【作者】 刘丽；

【导师】 陈琪； 范乐明；

【作者基本信息】 南京医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 目的本研究拟从CDH1基因序列再测序、miRNA表达及微卫星片段分析三个角度研究探讨胃癌的相关性分子标记物,并探讨研究方法的可行性,为进一步深入研究胃癌的机制提供参考。方法CDH1基因序列再测定的研究抽提汉族胃癌肿瘤组织和对应正常胃粘膜组织的基因组DNA,抽取正常健康人外周血基因组DNA。采用Applied Biosystems公司的CDH1基因再测序引物,分别进行PCR。琼脂糖凝胶电泳检测PCR产物。PCR产物纯化后再进行测序反应。NaAC/EDTA法进行测序反应产物纯化。甲酰胺变性,上测序仪进行毛细管电泳。电脑软件自动采集信号、分析原始并读出序列图谱。采用SeqScape v 2.1.1软件进行序列比对,寻找基因变异位点。miR-21、miR-145及miR-155的表达提取石蜡包埋的肿瘤组织和正常胃组织的总RNA,采用美国Applied Biosystems公司设计的特异茎环状引物进行逆转录反应,以U6为内参照基因,实时定量PCR技术检测miR-21、miR-145和miR-155的表达。根据实时获得的反应初始浓度的Ct值,分别计算各样本的ΔCt值,再以2^-ΔCt值进行t检验分析。微卫星不稳定研究提取冻存的胃癌组织和正常对照组织的基因组DNA。采用多重荧光PCR方法,在同一管内进行D2S123、D17S250、D5S346、Bat-25和Bat-26五个引物对的PCR反应。PCR产物变性后,在ABI3100测序分析仪上进行毛细管电泳。采用GeneMapper 3.0软件进行比对、判读微卫星不稳定。BAT25的调查性测序研究提取健康人群的基因组DNA进行BAT25的测序分析,调查BAT25在汉族人群中的真实数据情况,为病理诊断MSI提供参考依据。结果CDH1基因序列再测定的研究1)在散发性胃癌中发现11个CDH1基因变异位点,其中-49位点G＞T突变为新发现变异。-1030位点的A基因型和-284位点的A基因型与散发性胃癌的低分化显著相关。2)在家族性胃癌成员中除发现散发性胃癌具有的变异外,另外发现8处新突变,均未见报道。这8处位点都位于邻近编码区的内含子处。miR-21、miR-145及miR-155的表达1)miR-21和miR-145在胃癌和正常胃黏膜组织中的表达丰度高于miR-155在正常胃组织和肿瘤组织中的表达丰度。2)胃癌组织的miR-21表达显著高于正常组织的miR-21表达。3)胃癌组织的miR-155表达显著高于正常组织的miR-155表达。4)miR-145在肿瘤组织中的表达与正常组织无显著性差异,但总体来看,其在肿瘤中的表达低于正常组织。微卫星不稳定研究1)MSI胃癌在检测的散发性胃癌中阳性率达到35.1%,与文献报道的阳性率类似。五个位点中,BAT25在MSI中的阳性率最高,为85.0%;其次为BAT26,为45.0%;发生频率最低的位点是D17S250（5.0%）。2)MSI胃癌与MSS胃癌在性别、肿瘤分化程度、组织学分类、淋巴结转移、临床分期及TNM分期、Hp感染等方面无统计学差异。但MSI与MSS胃癌在年龄方面表现一定的差异。另外,MSI胃癌与hMSH2的低蛋白表达相关。BAT25的调查性测序研究1)BAT25在健康汉族人群中表现为单碱基准单态性。2)BAT25在作为微卫星位点判断肿瘤的微卫星不稳定时应同时设立正常对照进行实验。结论癌症基因组再测序技术能够发现并筛查出更多与癌症疾病相关的基因突变。我们应用基因序列再测序技术对CDH1基因在散发性胃癌和家族性胃癌成员中的变异进行筛查,共检测出19处CDH1基因变异,其中发现9个新的突变位点。-1030位点的A基因型和-284位点的A基因型趋向于胃癌的低分化。家族性胃癌成员比散发性胃癌具有更多的突变位点。CDH1基因多处变异的生物学意义有待进一步研究。miRNAs与肿瘤的发生、浸润和转移相关。在肿瘤研究中,miRNAs的研究应首先着眼于在肿瘤和正常组织中表达有差异的miRNAs。本研究采用石蜡包埋组织进行miRNAs表达研究,证明miRNAs在胃癌发生中起着重要的作用,发现胃癌与miR-21和miR-155的过表达相关,部分胃癌与miR-145的低表达相关。福尔马林固定、石蜡包埋的标本可以用来较好地进行大规模回顾性研究中miRNAs的表达研究。多重荧光PCR方法可以很好地用来检测胃癌的微卫星不稳定状态,较传统的聚丙烯酰胺凝胶电泳更方便、精确。汉族散发性胃癌的MSI与性别、肿瘤分化程度、组织学分类、淋巴结转移、临床分期及TNM分期、Hp感染等方面无显著性相关。胃癌MSI的发生与错配修复基因hMSH2的低表达相关。本研究中BAT25发生MSI的阳性率最高。本研究首次用测序方法证实中国健康人群BAT25具有单碱基准单态性,并且建议在利用BAT25进行MSI分析时,应同时设立正常对照。更多还原

【Abstract】 ObjectiveThe aims of this study were to study associated molecular markers of gastric cancer by CDH1 gene resequencing,miRNAs expression and MSI analysis.1.Resequencing anlysis of CDH1 geneDNAs were extracted from patients’ tissues,corresonding normal mucosa,and peripheral blood leucocytes of healthy donors.PCR was peoformed with 27 resequencing primer pairs of CDH1 gene synthesized by Applied Biosystems,respectively.The PCR products were detected by agarose electrophoresis.The purified PCR products were subjected to sequencing PCR reactions,in which purify of the products was made with a NaAC/EDTA method.After denaturated with Hi-Di formamide,the PCR products were sequenced via capillary electrophoresis on an ABI 3100-Avant Genetic Analyzer.The data of sequence were blasted with CDH1 gene reference sequence with SeqScape v 2.1.1 software（Applied Biosystems）to look for variant sites.We investigated 11 gene variant sites in CDH1 gene from patients with sporadic gastric cancer.Among these variants,intronl-49G＞T is a novel mutation site.Statistical analysis showed that there was a statistically significant correlation between intronl-1030 site A,-284 site A allele and tumor differentiation.We also found 8 novel germline mutations in 3 samples with familial gastric cancer history.All these new mutation sites locate in the intron adjacent to coding regions.2.Expression of miR-21,miR-145 and miR-155Total RNA was extracted from paraffin-embedded gastric cancer tissues and corresponding normal mucosa.Reverse transcription reactions were done with special looped primer from Applied Biosystems.The expression of miR-21、miR-145 and miR-155 were then detected by real time PCR with a reference gene U6.We calculatedΔCt values of each sample and analized on 2^-ΔCtby t-test statistical method.The results showed that the expression of miR-21 and miR-155 in gastric cancer tissues was higher significantly than that in adjacent normal tissues,respectively.The expression of miR-145 in most of cancer tissues was lower,but not statistically,than its expression in adjacent normal tissues.In addition,we found that the expression of miR-21 and miR-145 were higher than the expression of miR-155 in cancer tissues and normal tissues.The expression of miR-155 in gastric tissues is relatively low.3.Study on microsatellite instabilityTotal RNA was extracted from frozen gastric cancer tissues and the corresponding normal mucosa.Multiple fluorescent PCR reactions were done with 5 primer pairs including D2S123、D17S250、D5S346、Bat-25 and Bat-26 in one reaction tube.The PCR products were denatured and detected via capillary electrophoresis on an ABI 3100-Avant Genetic Analyzer.MSI was blasted and judged using GeneMapper 3.0 software.The rate of MSI in tested sporadic gastric cancer was 35.1%which was similar to previous report.Among these 5 loci,BAT25 was most sensitive （85.0%）,next was BAT26（45.0%）,and D17S250 was most unsensitive （5.0%）.MSI gastric cancer and MSS gastric cancer showed a different distribution in the different ages of population.Patients with MSI gastric cancer were significant higher in the group over 65 years as compared with those less 65 years.MSI gastric cancer was correlated significantly with lower hMSH2 expression.No significant difference between MSI gastric cancer and MSS gastric cancer was found to relate to sex, differetation,classification,lymphoid node metastasis,clinical staging, TNM staging,and Hp infection. 4.Investigation of BAT25 sequence in healthy populationWe extracted 431 DNAs from healthy people and detected BAT25 polymorphism by direct sequencing technique.We found that BAT-25 of Chinese people was quasi-mononucleotide in the size of the poly（T）tracts. We suggested that when BAT25 was used as a microsatellite analysis site, tumor samples should be done simultaneously with paired normal samples for the sake of correct microsatellite instability diagnoses.ConclusionResequencing whole gene technique could screen more mutations in cancer correlated gene.In patients with sporadic gastric cancer and people with familiar gastric cancer history,we detected 19 allele variations of CDH1 gene contained 9 novel mutations in all.-1030A allele and -284A allele were correlated to low differentiation of sporadic gastric cancer. CDH1 gene mutation rate in the subjects with familiar gastric cancer were higher than in the patients with sporadic cancer.More variations in CDH1 are still needed to be studied.Our study showed that the up-regulation of miR-21 and miR-155 were associated with the gastic carcinogenesis,whereas the down-regulation of miR-145 may partly play some roles.Formalin-fixed paraffin-embedded specimens could act as satisfactory resources for miRNA study.Multiple fluorescent PCR with 5 MS loci method was useful in detecting MSI in gastric cancer.Low expression of hMSH2 was correlated with MSI in gastric cancer.BAT25 was the most sensitive locus in MSI analysis in our study,and we confirmed that Bat25 of Chinese people was quasimonomorphic.更多还原