组蛋白去乙酰化酶1在胰腺癌发生机制中的作用研究
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摘要
胰腺癌(pancreatic carcinoma)是较常见而恶性程度高的消化系肿瘤,近几年来其发病率在国内外均有逐年上升趋势。由于胰腺癌在临床上缺乏特异表现,恶性程度高,极易出现转移,90%的胰腺癌患者于诊断后1年内死亡,诊断后未经治疗者平均生存时间为3个月,经化疗及介入治疗后仅能提高至6-9月。适合手术治疗者低于15%,手术切除病例的术后5年累积生存率不到4%。目前临床上仍缺乏有效的胰腺癌早期诊断方法,大多数患者确诊时往往已发生转移,丧失手术时机。且由于其转移倾向以及对细胞毒性化疗药物耐受,各种传统治疗手段如手术、放疗、化疗及这些方法的联合应用均未能显著改善患者的预后,因此迫切需要深入探讨胰腺癌发生发展的分子生物学机制,以寻找治疗胰腺癌的新途径,改善其预后。
表观遗传编码(Epigenetic code)是在基因DNA序列没有发生改变的情况下,基因功能发生了可遗传的变化,并最终导致了表型变化。其生理作用是通过组蛋白修饰和DNA甲基化,调控细胞在适当的时间和空间位置表达适当的基因,从而控制细胞的增殖分化。表观遗传编码紊乱常常参与肿瘤的发生。组蛋白修饰相关酶在表观遗传编码机制中具有重要作用。在哺乳动物细胞中,乙酰化和去乙酰化的平衡在基因转录和不同细胞蛋白功能中起到关键的作用。组蛋白乙酰化状态由组蛋白乙酰转移酶(Histone Acetyltransferases,HATs)和组蛋白去乙酰化酶(Histone Deacetylases,HDACs)调节。HATs和HDACs之间的动态平衡控制着染色质的结构和基因的表达,它们的功能紊乱是肿瘤发生发展的重要分子机制之一。
HDACs属于去乙酰化酶超家族,共有Ⅰ、Ⅱ、Ⅲ、Ⅳ四型。目前以Ⅰ型HDACs研究最多。Ⅰ型HDACs包括HDAC1、2、3、8。HDACs在染色质重塑、基因阻遏、调节细胞周期和分化中起到重要作用。肿瘤细胞中组蛋白去乙酰化酶功能异常可导致许多基因转录抑制,抑制抑癌基因表达。肿瘤细胞中HDAC1高表达可明显增加肿瘤细胞的增殖能力,并且HDAC1高表达可影响细胞外基质而使肿瘤细胞移行和侵袭力明显加强。有资料报道在胃癌、前列腺癌、结肠癌及肝癌组织中HDAC1mRNA水平和蛋白水平均高表达,并和肿瘤的TNM分期及淋巴结转移相关。GlaserKB等发现Ⅰ型HDACs中的HDAC1和HDAC3是肿瘤细胞存活和增殖所必需的。Senese S等发现用siRNA干扰HDAC1可引起人骨肉瘤细胞株U2OS、乳腺癌细胞株MCF7和正常乳腺细胞株MCF10细胞在G1或G2/M期细胞静止而不能进行有丝分裂从而导致细胞生长抑制,并增加细胞凋亡,而单独干扰HDAC2则无此效果。
许多HDACs抑制剂如曲古抑菌素A(Trichostatin A,TSA)、丁酸酯等通过诱导细胞生长抑制、分化和凋亡而起到抗肿瘤作用。HDAC1抑制剂还可激活死亡受体通路而诱导细胞凋亡。在胰腺癌中TSA可诱导胰腺癌细胞株凋亡和抑制其增殖,并且和吉西他滨或蛋白酶体抑制剂PS-341合用有协同作用。另有文献报道SAHA(suberoylanilide hydroxamic acid)能诱导胰腺癌生长抑制和诱导凋亡。并且在胰腺癌活体动物模型中SAHA可以抑制异位胰腺癌的生长。所以我们认为HDAC1在肿瘤的发生和发展中可能起到重要作用。
截止目前HDAC1在胰腺癌中的表达及其在胰腺癌的发生发展中的作用研究尚无报道。因此有必要利用分子生物学的方法对HDAC1基因在胰腺癌组织中表达水平进行检测,并通过siRNA干扰技术研究HDAC1在胰腺癌发生中的作用,及其对基因甲基化的影响,同时探讨HDAC1 siRNA在胰腺癌中的治疗作用,为胰腺癌的基因治疗提供一个可选的候选位点。
一、HDACs mRNA在胰腺癌组织中的表达的研究
目的:检测组蛋白去乙酰化酶家族在胰腺癌组织和癌旁组织中的表达情况,探讨组蛋白去乙酰化酶家族在胰腺癌中的作用。
材料和方法:收集手术切除的胰腺癌组织及相应的癌旁组织共22例,通过real-timePCR法(SYBR GREEN法)检测5例正常胰腺组织、22例胰腺癌患者癌组织和癌旁组织中组蛋白去乙酰化酶家族(HDAC1和HDAC3)的mRNA表达情况,分析组蛋白去乙酰化酶家族在胰腺癌组织和癌旁组织中的表达情况。
结果:以正常胰腺组织为对照,结果发现22例胰腺癌患者HDAC1和HDAC3在癌组织中的mRNA表达均较癌旁组织高(RQ值分别为2.60(1.07～6.24)vs 1.02(0.36～1.97),P=0.001;3.57(1.52～7.56)vs 1.67(0.42～2.91,),P=0.027)。
结论:胰腺癌组织中HDAC1基因和HDAC3基因在mRNA水平上较胰腺癌旁组织表达高,HDAC家族的高表达可能是胰腺癌的特征之一。
二、HDAC1蛋白在胰腺癌中的表达及临床病理学意义
目的:探讨HDAC1蛋白在胰腺癌组织和癌旁组织中表达情况,并明确胰腺癌组织中HDAC1表达水平与临床病理指标的关系。
材料和方法:收集2006年1月～2008年3月我院接受手术治疗的30例胰腺癌患者的胰腺癌组织和相应癌旁组织,同时收集患者临床资料。所有病例均经病理诊断为胰腺导管腺癌。采用链菌素抗生物素-过氧化酶(streptavidin peroxidase,SP)免疫组织化学法检测胰腺癌和相应癌旁组织HDAC1蛋白表达。胞核染色判定为阳性。随机计数高倍镜下至少500个细胞,如果病灶细胞数少于500个,则所有的细胞被计数。计算阳性细胞数占总细胞数的百分值,无肿瘤细胞着色为阴性。分析胰腺癌组织HDAC1表达强度和临床病理指标的关系。
结果:HDAC1在胰腺导管细胞癌细胞核染色,阳性细胞为棕黄色。在非肿瘤细胞中几乎没有阳性细胞发现。胰腺癌组HDAC1表达阳性率56.0%±23.2%,癌旁组织表达阳性率为7.4%±9.8%,两组相比HDAC1表达阳性率有显著性差异(P＜0.001)。胰腺癌HDAC1高表达组18例(≥56%),低表达组12例(＜56%)。分析HDAC1蛋白表达强度与临床病理参数的关系,结果示HDAC1表达强度和TNM分期(P=0.046)、淋巴结转移相关(P=0.035),而与年龄、性别、肿瘤位置、肿瘤大小、肿瘤分化、神经浸润与否、CEA和CA199浓度无关。
结论:HDAC1蛋白在胰腺癌表达明显较胰腺癌旁组织高,胰腺癌中HDAC1高表达在胰腺癌的侵袭力及淋巴结转移中可能起到重要的作用。
三、HDAC1 siRNA对胰腺癌细胞株增殖及凋亡的影响
目的:探讨HDAC1 siRNA对胰腺癌细胞株增殖及凋亡的影响,明确HDAC1在胰腺癌中的作用。
材料和方法:通过HDAC1 siRNA瞬间转染胰腺癌细胞株PaTu8988干扰HDAC1的表达。共分空白对照组,Negative siRNA组及HDAC1 siRNA组。采用real-timeRT-PCR检测HDAC1 siRNA瞬间转染对PaTu8988细胞HDAC1基因表达的影响;通过Western blotting检测HDAC1 siRNA瞬间转染对PaTu8988 HDAC1蛋白表达的影响;并通过WST-8法测定PaTu8988细胞瞬间转染HDAC1 siRNA后的增殖情况,绘制细胞生长曲线;应用流式细胞术检测HDAC1 siRNA瞬间转染PaTu8988细胞后细胞周期及凋亡的影响。
结果:与空白对照组real-time RT-PCR检测显示胰腺癌细胞株PaTu8988瞬间转染HDAC1 siRNA后HDAC1 mRNA表达量在HDAC1 siRNA干扰15nM、30nM组表达下降率分别为68%和54%,均显著低于空白对照组(P=0.013;P=0.025)和NegativesiRNA组(P=0.002;P=0.04);Westernblot检测显示PaTu898细胞8瞬间转染HDAC1siRNA后HDAC1蛋白的表达下降;WST-8法检测显示HDAC1 siRNA干扰48h时空白对照组、15nM Negative siRNA组、30nM Negative siRNA组、15nM HDAC1siRNA干扰组及30nM HDAC1 siRNA干扰组的细胞吸光度分别为2.21±0.33、1.91±0.18、1.79±0.07、1.68±0.16及1.55±0.15,30nM HDAC1 siRNA干扰组与其他各组相比,差异有显著性(P＜0.05)。流式细胞术检测显示上述5组胰腺癌细胞凋亡率分别为4.25%±1.34%、4.34%±1.67%、5.19%±0.31%、10.09%±1.36%和11.19%±6.07%,与空白对照组、Negative siRNA组相比,HDAC1 siRNA凋亡显著增高(P＜0.05),同时可见HDAC1 siRNA组出现细胞G0/G1和G2/M期阻滞(P＜0.05)。
结论:HDAC1 siRNA能有效地抑制胰腺癌细胞株PaTu8988 HDAC1基因mRNA及蛋白的表达;HDAC1 siRNA在体外能通过G0/G1和G2/M细胞周期阻滞有效抑制胰腺癌细胞的生长增殖,并诱导细胞凋亡。
四、HDAC1 siRNA对胰腺癌细胞甲基化水平及有关增殖凋亡基因的影响
目的:探讨HDAC1 siRNA干扰对胰腺癌细胞株甲基化水平及有关增殖凋亡基因的影响。
材料和方法:通过HDAC1 siRNA瞬间转染胰腺癌细胞株PaTu8988干扰HDAC1的表达。提取siRNA干扰前后细胞DNA,并进行亚硫酸盐处理。然后针对hLMH1基因进行亚硫酸氢盐测序PCR(bisulfite sequencing PCR,BSP)。回收纯化目的产物,将其分别插入pMD18-T载体,化学转化感受态大肠杆菌,在含氨苄青霉素、X-gal和IPTG的LB平板培养进行蓝白斑筛选。对挑选的菌落进行鉴定后摇菌,菌液送检测序,计算其甲基化位点的变化情况。同时采用real-time RT-PCR检测HDAC1siRNA干扰前后原癌基因Bcl-2及抑癌基因Bax、p21的mRNA水平的变化。
结果:胰腺癌细胞株PaTu8988瞬间转染HDAC1 siRNA后hLMH1基因启动子甲基化位点较空白对照组和Negative siRNA组减少,同时siRNA干扰48h时空白对照组、15 nM和30 nM Negative siRNA组、15 nM HDAC1 siRNA组和30 nM HDAC1siRNA组Bcl-2基因mRNA RQ值分别为1.00±0.11;0.82±0.07;0.67±0.02;0.57±0.02;0.77±0.01(P=0.002),Bax基因mRNA水平分别为1.02±0.22:1.37±0.20;2.64±0.39;1.49±0.27:3.83±0.27(P＜0.001),p21基因mRNA水平分别为1.05±0.36;5.19±0.17;7.35±0.07;7.12±0.67;9.29±1.22(P＜0.001)。
结论:HDAC1 siRNA干扰能降低胰腺癌细胞株hLMH1基因甲基化水平,同时能抑制原癌基因Bcl-2表达,增加抑癌基因Bax、p21的表达。
小结:HDAC1在胰腺癌的发生发展中起到重要的作用,尤其是对胰腺癌特异性基因甲基化有调控作用,在胰腺癌中是一个良好的抗癌作用靶位。HDAC1 siRNA可能是胰腺癌基因靶向治疗的方法之一。
通过上述研究,本课题得出以下结论:
1、胰腺癌组织中HDAC1基因和HDAC3基因在mRNA水平上较胰腺癌旁组织表达增高。
2、HDAC1蛋白在胰腺癌中的表达明显较胰腺癌旁组织高,胰腺癌中HDAC1高表达在胰腺癌的侵袭力及淋巴结转移中起到重要作用。
3、HDAC1 siRNA在体外能通过G0/G1和G2/M细胞周期阻滞有效抑制胰腺癌细胞的生长增殖,还可诱导细胞凋亡。
4、HDAC1 siRNA干扰能降低胰腺癌细胞株部分基因甲基化水平,同时能抑制原癌基因Bcl-2表达,增加抑癌基因Bax、p21的表达。
因此HDAC1有望成为胰腺癌基因治疗的新靶点,有关于其他HDAC家族成员的功能和治疗研究需要在今后的研究中进一步深入。
Pancreatic cancer is a common malignant tumor of gastrointestinal tract.In recent years,the prevalence of pancreatic cancer has been increasing all over the world. Pancreatic cancer which is absence of specific clinical symptoms and signs is a high-grade malignant tumor and prone to metastases.90%patients diagnosed with pancreatic cancer will die within 1 year and the average survival time is 3 months.Less than 15%patients with pancreatic cancer are suitable for operation and the accumulated 5-year survival rate after resection is less than 4%.Because of absence of early diagnosis techniques the patients already have metastases at diagnosis and surgical removal of the tumor has become impractical.Moreover traditional therapy such as surgery, radiotherapy,chemotherapy,combination therapy can not improve the prognosis of pancreatic cancer because of the susceptibility of metastases and drug resistance.It is likely that treatment of this fatal disease will require the detailed understanding of the molecular biology of pancreatic cancer.
Epigenetic code infers the DNA sequences are not changed but the functions of the gene are changed and could be inherited,finally,the phenotype was changed.Its physiological function is regulating the cells express the proper genes in the proper time and location by histone modifications and DNA methylation thereby controlling proliferation and differentiation.Epigenetic code usually is chaos in cancer cell.Histone modification enzymes play a major role in epigenetic code.In mammalian cells the balance of acetylation and deacetylation has a key role in gene transcription and regulation the function of cell proteins.The condition of acetylation are regulated by histone acetyltransferases(HATs) and c(HDACs).The dynamic balance between histone acetyltransferases and histone deacetylases controls the structure of chromatin and genes expression.The chaos of HATs and HDACs is one of the important molecular mechanism of carcinogenesis.
HDACs belonging to histone deacetylases superfamily have been classified into four distinct families(classesⅠ,Ⅱ,ⅢandⅣ).ClassⅠHDACs were studied at most.TypeⅠHDACs include HDAC1,HDAC2,HDAC3 and HDAC8.HDACs have important roles in chromatin remodeling,gene repression,regulating cell cycle progression and differentiation.The malfunction of HDACs can lead to genes transcription inhibition then repress the expression of anti-oncogene.Overexpression of human HDAC-1 can not only increase proliferation of cancer cells but also enhance the ability of cells to migrate or invade through extracellular matrix.Lots of reports showed overexpression of HDAC 1 on mRNA and protein levels in gastric cancer,prostate cancer,colonic cancer and hepatocellular carcinoma correlated significantly with a more advanced TNM stage and lymph node metastasis.Glaser KB et al.demonstrate that the ClassⅠHDACs such as HDACs 1 and 3 are important in the regulation of proliferation and survival in cancer cells. Senese S et al.showed that in the absence of HDAC1 cells using RNA interference-mediated protein knockdown can arrest either at the G1 phase of the cell cycle or at the G2/M transition,resulting in the loss of mitotic cells,cell growth inhibition,and an increase in the percentage of apoptotic cells.On the contrary,HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1.
HDAC inhibitors such as trichostatin A(TSA) and butylate express antitumor effects by inducing growth arrest,differentiation,and apoptosis.HDAC inhibitors can also activate the death receptor pathway and induce apoptosis.TSA can induce apoptosis and inhibit cell proliferation in pancreatic cancer cell lines moreover there were synergistic inhibition effects by using of TSA in combination with gemcitabine or proteasome inhibitor PS-341.Other literature reported suberoylanilide hydroxamic acid(SAHA) inhibited the growth of human pancreatic cancer cells by inducing apoptosis and growth inhibiting.In subcutaneous xenograft model,growth of xenografts was delayed with rejections of SAHA.So we think HDAC1 play an important role in carcinogenesis.
But there are no investigations about the expression of HDAC1 in pancreatic cancer and its role in tumorigenesis of pancreatic cancer.It is necessary to study the expression of HDAC1 in pancreatic cancer tissues using molecular biology techniques to research the role of HDAC1 in pancreatic cancer cells and its effects on methylation using siRNA, so we can know the effects of HDAC1 siRNA on pancreatic cancer for gene therapy.
1.The expression of HDACs mRNA in pancreatic cancer tissue
Objective To detect the expression of HDACs mRNA in pancreatic cancer and paracancerous tissues and investigate the effects of HDACs in pancreatic cancer.
Methods and materials 22 samples of pancreatic carcinoma tissues,paired paracancerous tissues and 5 cases normal pancreas tissues were collected,mRNA levels of HDAC1 and HDAC3 were detected by real-time RT-PCR(SYBR GREEN) then the expression of HDAC1 and HDAC3 mRNA were analyzed.
Results Compared with normal pancreas tissues the RQ values of HDAC1 mRNA and HDAC3 mRNA in human pancreatic carcinoma tissues were significant higher than in paracancerous tissues(2.60(1.07～6.24) vs 1.02(0.36～1.9),P=0.001;3.57(1.52～7.56) vs 1.67(0.42～2.91),P =0.027),respectively).
Conclusions The expression of HDAC1 and HDAC3 on mRNA levels were higher in human pancreatic carcinoma tissues than in paracancerous tissues.HDACs might play an important in tumorigenesis of pancreatic cancer.
2.Ciinicopathological significance and HDAC1 protein expression in pancreatic adenocarcinoma
Objective To detect the expression of HDAC1 on protein levels in pancreatic cancer and paracancerous tissues and investigate the relationship between clinicopathological significance and HDAC1 expression levels in patients with pancreatic carcinoma.
Methods and materials pancreatic carcinoma tumor tissue and paired paracancerous tissue samples were obtained from 30 pancreatic carcinoma patients who underwent curative pancreectomy at Changhai Hospital,from Jan.2006 to Mar.2008.Clinical data were collected.In all patients,the pancreatic ductal carcinoma was confirmed histologically,based mainly on the examination of sections stained with hematoxylin and eosin.HDAC1 expression was detected by streptavidin peroxidase immunohistochemistry techniques.The positive cells were nuclei staining.For each sample,at least 500 cells were randomly counted in high resolution.If the lesion was small with less than 500 cells,all the cells were counted.The incidence of HDAC1 immunoreactivity in each sample was expressed as a percentage of all the cells counted.If no tumor cells were stained,the result was judged negative.The relationships between clinicopathological findings and expression of HDAC1 were analyzed.
Results Positive cells were brown.Specific HDAC1 staining was seen in the nuclei of the cancer cells,while it was rarely seen in non-cancerous lesions.The index of HDAC1 expressions in human pancreatic carcinoma tissues and paracancerous tissues were 56.0%±23.2%and 7.4%±9.8%respectively(P＜0.01).High HDAC1 group(n =18) with 56%or more of the cancer cells stained HDAC1 positively and a low HDAC1 group(n =12) with less than 56%cancer cells stained HDAC1 positively.The high HDAC1 expression correlated significantly with more advanced TNM stage and lymph node metastasis.On the other hand,no association between HDAC1 expression and clinicopathological variables including age,gender,tumor location,tumor size,tumor differenciation,neural invasion,the concentration of CEA and CA-199.
Conclusions In pancreatic carcinoma tissues HDAC1 was highly expressed,high HDAC1 expression might have an important role in the aggressiveness and lymph node metastasis.
3.Effect of siRNA against HDAC1 on pancreatic cancer cell line proliferation and apoptosis
Objective To evaluate the effect of transient transfection in proliferation and apoptosis of pancreatic cancer cell line with siRNA against HDAC1.
Methods and materials transient transfection of human pancreatic cell line PaTu8988 cells with siRNA against HDAC1 were adopted.There were control group,Negative siRNA group,and HDAC1 siRNA group PaTu8988 cells were plated 24h prior to transfection then were transiently transfected HDAC1 siRNA or negative siRNA 48h after transfection,cells were collected and total mRNA and protein were extracted.Real-time RT-PCR(SYBR GREEN) was used to detect the mRNA expression of HDAC1 in each group.Western blotting was performed to evaluate the expression of HDAC1 protein of the above each groups.WST-8 assay was performed to measure cellular proliferation.Flow cytometry was performed to analyze cell cycle and apoptosis.
Results Real-time RT-PCR analysis showed that the mRNA expression inhibition rate of HDAC1 in HDAC1 siRNA 15nM,30nM group were 68%and 54%respectively.The expressions were significantly lower than control group(P=0.013;P=0.025) and negative siRNA group(P=0.002;P=0.04).Western blotting analysis showed that the expression of HDAC1 protein of PaTu8988 transfected with HDAC1 siRNA decreased either.WST-8 assay showed that at 48h after transfection,the growth of PaTu8988 cell lines in control group,15nM and 30nM Negative siRNA,15nM and 30nM HDAC1 siRNA group were 2.21±0.33,1.91±0.18,1.79±0.07,1.68±0.16 and 1.55±0.15 respectively.Compared with other groups 30nM HDAC1 siRNA group were significantly suppressed(P(0.05).Flow cytometry analysis showed the apoptosis rate control group,Negative siRNA group and HDAC1 siRNA group were 4.25%±1.34%,4.34%±1.67%,5.19%±0.31%,10.09%±1.36% and 11.19%±6.07%respectively(P(0.05).At the same time G0/G1 and G2/M cell cycle arrest was observed in HDAC1 siRNA group(P(0.05).
Conclusions Transfection of HDAC1 siRNA effectively inhibits the expression of HDAC1 mRNA and protein in human pancreatic cancer cell lines PaTu8988.In addition,HDAC1 siRNA induces G0/G1 and G2/M cell cycle arrest and apoptosis thereby suppresses the growth of PaTu8988 cell lines.
4.The effects of HDAC1 siRNA on methylation and gene concerning proliferation and apotosis in pancreatic cancer cells
Objective To evaluate the role of transient transfection of HDAC1 siRNA inducing gene concerning about proliferation and apotosis in pancreatic cancer cell line and the effect of HDAC1 on methylation in pancreatic cancer cell line.
Methods and materials Transient transfection of human pancreatic cell line PaTu8988 cells with siRNA against HDAC1 were adopted.PaTu8988 cells were plated 24h prior to transfection then were transiently transfected HDAC1 siRNA or Negative siRNA,cells were collected and DNA were extracted then sodium bisulfite modification and BSP of hMLH1 was performed based upon instrument.The production of BSP was purified and inserted into pMD18-T vector with ligase.The ligation mixture was used to chemotransform E.coliDH5a,after which the cells were plated on LB plate containing ampicillin,X-gal and IPTG and placed at 37℃.The next morning,the plate was examined and the white clones were picked and identified by PCR.Then the chemotransforrned E.coliDH5a was sequenced to analyze methylation of CpG islands of hMLH1.Real-time RT-PCR(SYBR GREEN) was used to detect the change of rnRNA expression of Bcl-2, Bax and p21 after HDAC1 siRNA interference.
Results Methylated CpG sites of hMLH1 in HDAC1 siRNA group were descreased than those in Negative siRNA group and control group.At 48h after transfection in control group,15nM and 30nM Negative siRNA group and HDAC1 siRNA group the mRNA expression of Bcl-2,Bax and p21 were(1.00±0.11;0.82±0.07;0.67±0.02;0.57±0.02; 0.77±0.01;P=0.002);(1.02±0.22;1.37±0.20;2.64±0.39;1.49±0.27;3.83±0.27;P＜0.001) and(1.05±0.36;5.19±0.17;7.35±0.07;7.12±0.67;9.29±1.22;P＜0.001) respectively.
Conclusions Transfection of HDAC1 siRNA effectively decreased the methylated CpG sites in promoter of hMLH1 in human pancreatic cancer cell lines PaTu8988.At the same HDAC1 siRNA interference can inhibit the expression of bcl-2 mRNA and increase the expression of Box and p21 mRNA
Based on the above experiments,the final conclusions were as followings:
1.The expression of HDAC1 and HDAC3 on mRNA levels were higher in human pancreatic carcinoma tissues than in paracancerous tissues.
2.In pancreatic carcinoma tissues HDAC1 was highly expressed,high HDAC1 expression might have an important role in the aggressiveness and lymph node metastasis.
3.Transfection of HDAC1 siRNA effectively inhibits the expression of HDAC1 mRNA and protein in human pancreatic cancer cell lines PaTu8988.In addition,HDAC1 siRNA induces G0/G1 and G2/M cell cycle arrest and apoptosis and thereby suppresses the growth of PaTu8988 cell lines.
4.Transfection of HDAC1 siRNA can decrease the methylated CpG sites in promoter of hMLH1 in human pancreatic cancer cell lines PaTu8988.At the same time HDAC1 siRNA interference can inhibit the expression of Bcl-2 and increase the expression of Bax and p21 on mRNA lever.
HDAC1 could be a new gene therapy target in pancreatic cancer,the role of other members of HDACs should be investigated in the future.
表观遗传编码(Epigenetic code)是在基因DNA序列没有发生改变的情况下,基因功能发生了可遗传的变化,并最终导致了表型变化。其生理作用是通过组蛋白修饰和DNA甲基化,调控细胞在适当的时间和空间位置表达适当的基因,从而控制细胞的增殖分化。表观遗传编码紊乱常常参与肿瘤的发生。组蛋白修饰相关酶在表观遗传编码机制中具有重要作用。在哺乳动物细胞中,乙酰化和去乙酰化的平衡在基因转录和不同细胞蛋白功能中起到关键的作用。组蛋白乙酰化状态由组蛋白乙酰转移酶(Histone Acetyltransferases,HATs)和组蛋白去乙酰化酶(Histone Deacetylases,HDACs)调节。HATs和HDACs之间的动态平衡控制着染色质的结构和基因的表达,它们的功能紊乱是肿瘤发生发展的重要分子机制之一。
HDACs属于去乙酰化酶超家族,共有Ⅰ、Ⅱ、Ⅲ、Ⅳ四型。目前以Ⅰ型HDACs研究最多。Ⅰ型HDACs包括HDAC1、2、3、8。HDACs在染色质重塑、基因阻遏、调节细胞周期和分化中起到重要作用。肿瘤细胞中组蛋白去乙酰化酶功能异常可导致许多基因转录抑制,抑制抑癌基因表达。肿瘤细胞中HDAC1高表达可明显增加肿瘤细胞的增殖能力,并且HDAC1高表达可影响细胞外基质而使肿瘤细胞移行和侵袭力明显加强。有资料报道在胃癌、前列腺癌、结肠癌及肝癌组织中HDAC1mRNA水平和蛋白水平均高表达,并和肿瘤的TNM分期及淋巴结转移相关。GlaserKB等发现Ⅰ型HDACs中的HDAC1和HDAC3是肿瘤细胞存活和增殖所必需的。Senese S等发现用siRNA干扰HDAC1可引起人骨肉瘤细胞株U2OS、乳腺癌细胞株MCF7和正常乳腺细胞株MCF10细胞在G1或G2/M期细胞静止而不能进行有丝分裂从而导致细胞生长抑制,并增加细胞凋亡,而单独干扰HDAC2则无此效果。
许多HDACs抑制剂如曲古抑菌素A(Trichostatin A,TSA)、丁酸酯等通过诱导细胞生长抑制、分化和凋亡而起到抗肿瘤作用。HDAC1抑制剂还可激活死亡受体通路而诱导细胞凋亡。在胰腺癌中TSA可诱导胰腺癌细胞株凋亡和抑制其增殖,并且和吉西他滨或蛋白酶体抑制剂PS-341合用有协同作用。另有文献报道SAHA(suberoylanilide hydroxamic acid)能诱导胰腺癌生长抑制和诱导凋亡。并且在胰腺癌活体动物模型中SAHA可以抑制异位胰腺癌的生长。所以我们认为HDAC1在肿瘤的发生和发展中可能起到重要作用。
截止目前HDAC1在胰腺癌中的表达及其在胰腺癌的发生发展中的作用研究尚无报道。因此有必要利用分子生物学的方法对HDAC1基因在胰腺癌组织中表达水平进行检测,并通过siRNA干扰技术研究HDAC1在胰腺癌发生中的作用,及其对基因甲基化的影响,同时探讨HDAC1 siRNA在胰腺癌中的治疗作用,为胰腺癌的基因治疗提供一个可选的候选位点。
一、HDACs mRNA在胰腺癌组织中的表达的研究
目的:检测组蛋白去乙酰化酶家族在胰腺癌组织和癌旁组织中的表达情况,探讨组蛋白去乙酰化酶家族在胰腺癌中的作用。
材料和方法:收集手术切除的胰腺癌组织及相应的癌旁组织共22例,通过real-timePCR法(SYBR GREEN法)检测5例正常胰腺组织、22例胰腺癌患者癌组织和癌旁组织中组蛋白去乙酰化酶家族(HDAC1和HDAC3)的mRNA表达情况,分析组蛋白去乙酰化酶家族在胰腺癌组织和癌旁组织中的表达情况。
结果:以正常胰腺组织为对照,结果发现22例胰腺癌患者HDAC1和HDAC3在癌组织中的mRNA表达均较癌旁组织高(RQ值分别为2.60(1.07～6.24)vs 1.02(0.36～1.97),P=0.001;3.57(1.52～7.56)vs 1.67(0.42～2.91,),P=0.027)。
结论:胰腺癌组织中HDAC1基因和HDAC3基因在mRNA水平上较胰腺癌旁组织表达高,HDAC家族的高表达可能是胰腺癌的特征之一。
二、HDAC1蛋白在胰腺癌中的表达及临床病理学意义
目的:探讨HDAC1蛋白在胰腺癌组织和癌旁组织中表达情况,并明确胰腺癌组织中HDAC1表达水平与临床病理指标的关系。
材料和方法:收集2006年1月～2008年3月我院接受手术治疗的30例胰腺癌患者的胰腺癌组织和相应癌旁组织,同时收集患者临床资料。所有病例均经病理诊断为胰腺导管腺癌。采用链菌素抗生物素-过氧化酶(streptavidin peroxidase,SP)免疫组织化学法检测胰腺癌和相应癌旁组织HDAC1蛋白表达。胞核染色判定为阳性。随机计数高倍镜下至少500个细胞,如果病灶细胞数少于500个,则所有的细胞被计数。计算阳性细胞数占总细胞数的百分值,无肿瘤细胞着色为阴性。分析胰腺癌组织HDAC1表达强度和临床病理指标的关系。
结果:HDAC1在胰腺导管细胞癌细胞核染色,阳性细胞为棕黄色。在非肿瘤细胞中几乎没有阳性细胞发现。胰腺癌组HDAC1表达阳性率56.0%±23.2%,癌旁组织表达阳性率为7.4%±9.8%,两组相比HDAC1表达阳性率有显著性差异(P＜0.001)。胰腺癌HDAC1高表达组18例(≥56%),低表达组12例(＜56%)。分析HDAC1蛋白表达强度与临床病理参数的关系,结果示HDAC1表达强度和TNM分期(P=0.046)、淋巴结转移相关(P=0.035),而与年龄、性别、肿瘤位置、肿瘤大小、肿瘤分化、神经浸润与否、CEA和CA199浓度无关。
结论:HDAC1蛋白在胰腺癌表达明显较胰腺癌旁组织高,胰腺癌中HDAC1高表达在胰腺癌的侵袭力及淋巴结转移中可能起到重要的作用。
三、HDAC1 siRNA对胰腺癌细胞株增殖及凋亡的影响
目的:探讨HDAC1 siRNA对胰腺癌细胞株增殖及凋亡的影响,明确HDAC1在胰腺癌中的作用。
材料和方法:通过HDAC1 siRNA瞬间转染胰腺癌细胞株PaTu8988干扰HDAC1的表达。共分空白对照组,Negative siRNA组及HDAC1 siRNA组。采用real-timeRT-PCR检测HDAC1 siRNA瞬间转染对PaTu8988细胞HDAC1基因表达的影响;通过Western blotting检测HDAC1 siRNA瞬间转染对PaTu8988 HDAC1蛋白表达的影响;并通过WST-8法测定PaTu8988细胞瞬间转染HDAC1 siRNA后的增殖情况,绘制细胞生长曲线;应用流式细胞术检测HDAC1 siRNA瞬间转染PaTu8988细胞后细胞周期及凋亡的影响。
结果:与空白对照组real-time RT-PCR检测显示胰腺癌细胞株PaTu8988瞬间转染HDAC1 siRNA后HDAC1 mRNA表达量在HDAC1 siRNA干扰15nM、30nM组表达下降率分别为68%和54%,均显著低于空白对照组(P=0.013;P=0.025)和NegativesiRNA组(P=0.002;P=0.04);Westernblot检测显示PaTu898细胞8瞬间转染HDAC1siRNA后HDAC1蛋白的表达下降;WST-8法检测显示HDAC1 siRNA干扰48h时空白对照组、15nM Negative siRNA组、30nM Negative siRNA组、15nM HDAC1siRNA干扰组及30nM HDAC1 siRNA干扰组的细胞吸光度分别为2.21±0.33、1.91±0.18、1.79±0.07、1.68±0.16及1.55±0.15,30nM HDAC1 siRNA干扰组与其他各组相比,差异有显著性(P＜0.05)。流式细胞术检测显示上述5组胰腺癌细胞凋亡率分别为4.25%±1.34%、4.34%±1.67%、5.19%±0.31%、10.09%±1.36%和11.19%±6.07%,与空白对照组、Negative siRNA组相比,HDAC1 siRNA凋亡显著增高(P＜0.05),同时可见HDAC1 siRNA组出现细胞G0/G1和G2/M期阻滞(P＜0.05)。
结论:HDAC1 siRNA能有效地抑制胰腺癌细胞株PaTu8988 HDAC1基因mRNA及蛋白的表达;HDAC1 siRNA在体外能通过G0/G1和G2/M细胞周期阻滞有效抑制胰腺癌细胞的生长增殖,并诱导细胞凋亡。
四、HDAC1 siRNA对胰腺癌细胞甲基化水平及有关增殖凋亡基因的影响
目的:探讨HDAC1 siRNA干扰对胰腺癌细胞株甲基化水平及有关增殖凋亡基因的影响。
材料和方法:通过HDAC1 siRNA瞬间转染胰腺癌细胞株PaTu8988干扰HDAC1的表达。提取siRNA干扰前后细胞DNA,并进行亚硫酸盐处理。然后针对hLMH1基因进行亚硫酸氢盐测序PCR(bisulfite sequencing PCR,BSP)。回收纯化目的产物,将其分别插入pMD18-T载体,化学转化感受态大肠杆菌,在含氨苄青霉素、X-gal和IPTG的LB平板培养进行蓝白斑筛选。对挑选的菌落进行鉴定后摇菌,菌液送检测序,计算其甲基化位点的变化情况。同时采用real-time RT-PCR检测HDAC1siRNA干扰前后原癌基因Bcl-2及抑癌基因Bax、p21的mRNA水平的变化。
结果:胰腺癌细胞株PaTu8988瞬间转染HDAC1 siRNA后hLMH1基因启动子甲基化位点较空白对照组和Negative siRNA组减少,同时siRNA干扰48h时空白对照组、15 nM和30 nM Negative siRNA组、15 nM HDAC1 siRNA组和30 nM HDAC1siRNA组Bcl-2基因mRNA RQ值分别为1.00±0.11;0.82±0.07;0.67±0.02;0.57±0.02;0.77±0.01(P=0.002),Bax基因mRNA水平分别为1.02±0.22:1.37±0.20;2.64±0.39;1.49±0.27:3.83±0.27(P＜0.001),p21基因mRNA水平分别为1.05±0.36;5.19±0.17;7.35±0.07;7.12±0.67;9.29±1.22(P＜0.001)。
结论:HDAC1 siRNA干扰能降低胰腺癌细胞株hLMH1基因甲基化水平,同时能抑制原癌基因Bcl-2表达,增加抑癌基因Bax、p21的表达。
小结:HDAC1在胰腺癌的发生发展中起到重要的作用,尤其是对胰腺癌特异性基因甲基化有调控作用,在胰腺癌中是一个良好的抗癌作用靶位。HDAC1 siRNA可能是胰腺癌基因靶向治疗的方法之一。
通过上述研究,本课题得出以下结论:
1、胰腺癌组织中HDAC1基因和HDAC3基因在mRNA水平上较胰腺癌旁组织表达增高。
2、HDAC1蛋白在胰腺癌中的表达明显较胰腺癌旁组织高,胰腺癌中HDAC1高表达在胰腺癌的侵袭力及淋巴结转移中起到重要作用。
3、HDAC1 siRNA在体外能通过G0/G1和G2/M细胞周期阻滞有效抑制胰腺癌细胞的生长增殖,还可诱导细胞凋亡。
4、HDAC1 siRNA干扰能降低胰腺癌细胞株部分基因甲基化水平,同时能抑制原癌基因Bcl-2表达,增加抑癌基因Bax、p21的表达。
因此HDAC1有望成为胰腺癌基因治疗的新靶点,有关于其他HDAC家族成员的功能和治疗研究需要在今后的研究中进一步深入。
Pancreatic cancer is a common malignant tumor of gastrointestinal tract.In recent years,the prevalence of pancreatic cancer has been increasing all over the world. Pancreatic cancer which is absence of specific clinical symptoms and signs is a high-grade malignant tumor and prone to metastases.90%patients diagnosed with pancreatic cancer will die within 1 year and the average survival time is 3 months.Less than 15%patients with pancreatic cancer are suitable for operation and the accumulated 5-year survival rate after resection is less than 4%.Because of absence of early diagnosis techniques the patients already have metastases at diagnosis and surgical removal of the tumor has become impractical.Moreover traditional therapy such as surgery, radiotherapy,chemotherapy,combination therapy can not improve the prognosis of pancreatic cancer because of the susceptibility of metastases and drug resistance.It is likely that treatment of this fatal disease will require the detailed understanding of the molecular biology of pancreatic cancer.
Epigenetic code infers the DNA sequences are not changed but the functions of the gene are changed and could be inherited,finally,the phenotype was changed.Its physiological function is regulating the cells express the proper genes in the proper time and location by histone modifications and DNA methylation thereby controlling proliferation and differentiation.Epigenetic code usually is chaos in cancer cell.Histone modification enzymes play a major role in epigenetic code.In mammalian cells the balance of acetylation and deacetylation has a key role in gene transcription and regulation the function of cell proteins.The condition of acetylation are regulated by histone acetyltransferases(HATs) and c(HDACs).The dynamic balance between histone acetyltransferases and histone deacetylases controls the structure of chromatin and genes expression.The chaos of HATs and HDACs is one of the important molecular mechanism of carcinogenesis.
HDACs belonging to histone deacetylases superfamily have been classified into four distinct families(classesⅠ,Ⅱ,ⅢandⅣ).ClassⅠHDACs were studied at most.TypeⅠHDACs include HDAC1,HDAC2,HDAC3 and HDAC8.HDACs have important roles in chromatin remodeling,gene repression,regulating cell cycle progression and differentiation.The malfunction of HDACs can lead to genes transcription inhibition then repress the expression of anti-oncogene.Overexpression of human HDAC-1 can not only increase proliferation of cancer cells but also enhance the ability of cells to migrate or invade through extracellular matrix.Lots of reports showed overexpression of HDAC 1 on mRNA and protein levels in gastric cancer,prostate cancer,colonic cancer and hepatocellular carcinoma correlated significantly with a more advanced TNM stage and lymph node metastasis.Glaser KB et al.demonstrate that the ClassⅠHDACs such as HDACs 1 and 3 are important in the regulation of proliferation and survival in cancer cells. Senese S et al.showed that in the absence of HDAC1 cells using RNA interference-mediated protein knockdown can arrest either at the G1 phase of the cell cycle or at the G2/M transition,resulting in the loss of mitotic cells,cell growth inhibition,and an increase in the percentage of apoptotic cells.On the contrary,HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1.
HDAC inhibitors such as trichostatin A(TSA) and butylate express antitumor effects by inducing growth arrest,differentiation,and apoptosis.HDAC inhibitors can also activate the death receptor pathway and induce apoptosis.TSA can induce apoptosis and inhibit cell proliferation in pancreatic cancer cell lines moreover there were synergistic inhibition effects by using of TSA in combination with gemcitabine or proteasome inhibitor PS-341.Other literature reported suberoylanilide hydroxamic acid(SAHA) inhibited the growth of human pancreatic cancer cells by inducing apoptosis and growth inhibiting.In subcutaneous xenograft model,growth of xenografts was delayed with rejections of SAHA.So we think HDAC1 play an important role in carcinogenesis.
But there are no investigations about the expression of HDAC1 in pancreatic cancer and its role in tumorigenesis of pancreatic cancer.It is necessary to study the expression of HDAC1 in pancreatic cancer tissues using molecular biology techniques to research the role of HDAC1 in pancreatic cancer cells and its effects on methylation using siRNA, so we can know the effects of HDAC1 siRNA on pancreatic cancer for gene therapy.
1.The expression of HDACs mRNA in pancreatic cancer tissue
Objective To detect the expression of HDACs mRNA in pancreatic cancer and paracancerous tissues and investigate the effects of HDACs in pancreatic cancer.
Methods and materials 22 samples of pancreatic carcinoma tissues,paired paracancerous tissues and 5 cases normal pancreas tissues were collected,mRNA levels of HDAC1 and HDAC3 were detected by real-time RT-PCR(SYBR GREEN) then the expression of HDAC1 and HDAC3 mRNA were analyzed.
Results Compared with normal pancreas tissues the RQ values of HDAC1 mRNA and HDAC3 mRNA in human pancreatic carcinoma tissues were significant higher than in paracancerous tissues(2.60(1.07～6.24) vs 1.02(0.36～1.9),P=0.001;3.57(1.52～7.56) vs 1.67(0.42～2.91),P =0.027),respectively).
Conclusions The expression of HDAC1 and HDAC3 on mRNA levels were higher in human pancreatic carcinoma tissues than in paracancerous tissues.HDACs might play an important in tumorigenesis of pancreatic cancer.
2.Ciinicopathological significance and HDAC1 protein expression in pancreatic adenocarcinoma
Objective To detect the expression of HDAC1 on protein levels in pancreatic cancer and paracancerous tissues and investigate the relationship between clinicopathological significance and HDAC1 expression levels in patients with pancreatic carcinoma.
Methods and materials pancreatic carcinoma tumor tissue and paired paracancerous tissue samples were obtained from 30 pancreatic carcinoma patients who underwent curative pancreectomy at Changhai Hospital,from Jan.2006 to Mar.2008.Clinical data were collected.In all patients,the pancreatic ductal carcinoma was confirmed histologically,based mainly on the examination of sections stained with hematoxylin and eosin.HDAC1 expression was detected by streptavidin peroxidase immunohistochemistry techniques.The positive cells were nuclei staining.For each sample,at least 500 cells were randomly counted in high resolution.If the lesion was small with less than 500 cells,all the cells were counted.The incidence of HDAC1 immunoreactivity in each sample was expressed as a percentage of all the cells counted.If no tumor cells were stained,the result was judged negative.The relationships between clinicopathological findings and expression of HDAC1 were analyzed.
Results Positive cells were brown.Specific HDAC1 staining was seen in the nuclei of the cancer cells,while it was rarely seen in non-cancerous lesions.The index of HDAC1 expressions in human pancreatic carcinoma tissues and paracancerous tissues were 56.0%±23.2%and 7.4%±9.8%respectively(P＜0.01).High HDAC1 group(n =18) with 56%or more of the cancer cells stained HDAC1 positively and a low HDAC1 group(n =12) with less than 56%cancer cells stained HDAC1 positively.The high HDAC1 expression correlated significantly with more advanced TNM stage and lymph node metastasis.On the other hand,no association between HDAC1 expression and clinicopathological variables including age,gender,tumor location,tumor size,tumor differenciation,neural invasion,the concentration of CEA and CA-199.
Conclusions In pancreatic carcinoma tissues HDAC1 was highly expressed,high HDAC1 expression might have an important role in the aggressiveness and lymph node metastasis.
3.Effect of siRNA against HDAC1 on pancreatic cancer cell line proliferation and apoptosis
Objective To evaluate the effect of transient transfection in proliferation and apoptosis of pancreatic cancer cell line with siRNA against HDAC1.
Methods and materials transient transfection of human pancreatic cell line PaTu8988 cells with siRNA against HDAC1 were adopted.There were control group,Negative siRNA group,and HDAC1 siRNA group PaTu8988 cells were plated 24h prior to transfection then were transiently transfected HDAC1 siRNA or negative siRNA 48h after transfection,cells were collected and total mRNA and protein were extracted.Real-time RT-PCR(SYBR GREEN) was used to detect the mRNA expression of HDAC1 in each group.Western blotting was performed to evaluate the expression of HDAC1 protein of the above each groups.WST-8 assay was performed to measure cellular proliferation.Flow cytometry was performed to analyze cell cycle and apoptosis.
Results Real-time RT-PCR analysis showed that the mRNA expression inhibition rate of HDAC1 in HDAC1 siRNA 15nM,30nM group were 68%and 54%respectively.The expressions were significantly lower than control group(P=0.013;P=0.025) and negative siRNA group(P=0.002;P=0.04).Western blotting analysis showed that the expression of HDAC1 protein of PaTu8988 transfected with HDAC1 siRNA decreased either.WST-8 assay showed that at 48h after transfection,the growth of PaTu8988 cell lines in control group,15nM and 30nM Negative siRNA,15nM and 30nM HDAC1 siRNA group were 2.21±0.33,1.91±0.18,1.79±0.07,1.68±0.16 and 1.55±0.15 respectively.Compared with other groups 30nM HDAC1 siRNA group were significantly suppressed(P(0.05).Flow cytometry analysis showed the apoptosis rate control group,Negative siRNA group and HDAC1 siRNA group were 4.25%±1.34%,4.34%±1.67%,5.19%±0.31%,10.09%±1.36% and 11.19%±6.07%respectively(P(0.05).At the same time G0/G1 and G2/M cell cycle arrest was observed in HDAC1 siRNA group(P(0.05).
Conclusions Transfection of HDAC1 siRNA effectively inhibits the expression of HDAC1 mRNA and protein in human pancreatic cancer cell lines PaTu8988.In addition,HDAC1 siRNA induces G0/G1 and G2/M cell cycle arrest and apoptosis thereby suppresses the growth of PaTu8988 cell lines.
4.The effects of HDAC1 siRNA on methylation and gene concerning proliferation and apotosis in pancreatic cancer cells
Objective To evaluate the role of transient transfection of HDAC1 siRNA inducing gene concerning about proliferation and apotosis in pancreatic cancer cell line and the effect of HDAC1 on methylation in pancreatic cancer cell line.
Methods and materials Transient transfection of human pancreatic cell line PaTu8988 cells with siRNA against HDAC1 were adopted.PaTu8988 cells were plated 24h prior to transfection then were transiently transfected HDAC1 siRNA or Negative siRNA,cells were collected and DNA were extracted then sodium bisulfite modification and BSP of hMLH1 was performed based upon instrument.The production of BSP was purified and inserted into pMD18-T vector with ligase.The ligation mixture was used to chemotransform E.coliDH5a,after which the cells were plated on LB plate containing ampicillin,X-gal and IPTG and placed at 37℃.The next morning,the plate was examined and the white clones were picked and identified by PCR.Then the chemotransforrned E.coliDH5a was sequenced to analyze methylation of CpG islands of hMLH1.Real-time RT-PCR(SYBR GREEN) was used to detect the change of rnRNA expression of Bcl-2, Bax and p21 after HDAC1 siRNA interference.
Results Methylated CpG sites of hMLH1 in HDAC1 siRNA group were descreased than those in Negative siRNA group and control group.At 48h after transfection in control group,15nM and 30nM Negative siRNA group and HDAC1 siRNA group the mRNA expression of Bcl-2,Bax and p21 were(1.00±0.11;0.82±0.07;0.67±0.02;0.57±0.02; 0.77±0.01;P=0.002);(1.02±0.22;1.37±0.20;2.64±0.39;1.49±0.27;3.83±0.27;P＜0.001) and(1.05±0.36;5.19±0.17;7.35±0.07;7.12±0.67;9.29±1.22;P＜0.001) respectively.
Conclusions Transfection of HDAC1 siRNA effectively decreased the methylated CpG sites in promoter of hMLH1 in human pancreatic cancer cell lines PaTu8988.At the same HDAC1 siRNA interference can inhibit the expression of bcl-2 mRNA and increase the expression of Box and p21 mRNA
Based on the above experiments,the final conclusions were as followings:
1.The expression of HDAC1 and HDAC3 on mRNA levels were higher in human pancreatic carcinoma tissues than in paracancerous tissues.
2.In pancreatic carcinoma tissues HDAC1 was highly expressed,high HDAC1 expression might have an important role in the aggressiveness and lymph node metastasis.
3.Transfection of HDAC1 siRNA effectively inhibits the expression of HDAC1 mRNA and protein in human pancreatic cancer cell lines PaTu8988.In addition,HDAC1 siRNA induces G0/G1 and G2/M cell cycle arrest and apoptosis and thereby suppresses the growth of PaTu8988 cell lines.
4.Transfection of HDAC1 siRNA can decrease the methylated CpG sites in promoter of hMLH1 in human pancreatic cancer cell lines PaTu8988.At the same time HDAC1 siRNA interference can inhibit the expression of Bcl-2 and increase the expression of Bax and p21 on mRNA lever.
HDAC1 could be a new gene therapy target in pancreatic cancer,the role of other members of HDACs should be investigated in the future.
引文
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