Delta mtDNA4977 is more common in non-tumoral cells from gastric cancer sample

BACKGROUND: The aim of this study was to determine the frequency of delta mtDNA4977 in tumoral cells as compared with adjacent normal cells in gastric cancer. METHODS: In order to investigate whether a high incidence of mutation exists in mitochondrial DNA of gastric cancer tissues, we screened one of common region of the mitochondrial genome by PCR amplification and Southern blot followed by DNA sequence analysis. DNA isolated from these cells was used to amplify hypervariable regions ATPase8/6, COXIII, ND3, ND4 and ND5 of delta mtDNA4977. RESULTS: In 107 cancer patients, delta mtDNA4977 was detected in 6 cases (5.60%) of the tumoral tissues and 18 cases (16.82%) of the non-tumoral tissues that were adjacent to the tumors. Levels of delta mtDNA4977 deletions were found to be more in non-tumoral tissues than in adjacent tumoral tissues. There was no correlation of patients with certain clinical parameters like age, sex, tumor location and tumor size; however, there was an obvious relationship with intestinal-type of gastric cancer. CONCLUSIONS: Unknown genetic aspects, ambiguous environmental factors and reactive oxygen species (ROS) can cause the delta mtDNA4977 mutation rate to be increased in gastric cancer. The results suggest that percentage level of delta mtDNA4977 is less common and intolerable in tumoral tissue, probably because of high metabolism and ROS generation. We supposed that the cells initially had delta mtDNA4977 transform to tumoral cells and the existed deletion conferred metabolic disadvantage; thus, cells containing such a mtDNA deletion would be overgrown by other cancer cells without this mtDNA deletion. As a result, the presence of delta mtDNA4977 will be low in tumoral cells.     

胃癌细胞系和胃癌组织中线粒体DNA4 977 bp缺失及其生物学意义

目的 明确线粒体DNA 4977bp大片段缺失在胃癌细胞系、胃癌组织及胃癌患者血清中的频率,为胃癌的早期临床诊断寻找简便准确的分子标记。方法 运用Primer-shift PCR和直接测序的方法对13个胃癌细胞系、52对胃癌组织及对应的癌旁正常组织（年龄从28-78岁）、40例胃癌患者血清及40名正常人血清进行筛查。取10例胃癌组织的石蜡切片,采用显微切割技术在同一患者的切片上同时分离3种组织（包括胃粘膜正常腺体、肠化上皮及胃癌组织）对mtDNA 4977bp缺失进行了分析比较。结果 在13个胃癌细胞系中12个有mtDNA4977bp缺失（缺失率为92.3%),52例胃癌组织中38例有缺失（缺失率为73.1%),52例癌旁正常组织中27例有缺失（缺失率为52%）,40名胃癌患者血清中17例有缺失（缺失率为42.5%),40名正常人血清中8例有缺失（缺失率为20%）。胃癌组织和癌旁正常组织mtDNA 4977bp缺失差异有显著意义,癌组织mtDNA 4977bp缺失率与胃癌的分型和患者的性别没有关联。胃癌患者血清与正常人血清mtDNA4977bp缺失差异也有显著意义。10例显微切割组织中2例肠化上皮及胃癌组织中有缺失,而胃粘膜正常腺体未见缺失。结论 线粒体DNA 4977bp大片段缺失可能在胃癌发生和胃粘膜病变演化及细胞癌变的过程中起重要作用,血清中可以检测到线粒体DNA 4977bp大片段缺失,而且在胃癌患者血清中检出率远高于正常人血清。检测胃癌患者血清中mtDNA 4977bp大片段缺失有望成为一种简便易行的胃癌生物学行为的分子标记物。     

线粒体脑肌病中线粒体DNA的部分缺失

在2例Kearns－Sayre综合征（KSS）和2例慢性进行性眼外肌麻痹（CPEO）患者的骨骼肌线粒体DNA（mtDNA）中发现存在单一的大片段缺失突变。缺失的长度2．5～5．5kb，突变只发生在部分线粒体DNA中，突变型mtDNA占全部mtDNA的60．5％～84．6％。在10例其它的线粒体脑肌病患者骨骼肌标本和外周血标本及数例正常骨骼肌和胎肝标本的mtDNA中均未发现缺失突变。上述发现支持mtDNA的缺失突变为KSS和CPEO主要病因的观点。     

Mutations in the D-loop region of mitochondrial DNA in g astric cancer

Objective: To investigate the mutations in the D-loop region of mitochondrial DNA (mtDNA) in gastric cancer.Methods: The mtDNA of D-loop region was amplified by PCR and ,sequenced in 20 samples from gastric cancer tissue and adjacent normal membrane. Results: There were 7/20(35% ) mutations in the mtDNA of D-loop region in gastric cancer patients. There were four microsatellite instabilities among the 18 mutations. Nine new polymorphisms were identified in 20 patients. Conclusion: The mtDNA of D-loop region might be highly polymorphoric and the mutation rate is high in patients with gastric cancer.     

结直肠癌线粒体DNA拷贝数改变及4977片段缺失

目的 探讨结直肠癌（colorectal cancer,CRC）线粒体DNA（mitochondrial DNA,mtDNA）拷贝数异常、线粒体DNA 4 977 bp大片段缺失与TNM分期和分化程度的关系.方法 选取118例石蜡标本大肠癌组织,分别提取总DNA.以ND1和β-actin为目的基因,进行SYBR Green荧光定量PCR扩增,并用PCR扩增方法检测4 977片段缺失情况,探讨它们与不同TNM分期和分化程度之间的关系.结果 CRCⅠ和Ⅱ期癌组织平均拷贝数（2ND1/β-actin）分别为128.42±31.25和115.12±47.15,Ⅲ和Ⅳ期癌组织平均拷贝数为105.22±16.35和99.45±28.46.Ⅰ和Ⅱ期的拷贝数明显高于Ⅲ和Ⅳ期的拷贝数（P〈0.01）,癌组织低、中、高分化的平均拷贝数分别为101.34±41.35、112.33±42.32和127.22±31.23（P〈0.01）,4 977片段缺失率为15.25%（18/118）,线粒体DNA4977bp缺失与患者的分期呈负相关,与分化程度无明显关系.结论 线粒体DNA 4 977 bp缺失在大肠癌的早期阶段起到了重要作用,随着肿瘤的进展,拷贝数逐渐减少,线粒体DNA拷贝数的变化及大片段缺失在肿瘤的发病机制中可能起一定作用.     

Cu/ZnSOD基因敲除小鼠耳蜗易于出现mtDNA缺失突变-ROS对组织损伤的特异性研究

目的　探讨ROS对各种组织mtDNA的损伤情况 ,耳蜗mtDNA是否为ROS损伤的标靶。方法　应用套式PCR及分子克隆测序技术对 10只Sod1基因敲除小鼠及 5只同系野生型小鼠耳蜗、脑、肝脏、肾脏、脾脏、心脏及皮肤组织进行研究。结果　 1 各组织mtDNA可检测到 3种缺失 ,常见的缺失为mtDNA386 7bp和mtDNA372 6bp缺失 ,mtDNA4 2 36bp缺失不常见。 2 mtDNA缺失在不同组织的含量有明显的不同 ,肝脏和肾脏组织含量最高 ,耳蜗、心脏和大脑组织其次 ,脾脏及皮肤组织含量最低。与野生型小鼠比较 ,Sod1基因敲除小鼠mtDNA在不同组织的缺失量是野生型小鼠的 3- 2 0倍 ,其中耳蜗组织mtDNA缺失量约是WT小鼠的 15倍。结论　缺乏Sod1的保护 ,ROS可以攻击各种组织mtDNA ,但具有明显的组织特异性 ,耳蜗mtDNA是其损害的敏感标靶。     

非小细胞肺癌线粒体基因组大片段缺失突变研究

目的 明确mtDNA 4 977 bp大片段缺失在肺癌组织、癌旁正常组织及非癌患者肺组织中的分布频率,探讨mtDNA 4 977 bp大片段缺失与肿瘤的关系.方法 采用长距离PCR方法对37例肺癌组织和相应的癌旁正常组织、20例非癌患者正常肺组织mtDNA 4 977 bp缺失进行了检测.结果 肺癌组织、癌旁正常肺组织及非癌患者正常肺组织中均检测出存在有线粒体DNA 4 977 bp片段缺失.37例肺癌组织标本中有20例(54.1%)检测到4 977 bp缺失,37例相应的癌旁肺组织22例(59.5%)有缺失, 其中8例在肺癌组织中未显示而却在癌旁正常组织检测到缺失,20例非癌患者正常肺组织中也有6例出现 4 977 bp片段缺失.线粒体DNA 4 977 bp缺失与年龄、吸烟因素有关.结论 线粒体DNA 4 977 bp缺失并非肺癌特异性突变,在癌变过程中不太可能起重要的作用,而可能仅仅是肿瘤发生发展过程中环境和遗传因素作用的反映.     

线粒体肌病和脑肌病患者线粒体基因缺失的研究

目的 探讨线粒体肌病和脑肌病的骨骼肌线粒体ＤＮＡ（ｍｔＤＮＡ）缺失情况。方法 从１例线粒体肌病和１例脑肌病（ＭＥＲＲＦ）患者骨骼肌活检标本中,提取总ＤＮＡ,以限制性内切酶ＰｖｕⅡ酶切,与ｍｔＤＮＡ全长探针进行分子杂交。结果 线粒体肌病和ＭＥＲＲＦ患者分别为１５ｋｂ和５ｋｂ和ｍｔＤＮＡ基因缺失。结论 ｍｔＤＮＡ基因缺失是线粒体肌病和脑肌病的重要病因之一。     

与老龄相关的豚鼠线粒体基因组缺失的定位研究

目的 准确定位与老龄相关的豚鼠mtDNA大片段缺失位点．方法 参照人类和大鼠线粒体DNA大片段缺失的共同规律，运用计算机软件寻找豚鼠mtDNA可能的缺失位点，采用聚合酶链反应(PCR)、套式PCR技术与DNA测序技术，扩增豚鼠mtDNA基因组可能的缺失区，并进行序列分析．检测耳蜗螺旋器组织、蜗神经组织、大脑颞叶组织中mtDNA缺失的位点和片段大小，及mtDNA大片段缺失的发生率。结果首次发现豚鼠mtDNA在nt8636～nt13203位点之间存在4568个碱基的大片段缺失；老年豚鼠各组织中mtDNA．缺失率与青年组比较均有显著差异，前者mtDNA缺失率明显高于后者．结论 豚鼠mtDNA．COⅢ与ND5基因间有4568个碱；基片段缺失．mtDNA^4568缺失的发生与老龄有关。     

内耳缺血及线粒体DNA缺失与老年性耳聋发病的关系

目的 探讨内耳缺血及ｍｔＤＮＡ４９７７缺失与老年性聋的关系,确定老年性耳聋颞骨中ｍｔＤＮＡ缺失的病理基础。方法 对４１例不同分组的６７侧颞骨应用巢式ＰＣＲ和三重巢式ＰＣＲ检测ｍｔＤＮＡ４９７７缺失,应用图像分析技术测量血管管腔与血管的截面积比值。结果 在老年性聋组检出ｍｔＤＮＡ４９７７缺失的总发生率为５０．０％（１７／３４,缺失阳性耳数／总耳数）。在老年对照组检出ｍｔＤＮＡ４９７７缺失总发生率为２１．１％（４／１９）,与病变组比较差异无显著意义（Ｐ〈０．０５）。非老年对照组１０例１４耳均未检出ｍｔＤＮＡ４９７７缺失,缺失率为０。ｍｔＤＮＡ４９７７缺失阳性的老年性耳聋颞骨的神经滋养血管管壁增厚,管腔狭窄程度较缺失阴性组严重。并且ｍｔＤＮＡ缺失与内耳血管管腔狭窄程度的关系较ｍｔＤＮＡ４９７７缺失与年龄的关系更为密切     

Deletions are easy detectable in cochlear mitochondrial DNA of Cu/Zn superoxide dismutase gene knockout mice

目的：探讨ROS对各种组织mtDNA的损伤情况,耳蜗mtDNA是否为ROS损伤的标靶。方法：应用套式PCR及分子克隆测序技术对10只Sod1基因敲除小鼠及5只同系野生型小鼠耳蜗、脑、肝脏、肾脏、心脏及皮肤组织进行研究。结果：1.各组mtDNA可检测到3种缺失,常见的缺失为mtDNA3867bp和mtDNA3726bp缺失,mtDNA4236bp缺失不常见。2.mtDNA缺失在不同组织的含量有明显的不同,肝脏和肾脏组织含量最高,耳蜗、心脏和大脑组织其次,脾脏及皮肤组织含量最低。与野生型小鼠比较,Sod1基因敲除小鼠mtDNA在不同组织的缺失量是野生型小鼠的3-20倍,其中耳蜗组织mtDNA缺失量约是WT小鼠的15倍。结论：缺乏Sod1的保护,ROS可以攻击各种组织mtDNA,但具有明显的组织特异性,耳蜗mtDNA是其损害的敏感标靶。     

胃癌中脆性组氨酸三联体基因异常的检测分析

目的:检测脆性组氨酸三联体基因在胃癌组织中等位基因缺失和突变情况,分析该异常在胃癌发生中的作用。方法:用PCR鄄SSCP的方法检测36例胃癌和自身正常组织中FHIT基因外显子5、8及多态性位点D3S1300、D3S1234、D3S1312、D3S1313的缺失和突变。结果:肿瘤组织中外显子5有2例(2/36,5.6%)发生缺失,外显子8没有发现缺失;D3S1300的LOH发生率为25%(7/28),MSI为22.2%(8/36);D3S1234的LOH发生率为31%(9/29),MSI为13.9%(5/36);外显子5、8及多态性位点D3S1300、D3S1234、D3S1312、D3S1313的SSCP分析未发现突变。结论:胃癌患者FHIT基因外显子5、8的缺失和突变频率并不高,这种缺失和突变对胃癌的发生到底起多大作用需要进一步探讨;胃癌患者D3S1300、D3S1234位点的LOH、MSI等遗传不稳定现象发生频率较高,能否用于胃癌高危人群筛查和早期诊断值得进一步研究。     

Mitochondrial gene defect in patients with chronic progressive external ophthalmoplegia

Objective To detect the gene defect of mitochondrial DNA(mtDNA) from skeletal muscles in 2 patients with chronic progressive external ophthalmoplegia (CPEO).Methods After extraction of mtDNA, Southern hybridization was performed after restrictive digestion by PvuⅡ, EcoRI, Hind Ⅲ, and SacI. Then, we carried out polymerase chain reaction(PCR) and the enzyme digestion of the PCR products. Finally, mtDNA sequencing was done by automatic DNA sequence analyzer. Results In case 1, a 5 kb deletion was found by Southern blot analysis and PCR. And dosage analysis showed a heteroplasmic change with 44% mtDNAs deleted. In case 2, PCR plus restriction endonuclease PvuⅡ digestion demonstrated a mutation which was confirmed by DNA sequencing to be a single base substitution (T→C) inducing a novel PvuⅡ site around 10909 on mtDNA sequence. The laser image analyzer measurement revealed the mutation was almost homologous (99.4% mutant).Conclusions In case 1, a 5 kb deletion found in mtDNA is called "common deletion" according to the literature. In case 2, a novel PvuⅡ site was found. It seems to be a de novo point mutation affecting ND4 in published CPEO research and is first reported in Chinese population. This point mutation does not induce an amino acid(Phe) change according to the published human mitochondrial genetic code as well as the mtDNA sequence. Whether it affects the translation efficiency or transportation of signals between mitochondrial and nuclear genome needs further studies.     

Relationship between alterations of p16INK4a and p14ARF genes of CDKN2A locus and gastric carcinogenesis

Objective To investigate the relationship between alterations of p16INK4a and p14ARF genes and gastric carcinogenesis. Methods The tumors and neighboring gastric tissues from 48 patients with gastric cancer were studied. The homozygous deletion, mutation, methylation of the CpG islands, and mRNA expression of p16INK4a and p14ARF genes were assessed by PCR, PCR-SSCP, PCR based methylation assay, and RT-PCR. Results ① The homozygous deletion rate of p16INK4a and p14ARF was 35.4% （17/48）, and no homozygous deletion was examined in any gastric tissue neighboring the tumor. ② There was no point mutation of p16INK4a and p14ARF in 31 gastric cancers without homozygous deletion or in the matched gastric tissues adjacent to the tumor. ③ Methylation of the CpG islands of p16INK4a and p14ARF was detected in 47.9% (23/48) of gastric cancers, while methylation was observed only in 2 of 48 gastric tissues neighboring the cancer with a significant difference (P<0.01). ④ The loss rate of p16INK4a mRNA was 47.9% (23/48) in gastric cancer, and the patients of the combined methylation of exons 1α and 2 had a higher loss rate (100%, 6/6) of p16INK4a mRNA than those of the methylation of the other exons (11.8%, 2/17, P<0.01); the loss rate of p14ARF mRNA was 45.8%(22/48) in gastric cancer, and patients with the combined methylation of exons 1β and 2 had a higher loss rate (100%, 3/3) of p14ARF mRNA than those of the methylation of the other exons (15%, 3/20, P<0.05). ⑤ The combined loss of p16INK4a and p14ARF mRNAs was examined in 1 (5.6%) of 18 patients of well and moderately-differentiated carcinomas, and 11 (36.7%) of 30 patients of poorly and not-differentiated carcinomas with a significant difference (P<0.05). Conclusion p16INK4a and p14ARF genes are frequently inactivated by homozygous deletion and methylation of the 5’CpG islands in gastric cancer, which may play an important role in the carcinogenesis of gastric cancer.     

宣威地区非小细胞肺癌患者线粒体DNA突变与多态性研究

【目的】寻找宣威地区肺癌患者肺组织中线粒体DNA突变和多态性情况,为进一步研究宣威地区女性肺癌高发机制提供参考。【方法】征集28例宣威籍肺癌患者,收集癌组织和相应癌旁正常肺组织,提取线粒体DNA ,实时荧光定量PCR和直接测序方法检测线粒体DNA突变和多态性改变。【结果】28例肺癌组织样本同对应癌旁组织比较,有21例（75．0％）发生mtDNA突变,15例有多种突变,突变发生在DLOOP 区,以及呼吸链编码区等区域。突变与患者年龄、性别,及组织学类型无明显联系。28例肺癌组织、相应癌旁组织同M t-DNA剑桥序列比较,有3例有mtDNA多态性改变。【结论】宣威肺癌患者线粒体DNA存在特异性的突变位点,以及多态性位点,可能和当地特殊的燃煤污染暴露及遗传易感性有关。     

Identification of Two Novel Mitochondrial DNA Deletions Induced by Ionizing Radiation

ObjectiveWe identify ionizing radiation-induced mitochondrial DNA (mtDNA) deletions in human lymphocytes and their distribution in normal populations.MethodsLong-range polymerase chain reactions (PCR) using two pairs of primers specific for the human mitochondrial genome were used to analyze the lymphoblastoid cell line following exposure to 10 Gy ⁶⁰Co γ-rays. Limited-condition PCR, cloning and sequencing techniques were applied to verify the mtDNA deletions detected with long-range PCR. Human peripheral blood samples were irradiated with 0, 2 and 6 Gy ⁶⁰Co γ-rays, and real-time PCR analysis was performed to validate the mtDNA deletions. In order to know the distribution of mtDNA deletions in normal population, 222 healthy Chinese adults were also investigated.ResultsTwo mtDNA deletions, a 7455-bp deletion (nt475-nt7929 in heavy strand) and a 9225-bp deletion (nt7714 -nt369 in heavy strand), occurring between two 8-bp direct repeats, were identified in lymphoblastoid cells using long-range PCR, limited-condition PCR and sequencing. These results were also observed for ⁶⁰Co γ-rays irradiated human peripheral blood cells.ConclusionTwo novel mtDNA deletions, a 7455-bp deletion and a 9225-bp deletion, were induced by ionizing radiation. The rate of the mtDNA deletions within a normal population was related to the donors' age, but was independent of gender.     

胃癌线粒体DNA D-loop区碱基突变的检测分析

目的 研究胃癌患者的癌组织线粒体DNA （mtDNA）D-loop区碱基突变情况,以探讨mtDNA D-loop区碱基突变与胃癌发生发展的相关性.方法 采用聚合酶链反应和DNA测序相结合的方法,对75例胃癌患者癌组织线粒体D-loop 区进行扩增并测序分析.结果 在75例胃癌组织中共有130个位点发生了变化,其中属于基因多态性有101个,基因突变有29个.75例胃癌组织中39例mtDNA D-loop区存在突变,突变率为52%.结论 mtDNA D-loop 区碱基突变可能在胃癌发生发展中发挥重要作用.     

Cochlear mitochondrial DNA3867bp deletion in aged mice

目的　探讨老龄小鼠耳蜗mtDNA缺失情况 ,明确老龄小鼠耳蜗mtDNA缺失的位置。方法　应用套式PCR和DNA测序技术对不同月龄 12 9/CD1杂交小鼠耳蜗 31个 (2月龄 7个 ,7- 10月龄 16个 ,17- 19月龄 8个 )进行定性及定量检测。结果　 1 小鼠耳蜗mtDNA386 7bp大片缺失 ,缺失发生在nt910 3-nt12 970之间 ,其两侧nt90 89-nt910 3和nt12 95 6-nt12 970为核苷酸完全相同的 15bp重复序列。 2 随着小鼠月龄的增加 ,耳蜗mtDNA386 7bp缺失发生率有明显增加的趋势 ,2月龄小鼠未发现缺失 (0 /7) ;17- 19月龄小鼠 (7/8)明显高于 7- 10月龄小鼠 (4/16 ) (P <0 0 1)。 3 缺失mtDNA/总mtDNA比值 ,17- 19月龄小鼠明显高于 7- 10月龄小鼠 (P <0 0 1)。结论　老龄小鼠耳蜗出现mtDNA386 7bp大片缺失可能与老年性聋发生相关     

胃粘膜线粒体DNA核内整合与IL-8活性的关系

目的 评价胃粘膜线粒体DNA核内整合与组织IL—8活性的关系。方法 采用原位杂交方法检测线粒体DNA核内整合；组织IL—8含量到定采用ELISA方法。结果 胃癌细胞核内mtDNA序列的检出率为20％(6／30)、异性增生为10％(1／10)、肠上皮化生粘膜为6．7％(1／15)、萎缩性胃炎为10％(1／10)，浅表性胃炎未发现有mtDNA序列整合。胃粘膜细胞IL—8活性在mtDNA序列的检出组显著高于mtDNA序列非检出组(P＜0．05)。结论 胃粘膜细胞mtDNA序列整合可能参与了胃癌的发生；胃粘膜细胞mtDNA序列核内整合可能与IL—8活性有关。