食管鳞癌组织p16基因调控区甲基化及其蛋白表达研究

目的探讨p16基因在食管癌变过程中表达缺失与其启动子区甲基化的关系。方法采用MSP免疫组化方法,检测环太行山地区45例食管鳞癌患者癌组织p16基因启动子区甲基化状态及蛋白表达情况。结果p16基因在癌组织中表达异常41例（91．1％）,间变组织中表达异常38例（84．4％）,发生纯合型甲基化的组织分别为33例（73．3％）（癌组织）和32例（71．1％）（间变组织）,而其周围正常组织26例（57．8％）均发生了p16启动子区的杂合型甲基化。p16基因纯合型甲基化与癌组织、间变组织、p16蛋白表达缺失相关（P〈0．05）。结论该地区食管癌组织p16基因在癌前病变中p16启动子区即发生了纯合型甲基化、食管癌变的早期事件。p16基因启动子区甲基化可单独影响p16蛋白的正常表达。     

Promoter hypermethylation of the CFTR gene and clinical/pathological features associated with non-small cell lung cancer

Background and objective: The exact role of the cystic fibrosis transmembrane conductance regulator (CFTR) in pathophysiology, and the mechanisms regulating its expression are poorly understood. The CFTR gene is known to be genetically or epigenetically associated with several cancers. In the present study, the methylation status of the promoter region of the CFTR gene and its expression in primary non‐small cell lung cancer (NSCLC) were investigated. Methods: The methylation status of the promoter region of the CFTR gene in NSCLC tissue was assessed by pyrosequencing and methylation‐specific PCR. Expression of the CFTR gene was analysed by real‐time PCR, and CFTR gene reactivation was investigated using 5‐aza‐2′‐deoxycytidine. The correlation between methylation of the CFTR gene and the clinical features of the patients was assessed. Results: Methylation of the CFTR gene in NSCLC was quantitatively high by pyrosequencing analysis and qualitatively frequent by methylation‐specific PCR analysis. Expression of the CFTR gene was significantly lower in NSCLC compared with normal lung tissue. In addition, the demethylating agent 5‐aza‐2′‐deoxycytidine increased CFTR gene expression. Methylation of the CFTR gene was significantly greater in squamous cell carcinomas than in adenocarcinomas. CFTR gene methylation was associated with significantly poorer survival in young patients, but not in elderly patients. Conclusions: These findings suggest that DNA methylation may be important for downregulation of CFTR gene expression in lung cancer. Promoter hypermethylation of the CFTR gene may be an important prognostic factor in younger patients with NSCLC.     

PTEN hypermethylation profiles of Chinese Kazakh patients with esophageal squamous cell carcinoma

Aberrant DNA methylation of promoter region CpG islands may serve as an alternative mechanism to genetic defects in the inactivation of tumor suppressor genes (TSGs) in human malignancies. The aim of this study was to examine the promoter methylation status of the PTEN TSG and its association with esophageal squamous cell carcinoma (ESCC) carcinogenesis in a Chinese Kazakh population, which is known to have a relatively high ESCC incidence and mortality. The methylation status of the PTEN promoter region was determined in patients with ESCC (n = 95) and healthy individuals (n = 65) using highly sensitive Sequenom Epityper assays. The methylation level of the PTEN gene was significantly higher in patients with ESCC than in healthy controls. The median methylation level was 10.0% (interquartile range [IQR]: 7.0–11.0%) in patients with ESCC and 6.0% in controls (IQR: 4.0–9.0%; P = 0.001). PTEN methylation levels were higher in male patients with ESCC than in male controls, whereas a trend toward significance was observed between female patients with ESCC and female controls (P = 0.005 and P = 0.086, respectively). The PTEN methylation level was associated with histopathological grade and lymph node metastasis in patients with ESCC (P = 0.002 and P = 0.009, respectively). To our knowledge, this is the first report to show the presence of PTEN promoter CpG hypermethylation in ESCC and its association with tumor metastasis.     

5-氮杂-2′-脱氧胞苷对胃癌AGS细胞CHFR基因去甲基化作用及意义

目的观察5-氮杂-2′-脱氧胞苷（5-Aza-CdR）对胃癌AGS细胞CHFR基因去甲基化的作用,并探讨其临床意义。方法分别采用BSP和RT-PCR技术检测5-Aza-CdR处理前后胃癌AGS细胞CHFR基因启动子甲基化状态及其mRNA。结果 AGS细胞CHFR基因启动子在5-Aza-CdR处理前呈现高甲基化状态（甲基化率≥60%）,其mRNA表达完全缺失;5-Aza-CdR处理后则表现为低或无甲基化状态（甲基化率≤20%）,其mRNA表达恢复正常。结论 CHFR基因启动子在AGS细胞中呈高甲基化状态,5-Aza-CdR能显著逆转其CHFR基因异常甲基化,诱导CHFR基因表达,为胃癌的治疗提供新思路。     

Original article: The expression of CFL1 and N‐WASP in esophageal squamous cell carcinoma and its correlation with clinicopathological features

Cofilin1 (CFL1) is an actin‐modulating protein, which belongs to the ADF/Cofilin family. Neural Wiskott–Aldrich syndrome protein (N‐WASP) is the key regulator of the actin cytoskeleton, a member of Wiskott‐Aldrich syndrome protein family. They have been suggested to be involved in cancer cell invasion and metastasis. In this study, the expression patterns of CFL1 and N‐WASP in normal esophageal mucosa and esophageal squamous cell carcinoma (ESCC) and their correlation with clinical characteristics were investigated. Immunohistochemical staining showed that CFL1 was expressed in nuclear and cytoplasm of cancer cells. However, N‐WASP was mainly found in the cytoplasm of the cancer cells. There were significant evidences that proved that CFL1 is correlated with clinicopathological factors in ESCC, such as infiltration depth, lymph node metastasis and pathological staging (P < 0.05). It is also proved that N‐WASP is related to lymph node metastasis and pathological staging in ESCC (P < 0.05). Kaplan–Meier analysis showed that there was no correlation between CFL1 and N‐WASP protein expression and survival (P > 0.05). Moreover, the mRNA expression of CFL1 and N‐WASP was detected by quantitative real time PCR in 70 tissue specimens. The results showed that CFL1 mRNA level was over‐expressed in ESCC tissue (P < 0.05), while N‐WASP mRNA expression level was not different between cancerous tissues and adjacent normal esophageal mucosa (P > 0.05). Also, CFL1 mRNA expression was significantly associated with regional lymph node metastasis and pathological staging (P < 0.05). Kaplan–Meier analysis showed that there was no correlation between CFL1 and N‐WASP mRNA expression and survival (P > 0.05). Our findings suggested that CFL1 and N‐WASP may play an important role in the tumorigenesis of ESCC, and to be the candidate novel biomarkers for the diagnosis and prognosis of ESCC. These findings may have implications for targeted therapies in patients with ESCC.     

结肠癌中心和边缘组织增殖能力的差异及相关基因的表达

目的探索结肠癌组织不同区域肿瘤细胞是否存在增殖能力的差异，且这种区域性差异和多重抑痛基因p16及其可变读码框架pl4基因的表达情况及其甲基化状态的相关性。方法免疫组化法测定癌块不同区域增殖蛋自Ki67和P16、P14的表达差异，激光显微切割技术结合甲基化特异性PCR、巢式逆转录PCR观察同一癌块不同区域癌细胞p16及p14基因甲基化状态的改变及其mRNA的表达情况。结果在42份癌组织标本的中心，增殖蛋自Ki67均呈强阳性表达，而P16和P14蛋白表达缺失；在癌组织标本的边缘．有13份Ki67表达阳性（30．1％）。不同区域癌细胞的增殖能力差异有统计学意义（P〈0．05）．且与年龄、性别、Dukes分期、p14的表达无关，但与p16的表达显著相关（X^2=25．37，P〈0．01）。且p16基因的甲基化状态和mRNA的表达也存在明冠的区域性差异（P〈0．05）。结论人结肠癌组织中存在肿瘤细胞增殖能力的区域性差异，在癌组织边缘肿瘤细胞侵袭力增强的同时增殖能力下降，癌边缘区P16基因去甲基化后的再表达可能是这一细胞事件的分子原因。     

Expression of klotho and β‐catenin in esophageal squamous cell carcinoma,and their clinicopathological and prognostic significance

Esophageal carcinoma is one of the most common types of cancers in the world; the molecular mechanism underlying its tumorigenesis is still not well understood. This study was aimed at investigating the expression of klotho and β‐catenin in patients with esophageal squamous cell carcinoma (ESCC) and analyzing their association with clinicopathological variables and their effects on prognosis. The expression patterns of klotho and β‐catenin were determined by tissue microarray and immunohistochemical technique in ESCC and normal tissues, and their correlations with clinicopathological characteristics were investigated using univariate and multivariate analysis. The serum klotho levels in 40 ESCC patients and controls were measured by sandwich enzyme‐linked immunosorbent assay system (ELISA). The expression level of klotho was significantly lower in ESCC than in the adjacent noncancerous tissues (30 vs. 50%, P < 0.000), and the protein level was negative correlated with clinical staging, histological grade, lymph node metastasis, and invasion depth (P < 0.05). Whereas, the expression of β‐catenin was much higher in ESCC than their corresponding normal mucosa tissues (78.3 vs. 11.5%, P < 0.000), and the level of protein correlated only with histological grade and invasion depth (P < 0.05). Correlation analysis showed the expression level of klotho inversely correlated with that of β‐catenin (r = ?0.214, P < 0.01). Patients with klotho‐positive tumors had longer survival than those with klotho‐negative tumors (P < 0.01). Cox proportional hazards model analysis demonstrated that positive expression of klotho was an important factor indicating good prognosis (hazard ratio, 0.371; 95% confidence interval, 0.201–0.685; P < 0.01). ELISA showed that the level of serum klotho was markedly higher (461.50 ± 43.30 pg/mL) than control group (239.37 ± 20.65 pg/mL) (P < 0.001). Receiver operating characteristic analysis gave a cut‐off value of 327.031 of serum klotho with a sensitivity of 81.3% and specificity of 81.2% (P < 0.000). Our present study demonstrated for the first time that klotho might be a novel biomarker candidate for predicting progression and prognosis in patients with ESCC.     

Protein kinase D1 mRNA level may predict cancer‐specific survival in heavy smokers with esophageal squamous cell cancers

Protein kinase D1 (PRKD1) is a kinase that regulates various pathways, which involve in cell proliferation, apoptosis, cell adhesion and invasion. Although PRKD1 expression has been observed in many cancers, its role in esophageal squamous cell cancer (ESCC) has not been well reported. As its dysregulation in cancers is organ specific, we sought to investigate the potential role of PRKD1 in the progression of ESCC. Samples were collected from 178 patients with completely resected ESCCs at Sun Yat‐sen University Cancer Center, including 47 pairs of tumorous and non‐tumorous tissues. PRKD1 mRNA expression was investigated by quantitative real‐time polymerase chain reaction. Receiver operating characteristic (ROC) curve analysis was used to search for a feasible cut‐off point of PRKD1 mRNA levels for predicting cancer‐specific survival. Kaplan–Meier and multivariate Cox regression analysis were used to assess the prognostic value of PRKD1 mRNA level in ESCC patients. In result, upregulation of PRKD1 mRNA was detected in 55.3% (26/47) of ESCC tissues compared with paired non‐tumorous ones (P = 0.011). ROC analysis indicated 3.28 as a cut‐off point, and thus 72 and 106 tumors with low and high PRKD1 mRNA expression were categorized. High‐PRKD1 mRNA expression in tumors appeared with more frequency in heavy smokers (P = 0.002) and patients with advanced pathological T category (P = 0.034). Kaplan–Meier analysis indicated that patients with low‐PRKD1 mRNA had a longer cancer‐specific survival than the ones with high‐PRKD1 level (P = 0.044). Multivariate analysis showed that tumorous PRKD1 mRNA expression was an independent prognostic factor (hazard ratio: 1.538, 95% confidence interval: 1.018–2.323, P = 0.041) in resected ESCC. Subgroup analysis revealed that the discernibility of PRKD1 mRNA level on ESCC outcomes was only pronounced in heavy smokers (P = 0.002), but not in non‐heavy smokers (P = 0.870). PRKD1 might play a potential oncogenic role in ESCC. It might be an independent biomarker to predict prognosis in heavy smokers with ESCC.     

MDM2 mRNA Expression in the p53 Pathway May Predict the Potential of Invasion and Liver Metastasis in Colorectal Cancer

Purpose  The p53/MDM2/p14ARF pathway is one of the major signaling cascades involved in the regulation of apoptosis. Although many tumors have been reported to show disruption of the p53/MDM2/p14ARF pathway, few studies have examined p53, MDM2, and p14ARF simultaneously in colorectal carcinoma. The present study was undertaken to clarify whether correlations exist among MDM2, p53, and p14ARF in colorectal cancer. Methods  We determined the presence of mutations in the p53 gene, MDM2 expression, and methylation status of the p14ARF in 97 primary colorectal carcinoma specimens. Associations with survival and clinicopathologic factors were investigated. Results  At least one abnormality of these three molecules was found in 82 (84 percent) tumors. We observed a significant inverse association between MDM2 expression and tumor invasion (P = 0.01). Furthermore, the presence of liver metastasis was also significantly associated with low MDM2 expression (P = 0.02). Conclusions  The results suggest that disruption of the p53/MDM2/p14ARF pathway may frequently participate in colonic carcinogenesis and that MDM2 expression status may be a factor in the prediction of potential invasion and liver metastasis of colorectal carcinomas. Reprints are not available.