摘要
胃癌中若干肿瘤抑制基因异常甲基化的研究
胃癌的发生与其他恶性肿瘤一样是多基因多阶段变异累积形成的病理过程,而抑癌基因的甲基化是一个研究的热点,目前已鉴定的易高甲基化的基因包括:参与细胞周期、DNA修复、耐药性形成、分化、凋亡的基因及参与肿瘤转移和血管生成的基因。此研究中选择了主要与细胞周期及细胞凋亡有关的基因Apaf-1,P27kip1,DAPK1,对胃癌组织中的DAPK1,Apaf-1,P27kip1基因甲基化状态、表达进行研究,以研究胃癌组织中此三种基因表达异常的原因,探讨基因甲基化在胃癌发病中的作用。在对胃癌细胞系的研究中除对此三个基因的甲基化状态进行进一步研究,在去甲基化药物5-aza-CdR作用细胞后,亦检测目的基因的甲基化状态、表达的改变及与细胞生长状态的关系,进一步探讨抑癌基因的表达与其甲基化状态的相关性。顺铂(CDDP)作为治疗多种实体瘤临床一线用药,是胃癌的常用化疗药物,5-aza-CdR是明确的去甲基化的药物,本实验从这两株胃腺癌细胞的抑癌基因的高甲基化状态入手,通过对低剂量的顺铂与5-aza-CdR的联合用药以观察两者对胃癌细胞生长的影响,探讨5-aza-CdR是否能够作为一线化疗药物顺铂的辅助用药,使顺铂在较低剂量时发挥较大的作用。
材料与方法
1、实验材料
中国医科大学附属第一医院及中国医科大学附属第二医院胃癌患者术后胃癌组织、胃癌旁组织;标本细胞株:胃癌BGC823细胞系,胃癌SGC7901细胞系(均购自细胞生物教研室);总RNA提取试剂:TRIZOL Reagent(GIBCO BRL公司),RNAout(TaKaRa公司),去甲基化试剂5-aza-2’-deoxycitydine(5-Aza-CdR)为Sigma产品,碘化丙啶(PI)、丫啶橙(AO)为Sigma产品,RT-PCR试剂盒购自TaKaRa公司,Wizard DNA Clean-up(Promega公司),Annexin-v-FITC凋亡检测试剂盒为北京宝赛生生物技术有限公司生产,RPMI 1640培养基为Hyclone产品,Apaf-1绵羊抗人单克隆抗体(Santa cruz产品),Taq酶及dNTP均为TaKaRa公司产品,Apaf-1绵羊抗人单克隆抗体(Santa cruz产品),p27kip1鼠抗人单克隆抗体(博士德公司产品),驴抗羊抗体(Santa cruz产品),羊抗鼠抗体(北京中杉金桥生物公司产品)。
2、实验方法
MTT法检测细胞生长活性,AO染色、Annexin-v-FITC凋亡流式细胞仪检测细胞凋亡;PI染色流式细胞仪检测细胞周期及凋亡的变化;用半定量RT-PCR方法,Western-Blot方法分析胃癌细胞及组织中各目的基因的表达,用MSP对胃癌细胞及组织中各基因的启动子区甲基化情况进行研究。用杂合性丢失(Loss of heterozygosity,LOH)分析胃癌患者Apaf-1基因所在的12q22-23区域出现缺失的情况。
3、统计分析
所有资料采用SPSS11.5分析。计量资料比较采用方差分析;两两比较采用LSD法;计数资料采用χ~2检验;凋亡率资料分析采用秩和检验,相关分析采用Spearman方法。
结果
一、胃癌组织中各基因的表达及甲基化状况
1、各目的基因在胃癌组织的表达
(1)RT-PCR检测基因mRNA表达:在胃癌组织中Apaf-1基因表达下调17/35例,p27kip1基因表达下调17/35例,DAPK1基因表达下调16/35例,此三组基因的癌旁组织的mRNA表达明显高于均癌组织,具有统计学意义(均P<0.05)。
(2)Western Blot检测基因蛋白表达:在胃癌组织中Apaf-1基因在胃癌组织中5例(5/35)有蛋白表达,癌旁组织则有29例蛋白表达(29/35),P<0.05,P27kip1基因在胃癌组织中3例有蛋白表达(3/35),癌旁组织有30例蛋白表达(30/35),此两组基因癌旁组织的蛋白表达显著高于癌组织,均具有统计学意义(P<0.05)。
(3)各基因启动子甲基化的情况DAPK1胃癌组织及癌旁组织中甲基化率均为100%,无统计学意义;P27kip1胃癌组织中甲基化率为51.42%明显高于癌旁组织(25.71%),具有统计学意义(P<0.05),Apaf-1胃癌组织及癌旁组织中甲基化率分别为48.57%,22.86%,胃癌组织的甲基化率明显高于癌旁组织,具有统计学意义(P<0.05)。
(4)胃癌中P27kip1、Apaf-1基因的甲基化状态与其表达的关系胃癌组织中17例具有Apaf-1基因甲基化的组织16例有mRNA表达下调,而在无该基因甲基化的胃癌组织中仅有1例表达下调(相关性分析r_s=0.886,P=10~(-6));而P27kip1基因在胃癌组织中则18例甲基化的组织中则有15例mRNA表达下调,而在无该基因甲基化的组织中有2例mRNA表达下调(相关性分析r_s=0.716,P=10~(-6))。分析结果显示此两种基因的在胃癌组织的低表达均与基因的高甲基化有关。
(5)胃癌中Apaf-1基因的5个多态位点的LOH与其表达下降的关系检出率分别是:D12S346为34.3%,D12S1706为8.3%,D12S327为57.1%,D12S1657为11.4%,D12S393为42.8%,而在两个位点以上均出现LOH的则占51.4%,其中有三个位点出现LOH的则占16.7%,出现两个位点LOH的18例组织中13例出现Apaf-1的mRNA表达下降,经相关性分析,r_s=0.487,P=0.003,有统计学意义,说明Apaf-1基因的多位点的LOH的出现与胃癌中该基因表达下降有关。
二、5-Aza-CdR对胃癌细胞系中多种肿瘤抑制基因甲基化的影响
1、5-aza-CdR对胃癌BGC823,SGC7901细胞生长的影响
MTT检测发现胃癌BGC823,SGC7901细胞在5-aza-CdR作用后,细胞生长活力明显减少,AO染色显示细胞形态亦明显改变,而流式细胞仪检测显示SGC7901细胞的细胞在用药后,10~(-6)与5×10~(-6)组中得G_0期细胞明显上升,而S期细胞数量下降(P<0.05),此细胞株的凋亡率亦明显上升(P<0.05),而BGC823细胞的细胞周期未受5-aza-CdR太多影响,但是在1×10~(-6),5×10~(-6)组细胞凋亡率均明显上升(P<0.05)。
2、5-aza-CdR对胃癌BGC823,SGC7901细胞的Apaf-1,P27kip1,DAPK1基因的表达影响
(1)mRNA的表达:在用药前胃癌BGC823,SGC7901细胞中Apaf-1基因及DAPK1基因是低表达状态,而经5-aza-CdR 1×10~(-6)mmol/L,5×10~(-6)mmol/L,1×10~(-5)mmol/L浓度作用下,48h、72h时各基因的表达较对照组均明显上调(P<0.05),但在此三个浓度组无明显的时间与剂量的依赖性,而p27kip1则在用药前为均失表达,而用药后则出现了表达的恢复(P<0.05),亦无明显的时间与剂量的依赖性。
(1) Western-blot检测胃癌BGC823,SGC7901细胞的Apaf-1,P27kip1蛋白表达:SGC7901中Apaf-1、P27kip1基因的表达在对照组中为失表达,而在药物作用后,基因的表达恢复,并且在5×10~(-7)mmol/L,1×10~(-6)mmol/L,5×10~(-6)mmol/L有一定的药物的剂量但无明显的时间依赖性;在BGC823中,Apaf-1基因的表达比较低,P27kip1基因的蛋白表达在对照组中为失表达,在用5-aza-CdR作用后基因表达明显上升(P<0.05)。
3、MSP检测胃癌BGC823,SGC7901细胞中目的基因的启动子甲基化状态
在经过5-aza-CdR药物作用后,二种细胞的各基因的甲基化状态均发生了改变。各基因在二种细胞中均为高甲基化状态并且通过去甲基化的药物作用后,高甲基化的状态均出现减少的改变,同时非甲基化的条带出现,甚至在药物浓度较大或作用时间过长时,高甲基化状态完全消失。
三、5-aza-CdR与低浓度CDDP联合用药对胃癌细胞的影响
1、MTT检测细胞活性
从生长曲线可以看出SGC7901细胞与BGC823细胞在5-aza-CdR(5×10~(-6)mmol/L)的作用下生长受到一定的抑制,而在低浓度的CDDP(5×10~(-7)mmol/L)的作用下生长为接近直线,无明显的对数生长期,而在两种药物的联合作用下生长曲线出现了下降的趋势,这说明此两种药物的联合作用有协同的作用。
2、Annexin-v-FITC凋亡检测各组凋亡情况
SGC7901细胞与BGC823细胞在未用药物作用的对照组中凋亡及坏死细胞很少,而在用药物5-aza-CdR(5×10~(-6)mmol/L)作用后,凋亡及坏死细胞均有所增加,在低浓度的CDDP(5×10~(-7)mmol/L)作用后,细胞的凋亡及坏死均明显增加,而在联合用药组则细胞的凋亡及坏死则较单独用药组有明显的上升。
3、各目的基因的表达的情况
(1)RT-PCR:目的基因的mRNA的表达情况
在SGC7901细胞中Apaf-1基因在与CDDP联合用药中虽然各基因的表达均较空白对照组明显上升(P<0.05),但相对于单独应用CDDP的对照组细胞的各基因表达并无明显上升,而BGC823细胞中则在联合用药组该基因的表达明显上升;P27kip1基因在此二株细胞中对照组表达均阴性,在5-aza-CdR处理后,基因均表达恢复,在单独应用CDDP后,亦出现基因表达的恢复,同样的在BGC823细胞中联合用药组亦见明显的该基因表达上升(与CDDP组比较)。而DAPK1基因的表达则在用药后表达均明显上升,尤其在联合用药组,二株细胞中该基因表达均明显上升(与CDDP组比较)。
(2) Western-Blot:目的基因蛋白的表达
二株细胞的目的基因的Apaf-1,p27kip1蛋白表达与mRNA的表达基本一致,用药后均可见蛋白的恢复表达与高表达(P<0.05vs对照组),但在联合用药组两株细胞的Apaf-1基因的蛋白表达均较对照组,5-aza-CdR组及CDDP组明显升高(均P<0.05);而P27kip1的蛋白表达则无此明显的变化。
结论
1、P27kip1基因在胃癌组织中表达下降与该基因启动子甲基化相关。
2、Apaf-1基因启动子甲基化以及多态位点的LOH均是该基因在胃癌组织中表达下降的主要原因。
3、5-Aza-CdR对SGC7901细胞和BGC823细胞具有增生抑制作用,其机制与其阻滞细胞周期、诱导凋亡有关。
4、5-Aza-CdR能改变细胞中Apaf-1,DAPK1,P27kip1基因的甲基化状态,Apaf-1、DAPK1、p27kip1基因的表达情况与其甲基化状态的改变有关。
5、5-aza-CdR能够增加胃癌细胞对低浓度CDDP的敏感性。
6、在胃癌细胞中,DAPK基因在5-aza-CdR增加CDDP药敏性有关。
Research on abnormal methylation of several tumor suppressors in human gastric cancer
Effects of 5-Aza-CdR on proliferation and abnormal methylation of tumor supressor genes of human gastric cancer cell lines Gastric cancer is a kind of malignant tumor which threatens human beings' health, and its occurance is a pathological process which is many different genes involved. The gene promoter CpG island methylation is a important reason in tumor growth. In the study, we detected the effects of 5-Aza-CdR on proliferation of human gastric cancer cell lines SGC7901 and BGC823, methylation and epression of Apaf-1, p27kipl, DAPK1 gene and discussed the relationship between the methylation and expression of Apaf-1, p27kip1, DAPK1. we detect the expression and methylation state of DAPK1, p27kip1, Apaf-1, and explore the role of these genes concurrent methylation in gastric cancer tissues. CDDP is one of the important clinical anticancer drugs, and 5-aza-CdR is a specific demethylating agents. Many studies have indicated that promoter hypermethylation is in relation to chemotherapy resistance of cisplatin in many tumors. The experiment is to study whether the 5-aza-CdR can increase the sensitivity of gastric cancer cells to low dose CDDP.
Materials and methods
Materials: 35 surgical specimens of gastric carcinoma and their adjacent nonmal gastic tissues resected at the First Affiliated Hospital of China Medical University and the second Affiliated Hospital of China Medical University. Gasric cancer lines: BGC823 cell line, SGC7901 cell line. TRIzol Reagent(GIBCOL BRL company), RNAout(TakaRa公司), demethylate agent 5-aza-2'-deoxycitydine(5-Aza-CdR) is the product of Sigma; PI is the product of Sigma; RT-PCR kit is the product of TaKaRa Company; Wizard DNA Clean-up(Promega company), Wizard DNA Clean-up(Promega company); RPMI 1640 culture medium is the product of Hyclone company. Apaf-1, p27kip1 monoclone antibody is the prduct of Santa cruz.
Methods: SGC7901 cells and BGC823 cells were cultured in RPMI1640 and was treated with different concentrations of 5-Aza-CdR. The proliferation of the cells was detected by MTT assay, OA stain, flow cytometry. The methylation of Apaf-1, DAPK1, P27kip1 in the two cell lines was detected by MSP, and the expression of Apaf-1, DAPK1, P27kip1 was detected by RT-PCR and westernblot. SGC7901 cells and BGC823 cells were cultured in RPMI1640 and was treated with different concentrations of 5-Aza-CdR and low dose CDDP. The proliferation of the cells was detected by MTT assay and flow cytometry. The expression of Apaf-1, DAPK1, P27kip1 was detected by RT-PCR and westernblot. Using the semi-quantitative RT-PCR and western-blot analyses the expression of the Apaf-1, p27kip1, DAPKlin gastric cancer. Using the Loss of heterozygosity(LOH) analyses whether there is the loss of Apaf-1 gene in the domain of 12q22-23. Using the methylation specific PCR(MSP) analysis the promoter methylation of Apaf-1, p27kip1, DAPK1 gene in gastric cancer. Statistics analysis: Adopting t test of Excel.
RESULTS
Ⅰ. Supressor genes methylation detection in gastric cancer
1. The expression of Apaf-1, DAPK1, p27kip1's mRNA: Apaf-1, p27kip1, DAPK1 gene low expression is 17/35, 17/35 and 16/35(P<0.05).
2. The expression of Apaf-1, DAPK1, p27kipl's protein: There was 5 pous expression of Apaf-1 in gastric cancer and 29 pous expression of Apaf-1 in cancer-adjacent tissues; There was 3 pou expression of Apaf-1 in gastric cancer and 30 pous expression of Apaf-1 in cancer-adjacent tissues.
3. The LOH frequencies of D12S346, D12S1706, D12S327, D12S1657 and D12S393is 34.3%, 8.3%, 57.1%, 11.4%, 42.8%. There was 50% LOH in two sites and 17% LOH in three sites.
4. Methylation of DAPK1 was present in both cancer-adjacent tissues and gastric cancer tissue. P27kip1 and Apaf-1 rate of methylation were 25.71% and 22.86% in cancer-adjacent tissues 51.42% and 48.57% in gastric cancer respectively(p<0.05).
Ⅱ. Effects of 5-Aza-CdR on proliferation of human gastric cancer cell lines and abnormal methylation ofApaf-1 gene
1. After SGC7901 and BGC823 cells were exposed to 5-Aza-CdR (1×10~(-7)mol/L, 5×10~(-7)mol/L, 1×10~(-6)mol/L, 5×10~(-6)mol/L), 5-Aza-CdR displayed a growth inhibitory effect on SGC7901 and BGC823 cells in a dose-and time-dependent manner, FCM analysis showed that G0/G1 phrase rate increased with exposing to 5-Aza-CdR (1×10~(-6)mol/L, 5×10~(-6)mol/L) for 72h(P<0.01), the apoptosis rate increased too(P<0.01).
2. The methylation and the loss of Apaf-1, P27kip1, DAPK1 mRNA and protein of Apaf-1,, P27kip1, DAPK1 in SGC7901 and BGC823 cells were changed with 5-Aza-CdR.
Ⅲ. The influence of 5-aza-CdR and low dose CDDP on gastric cell line
1. MTT assay: The proliferation of SGC7901 cell and BGC823 cell added 5-aza-CdR and low dose CDDP was more inactive than that only added low dose CDDP.
2. Annexin-v-FITC detecting the apoptosis: The apoptosis of SGC7901 cell and BGC823 cell added 5-aza-CdR and low dose CDDP was more than that only added low dose CDDP.
3. the expression of gene: Analysis of mRNA and protein indicated the DAPK is both increased more greatly in SGC7901 cell and BGC823 cell when they were added 5-aza-CdR and low dose CDDP than that only added low dose CDDP.
CONCLUSION
1. The mechanism of 5-Aza-CdR's growth inhibitory effect on SGC7901 and BGC823 cells is related with blocking cell cycles, inducing apoptosis, and changing the methylation ofApaf-1 gene.
2. The loss expression in SGC7901 and BGC823 cells is because of methylation states.
3. 5-aza-CdR can increase the gastric cells' sensitivity to low dose CDDP.
4. DAPK gene acts a important role in 5-aza-CdR increasing the gastric cells' sensitivity to low dose CDDP.
5. multiple genes concurrent methylation plays an important role in some gastric cancers, They happened at early stage.
6. The expression of Apaf-1, DAPK1, p27kip1 is low in gastric cancer.
7. gene's promoter Methylation of Apaf-1 and LOH of the gene in the domain of 12q22-23 both are the main reasons gene's promoter Methylation of p27kip1 is related with the in gastric cancer.
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