细胞自噬及其在肿瘤发生过程中的作用

细胞自噬性死亡是一种非凋亡性的程序性细胞死亡,其特征是在垂死细胞中利用自噬小体对细胞内容物进行降解。现对自噬的病理形态特征、生化改变、发生机制、分子调控、与凋亡的联系进行综述,以利于加深对细胞死亡方式的进一步认识,并介绍自噬在肿瘤发生过程中作用的研究进展。     

细胞自噬及其在肿瘤发生过程中的作用

细胞自噬性死亡是一种非凋亡性的程序性细胞死亡,其特征是在垂死细胞中利用自噬小体对细胞内容物进行降解。现对自噬的病理形态特征、生化改变、发生机制、分子调控、与凋亡的联系进行综述,以利于加深对细胞死亡方式的进一步认识,并介绍自噬在肿瘤发生过程中作用的研究进展。     

肉瘤样肝细胞癌中MET基因扩增与预后的相关性分析

背景与目的：肉瘤样肝细胞癌（sarcomatoid hepatocellular carcinoma,SHC）是一种罕见且高度恶性的肝脏肿瘤。MET基因异常具有肿瘤预后预测价值,以MET为靶点的抑制剂已成为晚期肿瘤治疗的重要方向,然而SHC组织中MET基因扩增状态尚不明确。探讨SHC中MET基因扩增与临床病理学因素的相关性及其预后预测价值。方法：收集2008年1月—2016年12月于复旦大学附属中山医院经病理学检查确诊的22例SHC患者资料。采用荧光原位杂交（fluorescence in situ hybridization,FISH）法检测上述患者的MET基因扩增情况,并结合临床病理学资料进行统计学分析。采用Kaplan-Meier模型分析总生存期（overall survival,OS）及无病生存期（disease-free survival,DFS）,采用log-rank检验比较生存曲线,采用多因素COX回归模型分析SHC中独立的预后因素。结果：22例SHC患者中,MET基因扩增5例（22.7%）。MET基因扩增存在异质性,主要位于分化差的梭形细胞区域（4例）。MET基因扩增的SHC患者中位OS明显短于MET阴性患者（6.8个月 vs 24.0个月,P=0.001）。SHC中具有完整肿瘤包膜的患者中位OS明显长于包膜不完整的患者（41.3个月 vs 8.5个月,P=0.001）。单灶肿瘤及中国肝癌分期（China Liver Cancer Staging,CNLC）Ⅰ期患者的中位DFS较多灶肿瘤及Ⅱ+Ⅲ+Ⅳ期患者明显延长（10.4个月 vs 3.8个月,12.4个月 vs 3.8个月,P=0.027和0.017）。MET基因扩增是SHC独立的预后因子。结论：22.7%（5/22）的SHC中存在MET基因扩增。MET基因扩增的SHC患者预后更差,是其预后的独立危险因素。该研究结果为该罕见肿瘤的治疗提供了新策略和临床依据。     

慢病毒介导shRNA特异性沉默livin基因促进SPC-A1细胞凋亡

目的：建立慢病毒介导的livin基因沉默系统,探讨其对肺癌细胞凋亡的影响。方法：Livin shRNA慢病毒感染肺腺癌细胞株SPCA1沉默livin基因表达。应用PI染色经荧光镜下观察SPCA1细胞凋亡形态,流式细胞术检测SPCA1细胞凋亡率及亚二倍体峰形成,Realtime PCR及Western blotting方法检测livin和caspase 3表达的改变。结果：livin基因在肺腺癌细胞株SPCA1中持续高表达。经慢病毒介导shRNA使livin基因表达沉默后,镜下可见肺腺癌细胞出现典型凋亡形态特征,流式细胞术检测出现亚二倍体峰,细胞凋亡率较空白对照及阴性病毒对照细胞明显增加（8.21％ vs 0.08％, 0.13％;P<0.05）,RTPCR及Western blotting 检测结果显示,caspase 3 mRNA表达无改变,但cleavedcaspase 3蛋白表达上调。结论：慢病毒载体介导的shRNA能抑制肺腺癌细胞株SPCA1中livin基因的表达,从而促进SPCA1细胞凋亡。     

Methylation and silencing of the Thrombospondin-1 promoter in human cancer.

Neovascularization is a common feature of many human cancers, but relatively few molecular defects have been demonstrated in genes regulating angiogenesis. Decreased expression of Thrombospondin-1 (THBS1), a P53 and Rb regulated angiogenesis inhibitor, has been observed in some human tumors, including glioblastoma multiforme (GBM). To study whether methylation-associated inactivation is involved in down-regulating THBS1 expression in cancer, we analysed the methylation status of THBS1 in several cell lines and primary tumors. Three cell lines (RKO, CEM and RAJI) were completely methylated at several CpG sites within the THBS1 5' CpG island, and had no detectable expression by RT-PCR. THBS1 expression was readily reactivated using the methylation-inhibitor 5-deoxy-azacytidine in all three lines. Furthermore, THBS1 methylation was present in 33% (14/42) of primary GBMs. Thus, de novo methylation may serve as a potential way to inactivate THBS1 expression in human neoplasms.     

Methylation of hMLH1 promoter correlates with the gene silencing with a region-specific manner in colorectal cancer

Microsatellite instability is present in over 80% of the hereditary non-polyposis colorectal carcinoma and about 15-20% of the sporadic cancer. Microsatellite instability is caused by the inactivation of the mismatch repair genes, such as primarily hMLH1, hMSH2. To study the mechanisms of the inactivation of mismatch repair genes in colorectal cancers, especially the region-specific methylation of hMLH1 promoter and its correlation with gene expression, we analysed microsatellite instability, expression and methylation of hMLH1 and loss of heterozygosity at hMLH1 locus in these samples. Microsatellite instability was present in 17 of 71 primary tumours of colorectal cancer, including 14 of 39 (36%) mucinous cancer and three of 32 (9%) non-mucinous cancer. Loss of hMLH1 and hMSH2 expression was detected in nine and three of 16 microsatellite instability tumours respectively. Methylation at CpG sites in a proximal region of hMLH1 promoter was detected in seven of nine tumours that showed no hMLH1 expression, while no methylation was present in normal mucosa and tumours which express hMLH1. However, methylation in the distal region was observed in all tissues including normal mucosa and hMLH1 expressing tumours. This observation indicates that methylation of hMLH1 promoter plays an important role in microsatellite instability with a region-specific manner in colorectal cancer. Loss of heterozygosity at hMLH1 locus was present in four of 17 cell lines and 16 of 54 tumours with normal hMLH1 status, while loss of heterozygosity was absent in all nine cell lines and nine tumours with abnormal hMLH1 status (mutation or loss of expression), showing loss of heterozygosity is not frequently involved in the inactivation of hMLH1 gene in sporadic colorectal cancer.     

胃癌ERCC1基因甲基化与蛋白表达的关系及其意义

  目的  检测胃癌组织、相应癌旁组织及正常胃组织中ERCC1蛋白表达情况,探讨ERCC1基因启动子CpG岛甲基化与蛋白表达的关系及其意义。  方法  采用甲基化特异性PCR技术,检测30例胃癌组织、相应癌旁组织、10例正常胃组织中ERCC1基因启动子CpG岛甲基化状态。采用免疫组织化学SP法检测胃癌组织、相应癌旁组织及正常胃组织中ERCC1蛋白的表达情况。  结果  胃癌组织中ERCC1基因启动子CpG岛完全甲基化率为46.7%（14/30）,部分甲基化率为30.0%（9/30）,总甲基化率为76.7%（23/30）,显著高于相应癌旁组织中该基因的甲基化率13.3%（4/30）,差异有统计学意义（P < 0.05）。10例正常胃组织中未检测到该基因的甲基化（0/10）。30例胃癌组织中ERCC1蛋白阴性表达率为70.0%（21/30）,显著高于相应癌旁组织中蛋白阴性表达率33.3%（10/30）,差异有统计学意义。10例正常胃组织中ERCC1蛋白均阳性表达。胃癌组织中发生ERCC1基因启动子CpG岛甲基化其ERCC1蛋白表达率明显低于胃癌组织中未发生ERCC1基因启动子CpG岛甲基化其ERCC1蛋白表达率。  结论  ERCC1基因启动子CpG岛甲基化与其蛋白表达呈负相关,ERCC1基因启动子CpG岛甲基化可能是导致其蛋白表达下调的主要原因之一。      

Methylation of the alpha-fetoprotein gene in productive and nonproductive rat hepatocellular carcinomas

The extent of methylation of Hpall-Mspl and Hhal sites in DNAs isolated from normal rat livers and from the transplantable hepatocellular carcinomas (THC) THC 7777 and THC 252 was compared. It was found that the overall level of methylation of the internal cytosine in CCGG sequences was lower in the THC DNAs than in the normal liver DNAs. This difference could also be detected in the extent of methylation of CCGG sites flanking a 400-base pair repetitive sequence. Examination of methylation of specific sites within the alpha-fetoprotein gene revealed differences between the DNAs that appear to reflect both the level of activity of the gene and the overall level of methylation of cellular DNA. This gene, which is repressed in normal adult liver and the nonproductive THC 252 and highly active in the productive THC 7777 (S. Sell et al., Cell Biol. Int. Rep., 4: 235-254, 1980), contains several CCGG sites that are methylated in both normal liver and THC 252 DNA but not in THC 7777 DNA. However, Hhal (GCGC) sites in the alpha-fetoprotein gene were less methylated in both hepatoma DNAs than in liver DNA, which the exception of one site in the productive tumor found to be no longer methylated.     

Expression of IGF-1R in colorectal polyps and its role in colorectal carcinogenesis

Insulin-like Growth Factor Receptor 1 (IGF-1R) may play a role in the neoplastic progression of colorectal cancer because it is related to both cellular proliferation and differentiation. The aim of this study was to further elucidate the role of IGF-1R in colorectal carcinogenesis by evaluating IGF-1R expression in different types of precancerous colorectal polyps and comparing its expression to normal mucosa and colorectal carcinoma. A total of 47 colorectal polyps and their respective adjacent normal mucosa were collected from 32 patients. In addition, 20 colorectal adenocarcinoma tissues were obtained from patients undergoing colorectal resection, and 12 normal non-malignant colorectal mucosal tissues collected from outpatients served as the control group. The pit patterns of polyps were classified by the Kudo classification scheme through magnifying chromoendoscopy. Immunohistochemistry and quantitative real-time RT-PCR were utilized for expression analysis of IGF-1R in colorectal mucosa, polyps, and adenocarcinoma tissue. The results of immunohistochemistry showed no significant differences in IGF-1R expression in inflammatory polyps compared with their surrounding normal mucosa by the Mann-Whitney U test (p=0.251); however, tubular adenoma and villous adenoma tissues exhibited significantly higher levels of IGF-1R expression (p=0.000). The results of real-time RT-PCR showed that IGF-1R was transcribed at a high level in colorectal adenomatous polyps and adenocarcinoma compared with their respective paired normal mucosa. Spearman's rank correlation two-variable analysis was used to demonstrate a significant correlation between the expression of IGF-1R and neoplastic progression from normal mucosa to adenomatous polyps and finally to colorectal cancer (r=0.574, p=0.000). This study suggests that the expression of IGF-1R correlates with the degree of carcinogenesis. In addition, these results demonstrated that there is a significant correlation between the level of IGF-1R expression and pit patterns of polyps (r=0.432, p=0.002). Thus, IGF-1R might be a factor in the morphological change of colorectal mucosal crypts, and it may play an important role in the growth and malignant transformation of precancerous polyps. These results suggest that IGF-1R can be considered a biomarker for the stage and risk of carcinogenesis during neoplastic initiation and progression along the colorectal normal mucosa-polyp-cancer sequence. Inhibitors of IGF-1R are not only a promising targeted anticancer strategy, but also a possible option for the chemoprevention of colorectal cancer.     

贲门腺癌中 DACT-1 基因的甲基化状态及其临床意义

目的:检测贲门腺癌(gastric cardia adenocarcinoma,GCA)及相应癌旁非肿瘤组织中β-环连蛋白抑制基因1(dishevelled-binding antagonist of beta-catenin 1,DACT-1)的甲基化状态,并探讨其临床意义.方法:应用甲基化特异性PCR(methylation specific PCR,MSP)、定量RT-PCR的方法分别检测112例贲门腺癌(河北医科大学第四医院外科和磁县肿瘤医院胸外科于2006-2014年收治)及相应癌旁非肿瘤组织中DACT-1基因的甲基化状态及其mRNA表达情况.结果:在贲门腺癌组织中,DACT-1基因的甲基化率为51.8％(58/112),癌旁非肿瘤组织中该基因的甲基化率为17.6％ (20/112),癌组织中DACT-1基因发生甲基化的频率明显高于癌旁非肿瘤组织(P＜0.01);癌组织中DACT-1基因mRNA的表达量为0.580±0.143,明显低于癌旁非肿瘤组织(0.654 ±0.110,P＜0.01);在DACT-1基因甲基化的贲门癌组织中该基因mRNA的表达量为0.488±0.097,明显低于该基因未甲基化的贲门癌组织(0.675 ±0.120),且该基因甲基化状态与其mRNA表达量相关(P＜0.01).癌组织中DACT-1基因的高甲基化状态与肿瘤患者的淋巴结转移情况及上消化道肿瘤家族史有关(P＜0.05),而与肿瘤患者的年龄、性别及肿瘤组织的病理分级、临床分期均无关(P＞0.05).结论:贲门腺癌中基因CpG岛的高甲基化可能是DACT-1基因表达下调的机制之一;DA CT-1基因启动子区的甲基化状态有望为贲门腺癌临床辅助诊断和预后评估提供新的指标.     

Study of the deletion mutation of glutathione transferase M1 gene and its role in susceptibility to hepatocellular carcinoma

Objection: To investigate the glutathione S transferase Ml (GSTM1) gene inherent deletion and its relation to prevalence of hepatocellular carcinoma (HCC) in Guangxi, China. Methods: The GSTM1 gene polymorphism of 120 HCC patients and 100 healthy subjects both from the same high aflatoxin B₁ (AFB₁) contaminated area were detected using PCR technique with special primers. Another 40 patients from AFB₁ low risk area were also tested. Results: In HCC high risk area, it was found that the frequencies of GSTM1 null genotype in HCC patients and healthy subjects were 59% and 51% respectively, with no significant difference. However, the frequency of GSTMl-null genotype in control group from AFB₁ low risk area was lower than those from high risk area (P<0.01). Conclusion: Populations in this HCC endemic region show a higher rate of GSTMl-null genotype, which may be partially responsible for the susceptibility to AFB₁ induced HCC. But the detoxification effect of GSTM1 alone is not sufficient to resist the genetic toxicity of AFB₁, especially in those people who expose to excess AFB₁. The GSTM1 gene deletion would not be suitable as an independent predictor of susceptibility to HCC. Foundation item: This work was supported by the National Science Foundation of China (No. 39860032) and a grant from the Department of Education of Guangxi Province (98-2-8). Biography: MA Yun (1963-), professor of pathology, physician in charge, Guangxi Medical University, majors in cancer etiology and fungi pathology.