PCR).
Results In the first batch of QBC_(939) cells,the expression of RASSF1A of the group whichwas transfected with siRNA was much weaker than that of the other two groups(P＜0.01).In the other batch of QBC_(939) cells the expression of RASSF1A of the group which wastreated with nicotinamide was much higher than that of the other group(P＜0.01).The statusof the promoter region of RASSF1A of the five groups was demethlated.
Conclusion Hydralazine and valproate reactivate the RASSF1A gene of QBC_(939) cells insome extent via repressing the expression of SIRT1.
Our research focused on the the effect of DNMTi and HDACi on the expression oftumor suppressor genes of QBC_(939) cells in vitro.Then we studied the mechanism of theeffect of hydralazine and valproate on SIRT1 gene of QBC_(939) cells by utilizing siRNAtechnology to knockdown HIC 1 expression.Finally we explored the the role of SIRT 1 genein re-expressing RASSF1A of QBC_(939) cells by using hydralazine and valproate incombination.According to the experimental results,we summarized the conclusions andingenuities as follows:
1.It is the first research that explored the effect of DNMTi and HDACi on QBC_(939)cells lines in vitro.The results of the experiment demonstrated that the two drugs cansynergistically reactivate the P16,RASSF1A and E-cardherin gene of QBC_(939) cells andmake the promoter region of the three genes to be demethylated,and the two drugs havesynergy effect on reducing the growth and invasiveness of the QBC_(939) cells.
2.Human bile duct carcinoma cell line QBC_(939) cells and human normal biliaryepithelial cell line HIBEC-HR cells were both treated with hydralazine and valproate.Theresult showed that the expression of SIRT1 of QBC_(939) cells was suppressed,while that ofHIBEC-HR cells did not change,which suggested that the expression of SIRT1 in QBC_(939)cells was abnormal.
3.QBC_(939) cells were transfected with siRNA/liposome complex.After treating thetransfected cells with hydralazine and valproate inhibitors for 48 hours,the expression ofSIRT1 and HIC1 gene of these cells was detected.Then it was found that hydralazine andvalproate decreased the expression of SIRT1 via re-expressing the HIC 1 gene.
4.QBC_(939) cells were transfected with siRNA/liposome complex.After treating thetransfected cells with hydralazine and valproate inhibitors and SIRTl-specific inhibitor for48 hours,the expression of RASSF1A gene of these cells was detected.The result of theexperiment demonstrated that hydralazine and valproate reactivated the RASSF1A gene ofQBC_(939) cells in some extent via repressing the expression of SIRT1.
5.This research revealed that DNA methylation and histone deacetylase inhibitorscould regulate the SIRT1 abnormal expression in QBC939 cells.At the same time,SIRT1was found to be the effector of DNMTi and HDACi,which would provide the fundamentalfor the further exploring new factors,and for epigenetic-therapy.
表遗传学主要研究没有DNA序列改变的可遗传表达改变，即提供何时、何地和如何应用遗传学信息的指令，以确保基因适当的表达或关闭。当人类进入后基因组时代，尤其是人类表观基因组计划的提出和实施后，表遗传学引起了越来越多的关注，并逐渐成为阐明基因组功能的关键研究领域，也为研究肿瘤的治疗提出一个全新的方向。表遗传的改变主要是对基因序列的修饰，而不改变基因序列本身，因此它是可逆的，这为利用表遗传学方法治疗肿瘤提供了理论依据。
现阶段，许多肿瘤亚型缺少染色体或者微卫星不稳定性的事实挑战了基因组不稳定在启动和维持肿瘤发生、发展上起主要作用的假说。肿瘤特异性的以表遗传学改变为基础的通过异常DNA甲基化、异常组蛋白修饰以及异常的核小体定位而导致的基因表达的改变被认可为肿瘤发生的驱动因素，其中肿瘤抑制基因的灭活和原癌基因的活化是癌变的中心事件。因此，通过表遗传药物逆转肿瘤细胞异常表遗传改变，恢复沉默抑癌基因表达，进而达到治疗肿瘤的目的，成为了我们课题组的研究方向。
在邹声泉教授的带领下，本课题组研究证实了DNA甲基化酶抑制剂5-Aza-CdR具有在体外抑制胆管癌细胞增殖、促进细胞调亡的作用；并证实联合下调DNA甲基转移酶DNMT1和DNMT3b能使RASSF1A基因启动子区域去甲基化并诱导其重新表达，表明抑制DNA甲基转移酶可使抑癌基因启动子区去甲基化，恢复表达。近年来，研究者逐步发现，抑癌基因沉默的根本原因是染色质结构变得致密，影响了转录起始因子与DNA上相应转录起始部位的结合，而正是抑癌基因启动子区的超甲基化和组蛋白的脱乙酰化决定了染色质的变构。根据这一发现，和以前研究基础，本试验研究了DNA甲基化酶抑制剂(肼屈嗪)与组蛋白脱乙酰化酶抑制剂(丙戊酸)联用对胆管癌细胞QBC_(939)增殖、细胞侵袭转移力以及细胞中抑癌基因表达的影响。
另外，近两年发现第三类组蛋白脱乙酰化酶Sir2家族中SIRT1蛋白在导致抑癌基因沉默以至肿瘤的发生中有非常重要的作用，为此我们专门探讨了DNA甲基化酶抑制剂、组蛋白脱乙酰化酶抑制剂与SIRT1之间的关联性。在研究中我们发现对于组蛋白脱乙酰化酶抑制剂不敏感的SIRT1在组蛋白脱乙酰化酶抑制剂与DNA甲基化酶抑制剂联合作用下表达减少。进一步研究后明确了恢复HIC1基因表达，抑制SIRT1表达是组蛋白脱乙酰化酶抑制剂与DNA甲基化酶抑制剂联合作用胆管癌细胞中抑癌基因表达的机制之一。本研究为进一步明确组蛋白脱乙酰化酶抑制剂与DNA甲基化酶抑制剂联合作用机制、明确表遗传学变化间的关系奠定了基础，并为利用表遗传学方法治疗胆管癌提供了实验依据。
第一部分：DNMTi与HDACi对胆管癌细胞的干预效应
论文1．DNMTi与HDACi对胆管癌细胞中抑癌基因表达的影响
目的研究肼屈嗪与丙戊酸联合作用对于体外胆管癌细胞QBC_(939)中P16、RASSF1A、E-Cadherin等抑癌基因mRNA及蛋白表达，以及启动子区甲基化状态的影响。
方法将胆管细胞QBC_(939)分为空白组、单用丙戊酸组、单用肼屈嗪组与两药合用组，相应给予丙戊酸、肼屈嗪以及两药联用，空白组给予等量RPIM-1640培养液，干预48h后，RT-PCR、Western-blot法检测各组细胞中P16、RASSF1A、E-Cadherin基因mRNA及其蛋白表达；MSP法检测各组细胞中P16、RASSF1A、E-Cadherin基因启动子区甲基化状态。
结果1．空白组胆管癌细胞QBC_(939)中P16基因表达量极低，RASSF1A与E-Cadherin基因均未见表达；单用丙戊酸组细胞中P16、RASSF1A及E-Cadherin基因表达量与其空白组一致；单用肼屈嗪组细胞中P16、RASSF1A及E-Cadherin基因表达量较空白组及单用丙戊酸组增多(P＜0．01)；两药联用组细胞中P16、RASSF1A及E-Cadherin基因表达量较其它三组明显增高(P＜0．01)。2．空白组中胆管癌细胞QBC_(939)P16蛋白表达量极低，未见RASSF1A与E-Cadherin蛋白表达；单用丙戊酸组细胞三种蛋白表达量与其空白组一致；单用肼屈嗪组细胞中三种蛋白表达量较空白组和单用丙戊酸组增多(P＜0．01)；两药联用组细胞中P16、RASSF1A及E-Cadherin蛋白表达量较其余三组明显增高(P＜0．01)。3．空白组胆管癌细胞QBC_(939)中，P16基因启动子区呈部分去甲基化；RASSF1A、E-Cadherin基因启动子区超甲基化；单用丙戊酸组细胞RASSF1A、E-Cadherin基因启动子区仍呈超甲基化状态，P16基因启动子区仍呈部分去甲基化状态；单用肼屈嗪组细胞中三种基因启动子区均呈部分去甲基化状态；两药联组细胞中三种抑癌基因启动子区呈完全去甲基化状态。
结论肼屈嗪与丙戊酸两药合用可诱导胆管癌细胞QBC_(939)中P16、RASSF1A、E-Cadherin三种抑癌基因启动子区完全去甲基化，并可促进三种基因mRNA及其蛋白大量恢复表达；而单用一种药物效果明显弱于两药合用。
论文2．DNMTi与HDACi对胆管癌细胞生长与侵袭转移力的影响
目的探讨肼屈嗪与丙戊酸联合作用对于体外胆管癌细胞QBC_(939)生长与侵袭转移力的影响。
方法将胆管细胞QBC_(939)分为空白组、单用丙戊酸组、单用肼屈嗪组与两药合用组，相应给予丙戊酸、肼屈嗪以及两药联用，空白组给予等量RPIM-1640培养液，48h后1．流式细胞术Annexin V／PI双标法检测各组细胞的凋亡情况；2．Transwell法检测各组细胞侵袭转移力；
结果1．空白组细胞凋亡率为(2．855±0．936)％，单用丙戊酸组细胞凋亡率为(1 1．56±1．590)％，单用肼屈嗪组细胞凋亡率为(24．65±2．330)％；两药合用组细胞凋亡率为(47．93±3．973)％。两药合用组凋亡细胞较单用药组调亡细胞数量明显增多(P＜0．01)。2．计数移至微孔下层的细胞，结果显示空白组细胞侵袭转移力最强(P＜0．01)；肼屈嗪与丙戊酸联合作用组细胞侵袭转移力最弱(P＜0．01)；肼屈嗪组细胞侵袭转移力较丙戊酸组弱(P＜0．01)。
结论单用肼屈嗪与单用丙戊酸均可引起体外胆管癌细胞QBC_(939)不同程度凋亡；但两药合用致使细胞凋亡数量远多于单用一种药物；单用肼屈嗪与单用丙戊酸均可引起体外胆管癌细胞QBC_(939)侵袭转移力不同程度减弱，但两药合用效果明显强于单用一种药物。表明两药合用在致细胞凋亡及降低胆管癌细胞QBC939侵袭转移力方面有增效作用。
第二部分：DNMTi与HDACi对胆管癌细胞中SIRT1基因表达的调节
论文3．DNMTi与HDACi通过恢复胆管癌细胞中HIC1基因表达抑制SIRT1基因表达
目的探讨肼屈嗪与丙戊酸对胆管癌细胞QBC_(939)中SIRT1基因表达的影响及调节机制。
方法将正常胆管上皮细胞HIBEC-HR和胆管癌细胞QBC_(939)均分别分为空白组、单用丙戊酸组、单用肼屈嗪组及两药合用组，相应给予丙戊酸、肼屈嗪以及两药联用，空白组给予等量RPIM-1640培养液，干预48h后，RT-PCR、Westen blot法检测各组胆管癌细胞QBC_(939)中SIRT1、HIC1基因mRNA及其蛋白表达；RT-PCR法检测各组正常胆管上皮细胞HIBEC-HR中SIRT1基因mRNA及其蛋白表达；构建含HIC1基因干扰片段质粒，将胆管癌细胞QBC_(939)分为两药合用组、两药合用+阴性对照组、两药合用+干扰质粒组，两药合用+阴性对照组与两药合用+干扰质粒组内分别转染阴性质粒和干扰质粒，两药合用组转染液中不含质粒，转染完成后以两药联用干预各组细胞，48h后，RT-PCR、Westenblot法检测干扰HIC1基因后两药联用对SIRT1基因mRNA及其蛋白表达的影响
结果1．空白组、单用肼屈嗪组与单用丙戊酸组胆管癌细胞QBC_(939)间SIRT1基因及其蛋白表达无统计学差异(P＞0．05)，两药联用组胆管癌细胞QBC_(939)内SIRT1基因及蛋白的表达较其余三组明显降低(P＜0．01)；两药联用组内HIC1基因及其蛋白表达较单用药组以及空白组明显增高(P＜0．01)，空白组与单用丙戊酸组内无HIC1基因及其蛋白表达，单用肼屈嗪组中可见HIC1基因及蛋白的少量表达。2．干预后各组间正常胆管上皮细胞HIBEC-HR内SIRT1基因及蛋白表达无统计学差异(P＞0．05)。3．抑制HIC1基因表达后，两药合用+干扰质粒组内细胞SIRT1基因及其蛋白表达较两药合用组和两药合用+阴性质粒组明显增强(P＜0．01)，而后两组间SIRT1表达无统计学差异(P＞0．05)。
结论肼屈嗪与丙戊酸联合作用通过上调HIC1基因表达降低胆管癌细胞QBC_(939)内SIRT1基因表达。
第三部分：DNMTi与HDACi联合作用抑制胆管癌细胞中SIRT1表达恢复RASSF1A基因表达
论文4．DNMTi与HDACi联合作用抑制胆管癌细胞中SIRT1表达恢复RASSF1A基因表达
目的探讨SIRT1在肼屈嗪与丙戊酸联合恢复胆管癌细胞QBC_(939)RASSF1A基因表达中的作用。
方法成功构建含HIC1基因干扰片段质粒，将培养的胆管癌细胞QBC939分为：两药合用组(H+V组)；两药合用+阴性对照组(H+V+pHK组)；两药合用+干扰质粒组(H+V+pSihHIC1-2组)，两药合用+阴性对照组与两药合用+干扰质粒组内分别转染阴性质粒和干扰质粒，两药合用组转染液中不含质粒，转染完成后以两药联用干预各组细胞，48h后，RT-PCR、Western blot法检测各组细胞中RASSF1A基因及其蛋白表达；另取胆管癌细胞分为两组，分别为：两药合用+干扰质粒组(H+V+pSihHIC1-2组)和两药合用+干扰质粒+烟酰胺组(H+V+pSihHIC1-2+NAM组)，转染含HIC1基因干扰片段质粒成功后给予相应药物干预48h后，RT-PCR、Western blot法检测各组细胞中RASSF1A基因mRNA及其蛋白表达；MSP法检测各组细胞中RASSF1A基因启动子区甲基化状态；
结果1．RT-PCR结果显示，两药合用组与两药合用+阴性对照组中RASSF1A基因mRNA表达较两药合用+干扰质粒组强(P＜0．01)；Western blot结果与RT-PCR一致，两药合用组与两药合用+阴性对照组中RASSF1A蛋白表达较两药合用+干扰质粒组高(P＜0．01)，MSP结果显示，三组细胞RASSF1A基因启动子区甲基化状态一致，都呈完全去甲基化状态。2．RT-PCR结果显示，两药合用+干扰质粒组中RASSF1A基因mRNA表达较两药合用+干扰质粒+烟酰胺组明显减弱(P＜0．01)；Western blot结果显示两药合用+干扰质粒组中RASSF1A蛋白表达较两药合用+干扰质粒+烟酰胺组明显减弱(P＜0．01)。3．各组细胞中RASSF1A基因启动子区均呈去甲基化状态
结论SIRT1参与了肼屈嗪与丙戊酸恢复胆管癌细胞QBC_(939)中抑癌基因RASSF1A表达的过程；恢复HIC1表达继而抑制SIRT1表达，是肼屈嗪与丙戊酸恢复胆管癌细胞QBC_(939)中抑癌基因RASSF1A表达的机制之一。SIRT1表达改变对抑癌基因启动子区甲基化状态无影响。
小结
本课题从利用DNA甲基转移酶抑制剂(肼屈嗪)与组蛋白脱乙酰化酶抑制剂(丙戊酸)对体外胆管癌细胞的干预效果入手，研究了两药对于体外胆管癌细胞QBC_(939)的干预效应；并通过RNA干扰技术抑制HIC1基因表达研究了肼屈嗪与丙戊酸对于SIRT1表达的调节作用，继而研究了SIRT1基因在肼屈嗪与丙戊酸联合作用恢复体外胆管癌细胞QBC939细胞抑癌基因表达中发挥的作用。根据以上研究，得出以下结果和创新点：
1．本课题首次研究了甲基化酶抑制剂(肼屈嗪)与组蛋白脱乙酰化酶抑制剂(丙戊酸)对于体外胆管细胞QBC_(939)的干预效应，发现两药在恢复QBC_(939)细胞中抑癌基因表达、诱导细胞调亡以及降低细胞侵袭转移力上具有增效作用，即1+1＞2的作用。显示出两药合用对于胆管癌的治疗有较好的前景。
2．同时用肼屈嗪与丙戊酸干预胆管癌细胞QBC_(939)与正常胆管上皮细胞HIBEC-HR，发现QBC_(939)细胞中SIRT1表达降低而HIBEC-HR细胞中SIRT1表达不变，表明QBC_(939)细胞中SIRT1发生了异常表达。
3．通过构建并转染含HIC1基因干扰片段质粒到胆管癌细胞QBC_(939)中，再给予两药同时干预转染后细胞，检测SIRT1表达发现，甲基化酶抑制剂与组蛋白脱乙酰化酶抑制剂联合作用可通过恢复HIC1基因表达降低第三类组蛋白脱乙酰化酶抑制剂(Sir2)成员SIRT1的表达。
4．通过转染含HIC1基因干扰片段质粒进QBC_(939)细胞内，同时给予肼屈嗪丙戊酸和烟酰胺干预转染后细胞，观察QBC_(939)细胞中RASSF1A基因表达变化，发现SIRT1参与了肼屈嗪与丙戊酸恢复胆管癌细胞QBC_(939)中抑癌基因RASSF1A表达的过程。这一结果表明，恢复HIC1表达继而抑制SIRT1表达，是肼屈嗪与丙戊酸联会作用恢复胆管癌细胞QBC_(939)中抑癌基因RASSF1A表达的机制之一。
5．本试验揭示了DNA甲基转移酶、组蛋白脱乙酰化酶对SIRT1异常表达的调节作用，阐明了SIRT1在DNA甲基转移酶与组蛋白脱乙酰化酶恢复胆管癌细胞QBC939抑癌基因RASSF1A表达中的作用。同时，发现了SIRT1这一DNA甲基转移酶与组蛋白脱乙酰化酶联合作用的效应因子，为今后继续发现其他效应因子及针对效应因子研究肿瘤的治疗奠定了基础。
Bile duct carcinoma is a malignancy originating from the biliary epithelium.To datethere is no imaging techniques or molecular markers that can aid in the accurate diagnosisand evaluation of bile duct carcinoma in the early stage of the disease.So many patientspresented with unresectable diseases when the diagnosis was definite.And also Bile ductcarcinoma is an aggressive malignancy typified by and unresponsiveness to the existingchemotherapy and radiotherapy regimes in the vast majority of cases.So,it is importantto explore new drugs for the treatment of bile duct carcinoma.
Epigenetics is a new science that studies the change of gene expression caused bynon-gene sequence alteration.It decides gene transcription and the way of gene expression.Since human being enters the post-genome era,epigeneitcs has attracted more and morescholars' attention and is becoming one of the major study fields for the function ofgenome.Epigenetics provides a brand-new research direction for the study of the tumortherapy.Epigenetics refers to alternate phenotypic states that are not based on differencesin genotype,and are potentially reversible,which provides the theoretical basis for the useof epigenetie therapy for tumors.
Nowadays,the fact that subsets of many cancers lack chromosomal or microsatelliteinstability argues against the hypothesis that genomic instability plays an essential role inthe initiation and maintenance of oncogenesis.Cancer-specific,epigenetically-basedchanges in gene expression caused by abnormalities in DNA methylation,histonemodifications,and nucleosome positioning are gaining recognition as driving events intumorigenesis,in which the epigenetic gene silencing is considered to be a key event.Soour research focuses on reversing the abnormal epigenetic pattern of tumor cells,especiallyreactivating the silent tumor suppressor gene by epigenetic drugs.
In our previous studies,we observed the cell cycle alteration of human biliary tractcarcinoma cell line QBC_(939) after treatment with DNMTs inhibitor 5-Aza-CdR and foundthat 5-Aza-CdR could suppress the growth and proliferation of QBC_(939) in vivo and in vitro and induce apoptosis,we also found that using antisense RNA technology to down-regulateDNMT1 and DNMT3b expression levels in human biliary tract carcinoma cell line QBC_(939)could make the promoter region of the RASSF1A gene to be demethylated and couldreactivate it.These experimental results suggested that DNA methyltransferases might playan important role in re-expressing tumor suppressor gene of QBC_(939) cells.In recent years,advances in research of chromatin structure,histone modification,transcriptional activityand DNA methylation have resulted in an increasingly integrated view of epigenetics.It isconfirmed that aberrant gene transcription resulting from epigenetic changes are frequentevents in the molecular pathogenesis of malignant transformation.DNA hypermethylationand histone deacetylation are critical for determining a closed chromatin structureresponsible for or related with aberrant gene transcription in malignancies.Based on suchobservations,in this research we focus on the effect of DNA methylationinhibitors(hydralazine) and histone deacetylase inhibitors(valproate) on the proliferationand invasiveness of the QBC_(939) cells and the expression of tumor suppressor gene of thecells.
In addition,it is reported that SIRT1,a member of silentin formation regulator 2family,plays an important role in the remodeling of chromatin and tumorigenesis.And inour research,we also found that using hydralazine and valproate in combination coulddown-regulate the SIRT1 expression of QBC939 cells,which was insensitive to histonedeacetylase inhibitors.So we further explored the mechanism of the effect of hydralazineand valproate on SIRT1 gene,and the role of SIRT1 gene in re-expressing RASSF1A byusing hydralazine and valproate in combination.This research may lay a foundation for theepigenetic therapy of bile duct carcinoma and clarify the mechanism of the effect of DNAmethylation inhibitors and histone deacetylase inhibitors on tumor cells.
PartⅠIntervention effect of DNMTi and HDACi on cells of humanbile duct carcinoma in vitro
Paper 1 The effect of DNMTi and HDACi on the expression of tumorsuppressor genes of QBC_(939) cells in vitro
Objective To investigate the effects of hydralazine and valproate on P16,RASSIF1A andE-cardherin gene of QBC_(939) cells in vitro.
Methods The QBC_(939) cells were divided into four groups.The experimental groups weretreated separately with hydralazine and valproate either alone or combined(the finalconcentration was 10mmol/L),and the control group was added with RPIM-1640 culturemedium.After 48hours,the expression of the mRNA and the protein of the three genes wasevaluated by reverse transcription-polymerase chain reaction and Western blot assay,andthe methylation status of promoter region of the three genes was detected byMSP(mehtylation specific PCR).
Results It was found that the promoter regions of P16,RASSIF 1A and E-cardherin gene ofQBC_(939) cells were hypermethylated;valproate alone could not contribute to demethyaltionof the three genes,while hydralazine could make them to be partially demethylated.However,the methylation status of the three genes could be thoroughly reversed by usingvalproate and hydralazine in combination.It was comfirmed that the three genes of QBC_(939)cells could not be transcriptionally reactivated by valproate alone,while hydralazine alonecould induce minimal re-expression of the three genes.However,using valproate andhydralazine in combination ccould result in robust re-expression of the threegenes(P＜0.01).
Conclusion The two drugs can synergistically reactivate the P16,RASSF1A andE-cardherin gene of QBC_(939) cells and make the promoter region of the three genes to bedemethylated.
Paper 2 The effect of DNMTi and HDACi on the growth and invasion ofQBC_(939) cells in vitro
Objective To investigate the effect of hydralazine and valproate on the growth and invasionof QBC_(939) cells in vitro
Methods The QBC_(939) cells were divided into four groups.The experimental groups weretreated separately with hydralazine and valproate either alone or combined(the finalconcentration was 10mmol/L),and the control group was added with RPIM-1640 culturemedium.After 48hours,the apoptosis of treated cells was detected by flow cytometry withAnnexin V/PI staining,and the in vitro invassiveness of the treated cells was detected byTranswell permeable assays.
Results 1.The flow cytometry assays showed that the treated cells were induced toapoptosis in different degree,but the number of apoptotic cells in both drugs group wasmuch higher than that of the other two groups(P＜0.01).It also showed that usinghydralazine alone could induce more cells to apoptosis than valproate could(P＜0.01).
2.The Transwell assays demonstrated that the invasiveness of treated cells wasreduced in different extent,but a combination of two drugs worked better than either drugalone(P＜0.01).It was also found that hydralazine worked better than valproate on reducingthe invasiveness of QBC_(939) cells.Conclusion The two drugs have synergy effect on reducing the growth and invasiveness ofthe QBC_(939) cells.
PartⅡMechanism of the effect of hydralazine and valproate onSIRT1 gene of QBC_(939) cells
Paper3 DNMTi and HDACi decrease the expression of SIRT1 viare-expressing the HIC 1 gene
Objective To investigate the mechanism of the effect of hydralazine and valproate onSIRT1 gene of QBC_(939).
Methods 1.The QBC_(939) cells were divided into four groups,and HIBEC-HR cells weretreated identically.Both of them were treated separately with hydralazine and valproateeither alone or combined(the final concentration was 10mmol/L),and the control groupwas added with RPIM-1640 culture medium.After 48hours,the expression of SIRT1 andHIC1 gene of the treated cells and cells in the contral group were detected with RT-PCRand Western blot.
2.One pair of HIC1 target sequence-specific small interfering RNA (siRNA) was designedand synthesized,then siRNA/liposome complex was used to transfect QBC cells.Aftertreating the transfected cells with hydralazine and valproate inhibitors for 48 hours,theexpression of SIRT1 and HIC1 gene of these cells was detected with RT-PCR and Westernblot.
Results The expression of SIRT1 gene was detected to be decreased only when the cellswere treated with hydralazine and valproate in combination(P＜0.01);the inhibition ofhydralazine and valproate on SIRT1 gene was weakened when HIC1 gene wasslieneed(P＜0.01).
Conclusion Hydralazine and valproate decrease the expression of SIRT1 via re-expressingthe HIC1 gene.
PartⅢUsing hydralazine and valproate in combination toreactivate the RASSF1A gene by repressing the SIRT1 gene ofQBC_(939) cells
Paper4 Using hydralazine and valproate in combination to reactivate theRASSF1A gene by repressing the SIRT1 gene of QBC_(939) cells
Objective To investigate the role of SIRT1 gene in re-expressing RASSF1A of QBC_(939)cells by using hydralazine and valproate in combination.
Methods a batch of QBC_(939) cells were divided into three groups,then siRNA/liposomecomplex was used to transfect one group of QBC_(939) cells,the other two groups of cellswere transfected with pHK or none.After treating the transfected cells with hydralazine andvalproate for 48 hours,the expression of RASSF1A was evaluated by reversetranscription-PCR and western blot.Another batch of QBC_(939) cells were divided into twogroups,After siRNA/liposome complex was used to transfect these cells,hydralazine andvalproate were added into the cells.Then one group of cells were added with nicotinamide,the other group were added with RPIM-1640.48h later,the expression of RASSF1A wasevaluated by reverse transcription-PCR and western blot.Finally the promoter regionmethylation status of RASSF1A of the five groups of cells was detected withMSP(mehtylation specific PCR).
Results In the first batch of QBC_(939) cells,the expression of RASSF1A of the group whichwas transfected with siRNA was much weaker than that of the other two groups(P＜0.01).In the other batch of QBC_(939) cells the expression of RASSF1A of the group which wastreated with nicotinamide was much higher than that of the other group(P＜0.01).The statusof the promoter region of RASSF1A of the five groups was demethlated.
Conclusion Hydralazine and valproate reactivate the RASSF1A gene of QBC_(939) cells insome extent via repressing the expression of SIRT1.
Our research focused on the the effect of DNMTi and HDACi on the expression oftumor suppressor genes of QBC_(939) cells in vitro.Then we studied the mechanism of theeffect of hydralazine and valproate on SIRT1 gene of QBC_(939) cells by utilizing siRNAtechnology to knockdown HIC 1 expression.Finally we explored the the role of SIRT 1 genein re-expressing RASSF1A of QBC_(939) cells by using hydralazine and valproate incombination.According to the experimental results,we summarized the conclusions andingenuities as follows:
1.It is the first research that explored the effect of DNMTi and HDACi on QBC_(939)cells lines in vitro.The results of the experiment demonstrated that the two drugs cansynergistically reactivate the P16,RASSF1A and E-cardherin gene of QBC_(939) cells andmake the promoter region of the three genes to be demethylated,and the two drugs havesynergy effect on reducing the growth and invasiveness of the QBC_(939) cells.
2.Human bile duct carcinoma cell line QBC_(939) cells and human normal biliaryepithelial cell line HIBEC-HR cells were both treated with hydralazine and valproate.Theresult showed that the expression of SIRT1 of QBC_(939) cells was suppressed,while that ofHIBEC-HR cells did not change,which suggested that the expression of SIRT1 in QBC_(939)cells was abnormal.
3.QBC_(939) cells were transfected with siRNA/liposome complex.After treating thetransfected cells with hydralazine and valproate inhibitors for 48 hours,the expression ofSIRT1 and HIC1 gene of these cells was detected.Then it was found that hydralazine andvalproate decreased the expression of SIRT1 via re-expressing the HIC 1 gene.
4.QBC_(939) cells were transfected with siRNA/liposome complex.After treating thetransfected cells with hydralazine and valproate inhibitors and SIRTl-specific inhibitor for48 hours,the expression of RASSF1A gene of these cells was detected.The result of theexperiment demonstrated that hydralazine and valproate reactivated the RASSF1A gene ofQBC_(939) cells in some extent via repressing the expression of SIRT1.
5.This research revealed that DNA methylation and histone deacetylase inhibitorscould regulate the SIRT1 abnormal expression in QBC939 cells.At the same time,SIRT1was found to be the effector of DNMTi and HDACi,which would provide the fundamentalfor the further exploring new factors,and for epigenetic-therapy.
引文
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