胃癌线粒体DNA拷贝量的变化

摘要

目的: 通过比较线粒体基因组(mitochondrial DNA, mtDNA)拷贝数在胃癌和癌旁胃黏膜组织间的差异, 阐述mtDNA与胃癌发生的关系.

方法: PCR分别扩增胃癌组织和癌旁胃黏膜组织各20例共40个样本的线粒体D-loop两个高变区HV₁ (hypervariable region)和HV₂; 并以核基因组的b-actin作为定量标准物. 聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis, PAGE)银染比较mtDNA拷贝数在癌和正常组织间的差异.

结果: HV₁和HV₂拷贝量(用b-actin标准化)在胃癌组织和癌旁组织间有显著的差异(P<0.01); 其拷贝量与组织类型, 癌组织浸润深度未发现有统计学联系(P>0.05); 而与核内一些重要的酶: 碱性磷酸酶(AKP)、环腺苷酸磷酸二脂酶(cAMP-PDE)和环鸟苷酸磷酸二脂酶(cGMP -PDE)表达有一定关系(P<0.05).

结论: 胃癌的发生与胃上皮细胞内mtDNA量的减少有着密切的关系. 有望成为一种新的肿瘤分子标志物.

关键词: N/A

Alterations of mtDNA copy number in gastric carcinoma

Cheng-Bo Han, Fan Li, Xue-Fei Yang, Xiao-Yun Mao, Dong-Ying Wu, Yan Xin

Cheng-Bo Han, Xue-Fei Yang, Xiao-Yun Mao, Dong-Ying Wu, Yan Xin, Fourth Laboratory of the Cancer Institute, First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China

Fan Li, Department of Geriatrics, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China

Supported by: the National Natural Science Foundation of China, No. 30371607.

Correspondence to: Yan Xin, The Fourth Laboratory of Tumor Institute, The First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China. yxin@hotmail.com

Received: August 7, 2003
Revised: August 20, 2003
Accepted: September 24, 2003
Published online: February 15, 2004

Abstract

AIM: To explore the relationship between mitochondrial DNA (mtDNA) and gastric cancer by comparing the difference of mtDNA copy number in gastric cancers and paracancerous tissues.

METHODS: Hypervariable reigon (HV)₁ and HV₂ of mitochondrial D-loop region from 20 cases of gastric cancer and 20 paracancerous tissues were amplified by PCR; meantime b-actin was served as a quantitative standard marker, followed by polyacrylamide gel electrophoresis (PAGE) and silver staining, in which the difference of mtDNA copy number was compared between gastric cancers and paracancerous tissues.

RESULTS: There existed significantly quantitative difference in HV₁, HV₂ (standardized with b-actin) between gastric cancers and paracancerous tissues (P < 0.01). mtDNA copy number was associated with important enzymes in nucleus such as AKP, cAMP-PDE and cGMP-PDE (P < 0.05), although not with tumor histological type and invasive depth (P > 0.05).

CONCLUSION: The occurrence of gastric cancer is closely associated with decreased mtDNA copy number, which may be a new tumor marker.

Key Words: N/A

0 引言

胃癌是世界上最常见的肿瘤之一[1-10], 过度的亚硝酸盐饮食和幽门螺旋杆菌感染等是胃癌发病的重要危险因素[11]. 线粒体DNA(mitochondrial DNA, mtDNA)与肿瘤的发生可能有一定的关系[12]. mtDNA是一长为16 569 bp双链闭环分子, 编码并参与了氧化磷酸化和ATP生成所必需的多肽. 线粒体内氧浓度很高, 易产生自由基(free radicals)及过氧化氢等活性氧簇(reactive oxygen species, ROS), 而线粒体本身又不能合成谷胱甘肽(glutathione)将其有效去除, 因此mtDNA易受活性氧损伤[13], 再之由于mtDNA缺乏组蛋白的保护以及线粒体内缺乏有效的DNA损伤修复系统, 因而mtDNA损伤要远高于核基因组[14].

1 材料和方法

1.1 材料

胃癌组织和癌旁胃黏膜组织各20例共40个样本取自于中国医科大学附属第一医院肿瘤科胃癌切除术后冰冻标本. 按照WHO组织学分型: 乳头管状腺癌3例, 高分化和中分化腺癌共8例, 低分化腺癌3例, 未分化癌2例, 黏液腺癌2例, 印戒细胞癌2例; 按照Lauren分型: 弥漫型胃癌11例, 肠型胃癌9例. 浸润至深肌层5例, 浆膜下6例, 透浆膜8例, 黏膜下1例. 所有胃癌组织标本经过酶组化和免疫组化染色分析.

1.2 方法

胃组织30 mg剪碎匀浆30 s, 用含有0.1 g/L 蛋白酶K和5 g/L SDS的10 mmol/L Tris-HCl/0.1 mol/L EDTA (pH7.4) 1 mL进行消化; DNA用体积比为25:24:1的酚/氯仿/异丙醇抽提2次, 氯仿/异丙醇(24:1) 1次; 再用1/10体积的3 mol/L乙酸钠(pH7.4)和2倍体积乙醇沉淀, 700 mL/L乙醇洗涤. 沉淀DNA重溶于50 ml 10 mmol/L Tris-HCl/0.1 mmol/L EDTA(pH8.0). 紫外分光光度仪定量后平衡DNA.

1.2.1 PCR扩增: 以D-loop上HV₁ (hypervariable region)和HV₂的2个片断的拷贝数来衡量mtDNA拷贝数. 设计的引物在HV区两侧保守区域内(12S rRNA和tRNA^pro), 以避免由于引物区变异导致假阴性的结果. 引物设计如下, HV₁: F15974 5'-ACTCCACCATTAGCACCCAAA- 3', R16564 5'-TGATGTCTTATTTAAGGGGAACGT-3'; HV₂: F4 5'-CACAGGTCTATCACCCTATTAACCA-3', R628 5'-GCCCGTCTAAACATTTTCAGTG625-3'. 同时为减少DNA模板量的差异给实验带来的假阴性结果, 扩增核内b-actin基因(引物F: 5'-AAGGGACTTCCTGTAACAATGCA-3', R: 5'-CTGGAACGGTGAAGGTGACA-3')作为内对照(定量标准物), 对HV₁、HV₂扩增量即mtDNA拷贝量进行标准化. 在50 ml扩增反应体系中包括总DNA 约50 ng, 引物各0.5 mmol/L, 每种dNTP各200 mmol/L, 2.5 U Taq酶(TakaRa ExTaq^TM). PCR反应条件: 起始94 ℃ 5 min; 接着94 ℃变性45 s, 60 ℃ 退火45 s, 72 ℃延伸1 min, 30循环; 最后72 ℃继续延伸3 min. 模板和引物等的量要严格保持一致(稀释后加入以减少实验的系统误差).

1.2.2 PAGE银染: 将PCR产物进行聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis, PAGE): 浓缩胶3%, 分离胶8%, 加产物3 ml和上样缓冲液2 ml至胶孔内, 8 mA等流电泳20 min, 然后调至15 mA继续电泳30 min. 取下凝胶进行银染: 100 mL/L乙醇内固定5 min; 11.3/mL硝酸5 min; 水洗1 min×2; 12 mmol/L硝酸银染色5 min×2; 水洗1min×2; 30 g/L碳酸钠/0.5 mL/L甲醛显影液显影数分钟; 100 mL/L乙酸后固定5 min; 30 mL/L甘油5 min; 封片保存, ChemiImager扫描; Image J软件对HV₁、HV₂和标准物b-actin进行定量分析, 计算HV₁/b-actin、HV₂ /b-actin比值.

统计学处理 计量资料以mean±SD表示, 以SPSS11.0统计软件包进行t检验、ANOVA及相关系数r检验. P<0.05有差异, P<0.01有显著性差异.

2 结果

2.1 胃癌组织mtDNA定量

在胃癌组织和癌旁胃黏膜正常组织均能扩增出产量比较一致的核内管家基因b-actin. 癌旁组织都可以扩增出HV₁和HV₂, 而癌组织则呈现明显的弱带甚至无条带(图1, 2), HV₁和HV₂扩增量在癌组织和癌旁组织间存在着显著性差异(P<0.01, 表1), 即mtDNA在胃癌组织和癌旁组织间的拷贝量是不同的, 癌组织mtDNA明显降低. HV₁与HV₂呈强相关(r = 0.91, P<0.01). 说明单独由于HV₁或HV₂引起的假阴性结果可能性非常小. mtDNA的拷贝量与胃癌WHO组织学分型之间没有关系, 也可能是由于样本量太少以及选取的样本类型过于分散有关. 但按Lauren分型, 定量结果同样没有统计学联系(P = 0.086).

表1 以mtDNA D-loop区HV₁为代表mtDNA定量分析结果.

组别	n	HV1/-actin	P
组织性质
正常	22	2.51±0.29	<0.01
癌组织	22	0.99±0.16
Lauren's
弥漫型	9	0.98±0.19	>0.05
肠型	11	1.02±0.25
浸润深度
深肌层	5	0.98±0.17	>0.05
浆膜下	6	1.09±0.13
透浆膜	8	0.95±0.11
AKP
_	9	0.83±0.10	<0.05
+-+++	11	0.98±0.13
LAP
_	14	0.90±0.13	>0.05
+-+++	6	0.91±0.18
cAMP-PDE
_	11	0.78±0.10	<0.05
+-+++	9	0.97±0.18
cGMP-PDE
_	14	0.80±0.17	<0.05
+-+++	6	1.90±0.20

图1 HV₁ 区 PCR 扩增PAGE 银染结果 M: 100 bp Ladder Marker; N: 正常组织; C: 癌组织.

图2 HV₂ 区 PCR 扩增PAGE 银染结果.

2.2 mtDNA定量与核内酶表达的关系

胃癌组织标本经酶组化和免疫组化染色分析显示, 碱性磷酸酶(alkaline phosphatase, AKP)表达11例: 亮氨酸氨基肽酶(Leu-aminopeptidase, LAP)表达6例; 环腺苷酸磷酸二脂酶(cyclic AMP phosphodiesterase, cAMP-PDE)表达9例; 环鸟苷酸磷酸二脂酶(cyclic GMP phosphodiesterase, cGMP-PDE)表达6例. 结果显示mtDNA定量与AKP、cAMP-PDE 和cGMP-PDE有统计学联系(P<0.05, 表1), 而与LAP无统计学联系(P>0.05).

3 讨论

胃癌的发生与多个癌基因和抑癌基因改变有关[1-10] 但确切的机制仍不十分清楚. 肿瘤的生物学特征不仅取决于核内遗传物质而且与核外的mtDNA 也有一定的关系. 线粒体基因组缺乏损伤修复系统加之线粒体内氧浓度很高易产生氧自由基而其本身又不能合成谷胱甘肽将其有效去除所以特别易遭受内源性损伤因子和外源性致癌物质的攻击[15-16]. 线粒体内脂肪/DNA的比值很高使具有嗜脂性的致癌物优先在占细胞总DNA 量很少的mtDNA上聚集[17]; mtDNA缺乏组蛋白的保护即是裸露的[18]. 因此mtDNA是致癌物作用的重要靶点其损伤程度和突变率显著高于核DNA(大约10 倍于核)[19].

mtDNA 非编码D-loop 区和高变(hypervariable region HV)是mtDNA 与线粒体内膜相结合的区域不仅暴露于脂质过氧化物而且其本身的三链结构对氧化损伤也高度敏感因此该区更易遭受ROS 的破坏[20]. Chomyn et al[21]认为mtDNA 对细胞凋亡启动并不是必需的但却会影响其发生的速率原因是mtDNA的减少或损伤增多会导致ROS 产生量增加.

细胞癌变的本质是增生不再受控制并呈现不同程度的去分化. cAMP 和cGMP 在调节细胞增生与分化这对矛盾中起着重要作用. cPDE 是降解cAMP和cGMP的关键酶其活性变化直接影响cAMP 和cGMP的代谢进而影响细胞的增生与分化[22-24]. 本研究组在以往的研究中发现cAMP-PDE 的表达与胃癌的病理生物学行为有一定的关系. 现研究表明特异性cPDE 抑制剂可以诱导慢性淋巴细胞白血病(CLL)细胞凋亡并且与cAMP水平成剂量依赖性[25-26]. 哈利普兰(rolipram)是cPDE4 抑制剂弗司扣林(forskolin)是腺苷酸环化酶(adenylate cyclase)激活剂Moon et al[27]认为二者诱导CLL 细胞凋亡可能是通过激活蛋白磷酸酶2A (protein phosphatase 2A PP2A) 诱导Bcl-2 家族成员Bad 去磷酸化使之转位至线粒体外膜电压依赖性离子通道(voltage dependent anion channel VDAC)处在Bax 等的共同作用下使线粒体膜去极化通过VDAC释放细胞色素c (cytochrome ccyt-c) 进而激活caspase-9 和caspase-3. 由此可见cPDE等所引起的核内一系列分子学改变影响了核外线粒体膜的通透性以至于细胞凋亡发生[28-31]. 这势必会对线粒体内的遗传物质的稳定性产生一定影响. 各种凋亡刺激因子直接或间接造成ROS 产生增加ROS 不仅会直接损伤mtDNA 尤其是D-loop 复制区导致其复制效率下降; 而且促进开启的VDAC 通道进一步打开释放凋亡促进因子cyt-c AIF 和Smac/Diablo 等. 此外一些类型胃癌从功能分化上不但产生黏液而且还不同程度表达小肠吸收细胞标志酶(如AKP 和LAP)有助于水解癌周间质并向周围正常组织浸润性生长是胃癌功能分化的重要标志酶. AKP 和(或)LAP 阳性而无黏液分泌的胃癌功能分类属于吸收功能分化型主要包括高分化的乳头状腺癌和管状腺癌; 而AKP 和LAP 阴性有黏液分泌的胃癌功能分类属于黏液分泌功能分化型主要包括黏液腺癌和一些未分化型胃癌. 本项研究结果显示AKP 阳性胃癌其mtDNA 拷贝数高于AKP 阴性胃癌(P<0.05)分析可能是分化程度低的胃癌mtDNA的受损程度更大所以拷贝数相对较低. mtDNA 定量与AKP cAMP-PDE和cGMP -PDE 有统计学联系进一步证实核内遗传物质或信号分子的改变对核外线粒体基因组有一定的影响为进一步研究其在疾病发生方面如何作用提供依据.

胃癌组织与正常胃黏膜mtDNA 定量存在显著差异mtDNA 定量有望成为一种新的肿瘤分子标志物但仍处于探索阶段具体的机制还有待于进一步研究.

志谢

中国医科大学生物芯片中心赵雨杰教授和教研室其他工作人员为本研究提供的帮助

备注

编辑: N/A

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