【作者】 高余佳；

【导师】 辛彦；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2008， 博士

【摘要】 胃癌是世界上最常见的恶性肿瘤之一。与其他恶性肿瘤一样,胃癌的发生是多基因、多阶段变异累积形成的病理过程。这些基因主要是癌基因、抑癌基因及DNA错配修复基因等。抑癌基因功能的丢失可以通过多种途径,除基因突变和杂合性丢失外还与其启动子发生甲基化修饰有关。DNA甲基化是指在DNA甲基转移酶(DNA methyltransferase,DNMT)催化下,以S-腺苷蛋氨酸(S-ademetionine,SAM)为甲基供体,将甲基转移到DNA特定碱基上去的过程。真核生物染色体DNA甲基化是基因表达调控的一种方式。DNA异常甲基化可引起染色体结构、DNA稳定性的改变和基因表达异常,影响细胞的增殖和分化。真核生物DNA甲基化水平与肿瘤关系密切。启动子区高甲基化能导致抑癌基因表达沉默,这是肿瘤发生的关键事件。新近研究证实,CHFR是重要的肿瘤抑制基因,其表达产物是Plk1的泛素化连接酶。Plk1调控cdc25和Weel激酶的磷酸化,在G2期进入M期时控制cdc2激酶的活性。而CHFR能引起Plk1泛素化和降解,阻断细胞进入有丝分裂中前期。CHFR基因在人正常组织中普遍表达,在一些肿瘤中表现为失活或低表达,因而不能阻断异常细胞通过G2期进入有丝分裂中前期,从而导致细胞的恶性增殖。另有研究证实,Runx3基因(runt-relatedtranscription factor 3 gene)编码的runx3蛋白是TGF-β信号通路下游的一个转录调节因子,TGF-β是多种细胞生长的有效抑制因子,TGF-β信号转导通路的紊乱可导致多种肿瘤的发生。本研究采用MSP、RT-PCR、Western blotting及免疫组化(IHC)技术,检测人胃癌组织及其配对正常胃黏膜CHFR及Runx3基因的甲基化状态、mRNA和蛋白的表达水平,观察分析CHFR及Runx3基因异常甲基化与其mRNA表达水平和蛋白表达的关系,探讨胃癌组织中CHFR及Runx3基因启动子甲基化和mRNA表达异常与胃癌病理生物学特征的关系,为阐释抑癌基因CHFR及Runx3在胃癌发生发展中的作用及其分子机理提供新的科学实验依据。实验材料和方法1、研究对象本实验研究对象收集2003-12至2004-05中国医科大学附属第一医院肿瘤科及辽宁省肿瘤医院外科手术切除胃癌常规病理组织标本151例。另外,收集2007年3月至9月辽宁省肿瘤医院肿瘤外科手术切除之新鲜胃癌组织及其配对远端的正常胃粘膜组织(距胃癌灶边缘＞5cm处)标本共36例,迅速置于液氮内冷冻,然后储存于-70℃冰箱中保存待用。所有病例均经病理检查确定诊断,所有胃癌患者术前均未经化疗和放疗。选择胃癌和其配对正常胃粘膜组织资料完整的20个病例用于实验。2、实验方法(1)胃癌组织芯片制作采用组织芯片制作仪(Microarrayer,Beecher Instruments,USA)制作组织芯片。构建完成胃癌及其癌前病变的组织微阵列石蜡块1、2、3、4和5(蜡块1:含117个组织芯;蜡块2:含109个组织芯;蜡块3:含110个组织芯;蜡块4:含101个组织芯;蜡块5:含124个组织芯)。将组织芯片蜡块行连续切片,切片厚度为4μm,用于免疫组织化学染色的切片裱于经0.1%多聚赖氨酸防脱片处理的载玻片上,60℃烤片1h,58℃中继续烤片18h,常温保存备用。(2)免疫组化染色检测CHFR和mP53蛋白表达采用Envision方法对胃癌及其癌前病变组织芯片进行免疫组化染色。Immuno-Bridge+试剂盒,鼠抗人CHFR单克隆抗体(工作浓度1:75)和鼠抗人P53单克隆抗体(即用型)分别购自Abnova公司和北京中杉金桥生物技术有限公司。所有步骤依据产品说明书进行,利用PBS(0.01mol/L,PH7.4)替代一抗作为阴性对照。免疫组化染色结果判定:CHFR和mP53蛋白免疫染色阳性信号分别定位于细胞浆和胞核,呈棕黄色颗粒,每个标本观察2个有代表性的高倍视野,每个视野计数100个细胞确定阳性细胞数所占的百分比并取它们的平均值,阳性细胞数≤20%为阴性(-),阳性细胞数＞20%为阳性(+)。(3)RT-PCR法检测CHFR和Runx3基因mRNA表达采用TRIZOL试剂提取标本总RNA,将总RNA反转录成cDNA,然后以CHFR和Runx3基因引物进行PCR扩增,以β-actin PCR为内参照。(4)MSP检测CHFR和Runx3基因甲基化状态采用TIANamp Genomic血液/细胞/组织基因组DNA提取试剂盒提取组织DNA。先用亚硫酸氢钠修饰基因组DNA,纯化DNA后,使用CHFR和Runx3基因甲基化及非甲基化引物进行扩增。(5)Western Blot检测CHFR和Runx3基因编码蛋白表达组织以1:5比例加入裂解缓冲液提取组织蛋白;样品(蛋白)浓度的定量采用考马斯亮蓝法;蛋白质进行12%浓度的SDS聚丙烯酰胺凝胶电泳;硝酸纤维素滤膜,转膜2h;与一抗(CHFR工作浓度1:400,Runx3工作浓度1:300)室温下孵育2h;辣根过氧化物酶标记的二抗(工作浓度1:1000)室温孵育1h;免疫印迹化学发光试剂(ECL Reagent)显示免疫反应条带;采用GEL-XR凝胶成像系统将将电泳结果成像;BANDSCAN5.0软件系统采集数据,采用目的蛋白条带光密度值与相应β-tubulin光密度值的比值作为相对表达水平指标,进行统计学分析。3、统计学分析数据用(?)±s表示,采用SPSS13.0统计软件包进行分析,组内资料以构成比描述,组间计数资料采用x~2检验(Fisher’精确概率法)和计量资料t检验,Spearman等级相关检查。P＜0.05为差异有统计学意义。P＜0.01为差异有显著统计学意义。结果1、胃癌组织中CHFR和mP53基因编码蛋白的表达CHFR蛋白在非癌胃黏膜组织中阳性表达率为85.25%(52/61),在胃癌组织中阳性表达率显著降低(49.67%,75/151,P＜0.05);CHFR表达下调或缺失与胃癌患者的性别显著相关,女性患者胃癌组织中CHFR表达缺失率显著高于男性患者(64.00%vs43.56%,P＜0.05)。BorrmanⅢ+Ⅳ型胃癌组织中CHFR表达缺失率显著高于BorrmanⅠ+Ⅱ型胃癌(57.14%vs34.78%,P＜0.05)。虽然不同组织学类型胃癌之间CHFR的表达无统计学差异,但本研究发现胃印戒细胞癌组CHFR表达缺失率最高(71.43%,5/7)。胃癌组织中CHFR蛋白的表达缺失与肿瘤浸润深度、淋巴结转移以及mP53蛋白表达未见显著相关性(P＞0.05)。2、胃癌组织中CHFR和Runx3基因的异常表达CHFR、Runx3基因mRNA在胃癌组织中均较其配对正常胃粘膜中的目的基因表达下调/缺失,CHFR mRNA表达量相对值在胃癌组织和其配对正常胃粘膜中分别是(0.2186±0.2113)和(0.7020±0.2163),两者差异有统计学意义(t=7.148,P＜0.001);20例胃癌组织中,低分化组CHFR mRNA表达量相对值(0.1364±1.772)显著低于高分化组(0.4106±0.1574),两组间差异有统计学意义(t=3.276,P＜0.01)。Runx3 mRNA表达量相对值在胃癌组织和其配对正常胃粘膜中分别是(0.1755±0.1877)和(0.6195±0.2225),两者差异有统计学意义(t=6.822,P＜0.001);其中低分化组中Runx3 mRNA表达量相对值(0.1114±0.1863)显著低于高分化组表达量(0.3250±0.0725),两组间的差异具有统计学意义(t=2.514,P＜0.05);浸润生长穿出肌层胃癌组Runx3 mRNA表达量相对值(0.1113±0.1329)显著低于未穿出肌层组(0.3660±0.2084),t=3.224,P＜0.05:伴有淋巴结转移胃癌组Runx3 mRNA表达量(0.0750±0.1054)明显低于无淋巴结转移组(0.3263±0.1867),t=3.850,P＜0.01。胃癌组织中CHFR与Runx3基因mRNA表达未见显著相关性(P＞0.05)。3、胃癌组织中CHFR和Runx3基因编码蛋白的表达BANDSCAN5.0软件系统采集数据,采用目的蛋白条带光密度值与相应β-tubulin光密度值的比值作为相对表达水平指标,进行统计学分析。胃癌组织中CHFR基因编码的蛋白表达下调/缺失率(70.00%,14/20),胃癌组织及其配对正常胃粘膜相对光密度值分别为(0.2435±0.2620)和(0.5955±0.2196),t=4.605,P＜0.001。其中,低分化组显著低于高分化组,两组相对光密度值分别为(0.1143±0.122vs0.5447±0.2573)两组间差异具有统计学意义(t=5.162,P＜0.01)。胃癌组织中Runx3基因编码的蛋白表达下调/缺失率(65.00%,13/20)显著低于其配对正常胃粘膜(5.00%,1/20),胃癌组织及其配对正常胃粘膜相对光密度值分别为(0.2700±0.3877)和(0.5860±0.2784)t=2.961,P＜0.05。高分化组Runx3基因编码蛋白表达相对光密度值(0.6930±0.3449)显著高于低分化组(0.0893±0.238)(t=4.546,P＜0.01);肿瘤侵袭深度:穿出肌层组Runx3基因编码蛋白表达相对光密度值(0.1485±0.3231)显著低于未穿出肌层组(0.6360±0.3576)(t=2.851,P＜0.05);伴淋巴结转移组Runx3基因编码蛋白表达相对光密度值(0.0683±0.2107)显著低于无淋巴结转移组(0.5848±0.3887)(t=3.916,P＜0.01)。4、胃癌组织中CHFR和Runx3基因的异常甲基化状态胃癌组织中CHFR基因甲基化率为45.00%(9/20),而正常胃粘膜未检出甲基化,两组间差异具有统计学意义(P＜0.001);低分化胃癌组CHFR基因甲基化率显著高于高分化胃癌组(P＜0.05)。Runx3基因胃癌组织及其配对正常胃粘膜中甲基化率分别为65.00%(13/20)和5.00%(1/20),胃癌组织中的甲基化率显著高于其配对正常胃粘膜,两组间差异具有统计学意义(P＜0.001);Runx3基因异常甲基化与胃癌组织分化程度、肿瘤侵袭深度及淋巴结转移密切相关(P＜0.05)。5、胃癌组织CHFR和Runx3基因甲基化与其mRNA和蛋白表达的相关性CHFR基因甲基化的9例胃癌组织中其mRNA表达均下调或缺失;而11例非甲基化者中仅有5例mRNA表达下调或缺失。相关分析:r=0.592,P＜0.05。13例Runx3基因甲基化的胃癌组织中12例其mRNA表达下调或缺失,而7例非甲基化者中仅有1例mRNA表达下调。相关分析:r=0.780,P＜0.001。CHFR基因甲基化的9例胃癌组织均表现为相应编码蛋白表达下调或缺失,而非甲基化的11例胃癌组织中仅有5例其编码蛋白表达下调或缺失(相关性分析:r=0.592,P＜0.05)。13例Runx3基因异常甲基化的胃癌组织中其编码蛋白表达均下调或缺失,而7例Runx3基因非甲基化的胃癌组织中均未见其编码蛋白表达下调或缺失(相关性分析:r=1)。本研究结果提示,CHFR和Runx3基因的异常甲基化可能是其mRNA和编码蛋白在胃癌组织中表达下调或缺失的主要原因。结论1、151例胃癌组织中CHFR基因编码蛋白表达结果显示,有丝分裂前期检查点CHFR基因表达下调或缺失在胃癌中是频发事件。可能参与胃癌的发生,其与女性、弥漫浸润型胃癌发生发展的关系可能更为密切。2、CHFR和Runx3基因在胃癌组织中的甲基化率均显著高于其配对正常胃粘膜,提示CHFR和Runx3基因异常甲基化可能参与胃癌的发生发展;胃癌组织中CHFR和Runx3基因启动子甲基化是其mRNA及编码蛋白表达下调或缺失的主要原因。对二者的检测可有助于胃癌的早期诊断。3、胃癌组织中CHFR与Runx3 mRNA异常表达无密切相关关系,但均与组织学分化程度有关,二者可能通过不同作用机制参与胃癌的组织学分化。Runx3mRNA表达下调或缺失与胃癌的侵袭深度及淋巴结转移密切相关,对客观判断胃癌患者预后,指导临床治疗具有重要意义。更多还原

【Abstract】 ObjectivesGastric carcinoma is one of the most common malignancies in the world.As the other malignant tumors,gastric carcinogenisis is a multigene and multistep involved pathological course.The correlated genes are mostly oncogenes,tumor suppressor genes,and DNA mismatch repair genes.Tumor suppressor genes may loss their functions by the way of gene mutation,loss of heterozygosity and methylation of promoters.DNA methylation is the methyl transfer,which is supplied by Sademetionine (SAM),to the specific bases under the catalysis of DNA methyltransferase(DNMT).DNA methylation of eukaryote is a way of regulating gene expression.DNA aberrant methylation can change the chromosomal structure,DNA stability,cause gene expression abnormalities,and affect cellular proliferation and differentiation.The level DNA mehtylation of eukaryote is closely correlated with tumor.Hyermethylation in promoter region causes silencing of suppressor genes,which is the key factor in carcinogenesis.Recently,some studies have shown that CHFR is an important tumor suppressor gene and its encoding product is ubiquitin ligase of Plk1. Plk1 regulates both the Weel kinase and the Cdc25C phosphatase,which in turn control the Cdc2 kinase activity at the G2 to M transition.The CHFR gene can ubiquitinate and degenerate the Plk1,which prevents cells from entering prophase and metaphase.CHFR gene expresses in normal tissue while it is low expressed or inactivated in cancer tissue, in which losses the ability to prevent abnormal cells proliferating from G2 to M phase, thus cells abnormal differentiation and proliferation occur.Some studies also show that runx3 protein coded by Runx3 gene(runt-related transcription factor 3 gene) is a regulator in the downstream of TGF-βsignal pathway.TGF-βis the effective suppressor of many cells,and the abnormality of TGF-βsignal pathway can cause carcinogenesis of many cancers.In this study we employed MSP,RT-PCR,Western blotting and immunohistochemical methods to detect the methylaion status of CHFR gene and Runx3 gene and the expression of mRNA and protein in gastric cancer tissue and normal gastric mucosa.We analyzed aberrant methylaion of the CHFR and Runx3 gene and their possible correlations to the mRNA and protein expression,further discussed the correlation between promoter methylaion of CHFR gene and Runx3 gene and mRNA abnormal expression,as well as pathologic characteristics in human gastric cancer.The studies may offer experimental basis to elucidate the effect of CHFR gene and Run3 gene in occurrence and development of human gastric cancer.Samples and Methods1.SamplesSamples:One hundred and fifty-one samples of gastric cancer tissues were obtained from Department of Surgical Oncology of the First Affiliated Hospital of China Medical University and Liaoning Cancer Hospital during December 2003 to May 2004.Besides,fresh gastric cancer tissues of thirty-six cases were taken and paired normal gastric mucosa taken at least 5 cm away from the cancer margin were collected simultaneously from Department of Surgical Oncology of Liaoning Cancer Hospital during March to September 2007.These samples were placed in liquid nitrogen immediately and then stored at -70℃refrigerator until analysis.All the samples were diagnosed pathologically,and all patients didn’t receive either chemical treatment or radiotherapy before operations.The gastric cancer tissues and paired normal gastric mucosas of twenty cases with integrated data were used for experiment.2.Methods (1) Constructing gastric cancer tissue chipTissue chips were made by tissue chip maker(Miroarrayer,Beecher Instruments, USA).Five TMA blocks numbered 1 to 5 containing gastric cancer and precancerous lesions were constructed.Block 1 contains 117 tissue samples;Block 2 contains 109 tissue samples;Block 3 contains 110 tissue samples;Block 4:contains 101 tissue samples;Block 5 contains 124 tissue samples.The TMA blocks were cut continuously and all sections were within 4μm.Sections for immunohistochemical stain were fixed on the slides processed by 0.1%polylysine to prevent slipping.Sections were baked in 60℃for 1 hour,and then continued baking in 58℃for 18 hours.After baking,the sections were kept in room temperature for further use.(2) Immunohistochemical staining for CHFR and mP53 protein expressionTissue chips of gastric cancer and precancerous lesions were immunohistochemical stained by Envision method.Immuno-Bridge kits,rat anti- human CHFR monoclone antibody(working dilution 1:75) and rat anti-human P53 monoclone antibody(ready to use) were bought from Abnova Company and Beijing Zhongshan Jinqiao biothchnique limited company,respectively.All steps were accomplished in accordance with the instructions.PBS(0.01mol/L,pH7.4 used instead of specific antibodies for negative control.Gradingof immunohistochemical stain:the positive staining signals of the CHFR and mP53 protein were yellow brown granules and located in cytoplasm and nucleus respectively.For each sample,two representative high power fields were observed.Positive rate was assessed by the percent of positive cells in all counted 100 cells,which was taken their average value together from two representative high power fields.Positive cells≤20%of total counted cells were determined negative while positive cells＞20%of total counted cells were determined positive.(3) RT-PCR of CHFR and Runx3 genesTotal RNA extracted by using TRIZOL reagent was used for cDNA synthesis.RTPCR was performed with primers of CHFR,Runx3 gene orβ-actin.(4) Methylation -specific PCR for methylation level TIANamp Genomic blood/cell/tissue genomic DNA kit was used to extract DNA. Genomic DNA was modified by sodium bisulfite.After DNA purification,PCR was performed by using methylated and unmethyiated product of CHFR and Runx3 gene as primers.(5) Western blotting for protein encoded by CHFR and Runx3 genesLysis buffer solution was added into tissue at the proportion of 5:1 to extract protein.Protein density was determined by Coomassie brilliant blue.And then12% SDS polyacrylamide gel electrophoresis was performed.Nitrocellulose membrane sheet transferred for 2 hours,and incubated with primary antibody(CHFR1:400,Runx3 1:300) for 2 hours,further incubated with horseradish peroxidase signed secondary antibodies in room temperature for 2 hours.Immunological straps were finally shown with immunoblotting chemoluminescene reagent(ECL reagent).The electrophoretic results were scanned into images.Data were collected by BANDSCAN5.0 software and the ratio of strip density toβ-tubulin density served as index for statistical analysis.3.Statistical analysisThe data was processed using SPSS 13.0 statistical software.Quantitative data were expressed with mean±standard deviation(SD).Data were analyzed by Fisher’s exact x~2 test,independent-samples t-test and Spearman rank related test.P values less than 0.05 were considered to be statistically significant,and P values less than 0.01 were remarkably significant.Results1.The expressin of CHFR and mP53 gene coded protein in human gastric cancerThe positive rate of CHFR protein in gastric cancer tissues(49.67%,75/151) was significantly lower than in normal gastric mucosa(85.25%,52/61)(P＜0.05).The down-regulation or loss of mitotic CHRF protein expression in gastric cancer was closely correlated with gender of gastric cancer patients.The negative rate of CHFR protein expression in the female gastric cancer patients was significantly higher than that in the male gastric cancer ones(64.00%vs 43.56%,P＜0.05).The negative rates of CHRF protein expression were also significantly different between gastric cancer patients of BorrmanⅢ+Ⅳand BorrmanⅠ+Ⅱtypes(57.14%vs 34.78%,P＜0.05).In the present study,although CHFR protein expression showed no significant differences among various histological types of gastric cancer,the negative rate of CHRF protein expression was the highest in signet ring cell carcinoma(71.43%,5/7).The negative rate of CHFR protein expression was not correlated with the depth of invasion and lymph node metastasisof gastric cancer.In addition,no correlation was found between the expressionof CHFR protein and mP53 protein expression in gastric cancer(P＞0.05).2.Aberrant expressions of the CHFR and Runx3 gene in gastric cancer tissuesCHFR mRNA expression was down-regulated in gastric cancer tissue (0.2186±0.2113) compared with that in normal gastric mucosa(0.7020±0.2163) and the difference between them was significant(t=7.148,P＜0.001).Among them,CHFR mRNA expression in 14 cases of poorly differentiated gastric cancers tissues (0.1364±1.772) was significantly lower than that(0.4106±0.1574) in 6 cases of well-differentiated group(t=3.276,P＜0.01).Runx3 mRNA expression in gastric cancer (0.1755±0.1877) was significantly lower than that in normal gastric mucosa (0.6195±0.2225),t=6.822,P＜0.001.Among them,Runx3 mRNA expression in 14 cases of poorly differentiated gastric cancers(0.1114±0.1863) was significantly lower than that(0.3250±0.0725) in 6 cases of well-differentiated group(t=t=2.514,P＜0.05).In addition,Runx3 mRNA expression in gastric cancers which infiltrated throughout muscular layer was significantly lower than non-infiltrated group (0.1113±0.1329 vs 0.3660±0.2084,t=3.224,P＜0.05).Runx3 mRNA expression in gastric cancer tissues with lymph node metastasis was significantly lower than that without lymph node metastasis(0.0750±0.1054 vs 0.3263±0.1867,t=3.850,P＜0.01). However,the mRNA expression levels between the CHFR and Runx3 genes showed no significant correlation(P＞0.05).3.The CHFR and Runx3 protein expressions in gastric cancer tissuresData were collected by BANDSCAN5.0 software,the ratio of strip density toβ-tubulin density served as index for statistical analysis.The down-regulation or loss rate of CHFR gene coded protein was 70.00%(14/20).The difference in relative optical density value between gastric cancer and normal gastric mucosa was significant (0.2435±0.2620 vs0.5955±0.2196,t=4.605,P＜0.001).Among them,the level of CHFR protein expression in poorly differentiated group was significantly lower than that in well-differentiated group.Their relative optical density values were respective 0.1143±0.1224 vs 0.5447±0.2573,and the difference between them was significant (t=5.162,P＜0.01).The down-regulation or loss rate of Runx3 protein expression in gastric cancer was significantly higher than that in normal gastric mucosa(65.00%, 13/20 vs 5.00%,1/20).The difference in relative optical density value between gastric cancer and normal gastric mucosa was significant(0.2700±0.3877 vs 0.5860±0.2784, t=2.961 P＜0.05).The relative optical density value of Runx3 protein expression in well differentiated group(0.6930±0.3449) was significant higher than that in poorly differentiated group(0.0893±0.238),and the difference between them was significant(t =4.546,P＜0.01).The relative optical density value of Runx3 protein expression in gastric cancers which infiltrated through muscular layer was significantly lower than non-infiltrated group(0.1485±0.3 vs 0.6360±0.3576,t=2.851,P＜0.05);Runx3 protein expression in gastric cancer with lymph node metastasis was significantly lower than that without lymph node metastasis(0.0683±0.2107 vs 0.5848±0.3887,t=3.916, P＜0.01).4.The aberrant methylation of CHFR and Runx3 promoters in human gastric cancerThe methylation rate of CHFR gene in gastric cancer tissue was 45.00%(9/20).As a control,CHFR methylation was screened for the corresponding normal gastric mucosas of these same patients.No methylation was detected in these tissues.The difference between gastric cancer tissue and normal gastric mucosa was significant (P＜0.001).The CHFR gene methylation rate in poorly differentiated gastric cancer was significantly higher than that in well-differentiated group(P＜0.05).The Runx3 gene methylation rate(65.00%,13/20) in gastric cancer tissue was higher than that(5.00%, 1/20) in normal gastric mucosa(P＜0.001).The aberrant methylation of the Runx3 gene was significantly correlated with differentiation degree,depth of tumor invasion and lymph node metastasis(P＜0.05).5.The correlation of CHFR and Runx3 gene methylation with their mRNA and protein expressionCHFR mRNA expressive levels in 9 gastric cancer samples with CHFR methylation were all down-regulated or lost,while in 11 gastric cancer samples with unmethylated CHFR gene,the mRNA expressions were down-regulated only in 5 samples(r=0.592,P＜0.05).The mRNA expressions in 12 out of 13 gastric cancer samples with the aberrant methylation of Runx3 gene were down-regulated or lost, while in 7 gastric cancer samples with unmethylated Runx3 gene,the mRNA expressive level was down-regulated only in 1 sample(r=0.780,P＜0.001).CHFR protein expressions in 9 gastric cancer samples with aberrant methylation of CHFR gene were all down-regulated or lost,while in 11 gastric cancer samples with unmethylated CHFR gene,only 5 samples were down-regulated(r=0.592,P＜0.05). Runx3 gene coded proteins in 13 gastric cancer samples with aberrant methylation of the gene were all down-regulated or lost,while in 7 gastric cancer samples with unmethylated Runx3 gene,none was down-regulated(r=1).The results of this study suggested that aberrant methylation of the CHFR and Runx3 gene is the main cause of down-regulation or loss of their mRNA or coded protein expression.Conclusions1.The results of the CHRF protein expression in gastric cancer tissues of 151 cases show that the down-regulation or loss of the mitotic CHRF protein expression occurs frequently in gastric cancer.It may involve in the carcinogenesis of gastric cancer and closely correlate with gender or more invasive type of human gastric cancer.2.The methylation rates of CHFR and Runx3 genes in gastric cancer tissues were all significantly higher than those in their normal gastric mucosas,suggesting that they may involve in gastric carcinogenesis and development.In addition,the aberrant methylation of CHFR and Runx3 promoters in gastric cancer tissues is the main cause of down-regulation or loss of their mRNA or coded protein expression.Thus,the detection of CHFR and Runx3 genes will be helpful to early diagnosis of gastric cancer.3.The mRNA expression levels between the CHFR and Runx3 genes showed no significant correlation in gastric cancer tissues,but they were all correlated with differentiation degree of the gastric cancers.Both them may participate the histological differentiation of gastric cancer by various mechanism of action.Besides,down-regulation or loss of the Runx3 mRNA expression in gastric cancer tissures was significantly correlated with the depth of tumor invasion and lymph node metastasis, which can help predicting the patient’s prognosis and for better clinic treatment.更多还原