DNA甲基转移酶在肝癌中的表达及其临床意义

目的 探讨DNA甲基转移酶(DNMTs)在肝癌中的表达及其临床意义.方法 收集2007年7月至2008年4月中山大学附属第一医院行根治性切除的50例肝癌患者的肝癌组织、癌旁组织、肝硬化组织和慢性肝炎组织.采用RT-PCR和免疫组织化学方法检测肝癌组织及癌旁组织中DNMT1、DNMT3a、DNMT3b mRNA与蛋白表达水平,采用配对t检验和Mann-Whitney U检验比较肝癌组织与癌旁组织,肝硬化组织与慢性肝炎组织DNMTs mRNA表达的差异,x2检验和Fisher确切概率法分析肝癌组织DNMTs蛋白表达与临床病理因素的相关性,Kaplan-Meier法分析患者无瘤生存时间,不同患者无瘤生存时间的比较采用Log-rank检验.结果 肝癌组织中DNMT1、DNMT3a、DNMT3b mRNA的表达水平分别为癌旁组织的2.57、2.29和4.86倍(t=3.94,2.72,4.06,P＜0.05).肝癌组织中DNMT1、DNMT3a、DNMT3b mRNA的表达分别是肝硬化组织和慢性肝炎组织的2.38、2.14、4.66倍和6.12、4.58、12.99倍.DNMT1、DNMT3a、DNMT3b mRNA在肝癌组织的表达显著高于其在肝硬化组织和慢性肝炎组织中的表达(U=587.5、730.0、562.5,65.5、64.5、71.0,P＜0.05).DNMT1蛋白表达与肿瘤大小、数目、有无血管侵犯、TNM分期有关(x2=5.24,4.08,4.08,14.13,P＜0.05);DNMT3a蛋白表达与肿瘤大小、数目、TNM分期有关(x2=4.08,5.95,4.08,P＜0.05).DNMT1和DNMT3a低表达者平均肿瘤复发时间分别为9.4个月和8.7个月,显著长于高表达者的5.0个月和3.2个月,两者比较,差异有统计学意义(x2=3.89,9.91,P＜0.05).结论 DNMTs在肝癌发生、发展中起重要作用;DNMT1与DNMT3a高表达与肿瘤切除术后复发密切相关,可望成为肝癌预后的重要预测因子.

Abstract：

Objective To investigate the expression of DNA methyltransferases (DNMTs) in liver cancer and its clinical significance. Methods The specimens of liver cancer tissues, adjacent tissues, cirrhotic tissues and chronic hepatitis tissues were collected from 50 patients who received radical resection at the First Affiliated Hospital of Sun Yat-Sen University from July 2007 to April 2008. The mRNA and protein expressions of DNMT1,DNMT3a and DNMT3b in liver cancer tissues, adjacent tissues, cirrhotic tissues and chronic hepatitis tissues were detected by real-time quantitative PCR and immunohistochemical staining. The mRNA expression of DNMTs in the liver cancer tissues was compared with those in the adjacent tissues, cirrhotic tissues and chronic hepatitis tissues by using t test and Mann-Whitney U test. The correlation between the protein expression of DNMTs in the liver cancer tissue and the clinicopathological features was analyzed by chi-square test or Fisher exact test, and the tumor-free survival time was analyzed by using Kaplan-Meier method and the difference in tumor-free survival rate between different patients was analyzed by Log-rank test. Results The mRNA expressions of DNMT1, DNMT3a and DNMT3b in the liver cancer tissue were 2.57, 2.29 and 4.86 times higher than those in the adjacent tissues (t = 3.94, 2. 72, 4. 06, P ＜ 0.05 ). The mRNA expressions of DNMT1, DNMT3a and DNMT3b were 2.38,2.14 and 4.66 times higher than those in the cirrhotic tissues, and 6.12, 4.58 and 12.99 times higher than those in the chronic hepatitis tissues. The mRNA expressions of DNMT1, DNMT3a and DNMT3b in the liver cancer tissue were significantly higher than those in the cirrhotic tissues and chronic hepatitis tissues ( U = 587.5,730. 0,562.5; 65.5, 64.5, 71.0, P ＜ 0.05). The protein expression of DNMT1 was correlated with the size, number,TNM stages and vascular invasion of tumors ( x2 = 4.08, 5.95, 4.08, P ＜ 0.05 ). The protein expression of DNMT3a was correlated with the size, number and TNM stages of tumors (x2 = 4.08, 5.95, 4.08, P ＜ 0.05 ).The mean tumor recurrence time of patients with low expressions of DNMT1 and DNMT3a were 9.4 and 8.7 months, which were significantly longer than 5.0 and 3.2 months of those with high expressions of DNMT1 and DNMT3a (x2 =3.89, 9.91, P＜0.05). Conclusions DNMTs play an important role in hepatocarcinogenesis.High expressions of DNMT1 and DNMT3a are correlated with the postoperative recurrence of liver cancer, which are valuable prognostic factors for liver cancer.     

ABCG2蛋白在肝癌中表达的临床意义

Objective To investigate the expression and clinical significance of ABCG2 protein in hepato-cellular carcinoma (HCC). Methods Specimens of HCC were collected at The First Aifiliated Hospital of Sun Yat-sen University from January 2005 to December 2006. The expression of ABCG2 protein in 165 samples of HCC tissue, 25 samples of normal liver tissue and 40 samples of cirrhotic liver tissue was detected using immunohisto-chemistry. The correlation between the expression of ABCG2 protein and clinicopathological characters was then analyzed. Enumeration data, survival rate and the difference between groups were analyzed with a chi-square test, the Kaplan-Meier method and Log-rank test, respectively. Results ABCG2 protein expression was weakly posi-tive in all normal and cirrhotic liver tissues. In HCC tissues, the expression of ABCG2 protein was strongly positive in 66 cases and weakly positive in 99 cases. The expression of ABCG2 protein was related to tumor diameter, tumor number, adjacent organ invasion and TNM stages (χ2 =8. 130, 14. 279, 4. 820, 21. 179, P <0. 05). Kaplan-Meier survival analysis revealed that patients with strongly positive ABCG2 protein had a significantly lower 3-year overall survival (24. 1%) compared with those with weakly positive ABCG2 protein (39. 4%) (χ2 = 15.716, P<0.05). Conclusions The expression level of ABCG2 protein is related to tumor invasiveness, TNM stage and prognosis. ABCG2 has the potential to become a new target for HCC treatment.     

ABCG2蛋白在肝癌中表达的临床意义

Objective To investigate the expression and clinical significance of ABCG2 protein in hepato-cellular carcinoma (HCC). Methods Specimens of HCC were collected at The First Aifiliated Hospital of Sun Yat-sen University from January 2005 to December 2006. The expression of ABCG2 protein in 165 samples of HCC tissue, 25 samples of normal liver tissue and 40 samples of cirrhotic liver tissue was detected using immunohisto-chemistry. The correlation between the expression of ABCG2 protein and clinicopathological characters was then analyzed. Enumeration data, survival rate and the difference between groups were analyzed with a chi-square test, the Kaplan-Meier method and Log-rank test, respectively. Results ABCG2 protein expression was weakly posi-tive in all normal and cirrhotic liver tissues. In HCC tissues, the expression of ABCG2 protein was strongly positive in 66 cases and weakly positive in 99 cases. The expression of ABCG2 protein was related to tumor diameter, tumor number, adjacent organ invasion and TNM stages (χ2 =8. 130, 14. 279, 4. 820, 21. 179, P <0. 05). Kaplan-Meier survival analysis revealed that patients with strongly positive ABCG2 protein had a significantly lower 3-year overall survival (24. 1%) compared with those with weakly positive ABCG2 protein (39. 4%) (χ2 = 15.716, P<0.05). Conclusions The expression level of ABCG2 protein is related to tumor invasiveness, TNM stage and prognosis. ABCG2 has the potential to become a new target for HCC treatment.     

Association of hypoxia-inducible factor 1α expression with microlymphatic vessel density and lymph node micrometastasis in rectal cancer

Objective To explore the association of the expression of hypoxia-inducible factor 1α (HIF-1α) with microlymphatic vessel density (MLVD) and lymph node micro-metastasis in rectal cancer.Methods The experimental group consisted of 40 middle-low rectal cancer specimens pathologically confirmed at the First Affiliated Hospital of Anhui Medical University between 2000 and 2003.Forty samples of normal tissues taken from the corresponding area around the cancer were used as the control group. Immunohistochemistry was used to detect HIF-1α expression and MLVD in both the tumor tissues and the adjacent normal tissues. Lymph node micrometastasis was ascertained using immunohistochemical staining with CK20. Results In rectal cancer tissues, the HIF-lα expression was 77 386±14 911 and MLVD was 7.3±0.7, significantly higher than those in normal adjacent tissues(33 092±5877 and 0.3±0.2, both P＜0.01). The HIF-1α expression was positively correlated with MLVD in rectal cancer (r=0.781, P＜0.01). Thirty-one patients had no lymph nodes metastasis and 10 had micrometastasis. The HIF-1α expression and MLVD in specimens with lymph node micrometastasis was significantly higher than that in those without lymph node micrometastasis(P＜0.05). Conclusion HIF-1α and MLVD play important roles in the development of rectal cancer, which may promote lymphatic micrometastasis in rectal cancer.     

KiSS-1、KiSS-1R及MMP-9表达与肝细胞癌侵袭转移的关系及意义

目的 检测肿瘤转移抑制基因KiSS-1及其配体KiSS-1R和基质金属蛋白酶-9(MMP-9)在肝细胞癌(HCC)中的表达,探讨KiSS-1表达与肝细胞癌侵袭转移的关系及其机制.方法 利用逆转录聚合酶链反应(RT-PCR)技术,检测HCC、癌旁肝组织及远癌肝组织中KiSS-1、KiSS-1R mRNA的表达情况;应用HCC组织芯片,结合免疫组化及彩色图像分析技术检测了KiSS-1、MMP-9蛋白在HCC芯片中各组织的相对表达量.结果 KiSS-1 mRNA在HCC中表达明显低于癌旁、远癌肝组织(P＜0.01).在转移组和临床Ⅲ期HCC中,KiSS-1蛋白的表达明显低于无转移及临床分期Ⅰ和Ⅱ期HCC(P＜0.01);KiSS-1在肝内转移灶中的表达量显著低于原发灶(P＜0.01).在转移组的HCC中,MMP-9表达量显著高于无转移的HCC(P＜0.01).在HCC组织中,KiSS-1和MMP-9的表达呈负相关性(r=-0.340,P＜0.01).结论 KiSS-1和MMP-9的表达失衡与HCC侵袭转移有关,KiSS-1的缺失表达可作为预测HCC转移潜能的有价值的参考指标.

Abstract：

Objective To detect the expression of KiSS-1, KiSS-1R and MMP-9 in hepatocellular carcinoma (HCC). To study the correlation of KiSS-1, KiSS-1R and MMP-9 expression with invasion and metastasis of HCC, and to explore the underlying mechanisms. Methods The expression of KiSS-1 , KiSS-1R mRNA in 33 HCC samples, 26 non-neoplastic adjacent liver tissue samples and 13 non-neoplastic distant liver tissue samples were detected by RT-PCR. Tissue chips were constructed by modified manual tools, which contained HCC, non-neoplastic adjacent liver tissues, non-neoplastic distant liver tissues, normal liver tissues and intrahepatic metastasis lesions. The expression of KiSS-1 and MMP-9 protein was determined by tissue chips, immunohistochemistry and semi-quantitative image analysis in 150 HCC, 137 non-neoplastic adjacent liver tissue, 98 non-neoplastic distant liver tissues, 16 normal liver tissues and 37 intrahepatic metastasis lesion samples. Results The results of RT-PCR showed that compared with the non-neoplastic adjacent liver tissues and the non-neoplastic distant liver tissues, the expression of KiSS-1 mRNA in HCC was significantly lower (P＜0.01). The expression of KiSS-1R mRNA did not changed in HCC and non-neoplastic liver tissues (P＞0.05). The expression of KiSS-1 protein was lower in HCC with metastasis and in clinical stage Ⅲ than that in those with non-metastasis, and in clinical stages Ⅰ and Ⅱ . It was also higher in the primary than in the metastasis lesions (P＜0.01, respectively). The expression of MMP-9 was higher in tumors having peplos invasion and metastasis than in those with negative peplos invasion and non-metastasis. It was lower in the primary than the metastasis lesions (P＜0. 01, respectively).Negative correlation between KiSS-1 and MMP-9 expression was found in HCC(r=- 0.340,P＜0.01). Conclusions The imbalance between KiSS-1 and MMP-9 expression might play an important role in enhancing the invasive and metastatic capacity of HCC. Loss of KiSS-1 expression might predict an aggressive clinical behavior and was associated with metastatic potential in HCC.     

胰腺癌组织中程序性细胞死亡因子4与细胞色素C的关系

Objective To study the relationship between the expression of cytochrome c ( Cyt c) and programmed cell death 4 (PDCD4) in pancreatic cancer, and investigate the pathway of PDCD4 inducing the apoptosis of pancreatic cancer cells. Methods Pancreatic cancer specimens from 69 patients who received pancreatic resection from 1990 to 2002 in First Affiliated Hospital of China Medical University were collected. The expression of Cyt c in the 69 paraffin specimens of pancreatic cancer was detected by immunohistochemistry, and the expression of Cyt c in 8 samples of cold-preserved fresh pancreatic cancer and normal pancreatic tissues were detected by Western blot. The expression of PDCD4 and Cyt c in pancreatic cancer was analyzed by paired t test and chi-square test. Results Compared with normal pancreatic tissues, the expression of Cyt c in pancreatic cancer was significantly decreased. The positive expression rate of Cyt c in 69 samples of pancreatic cancer was 41% (28/69). The expression of Cyt c was positive in most patients with positive expression of PDCD4, and the expression of PDCD4 was negative in most patients with negative expression of Cyt c. The expression of PDCD4 and Cyt c was closely correlated with each other (χ2= 10.52, P < 0.05). Conclusions There is a close relationship between the expression of PDCD4 and Cyt c in pancreatic cancer. PDCD4 may induce the apoptosis of pancreatic cancer cells through mitochondrial pathway.     

Increased expressions of vascular endothelial growth factor （VEGF）, VEGF-C and VEGF receptor-3 in prostate cancer tissue are associated with tumor progression

Aim： To investigate the differences in microvessel densities （MVD） and the expressions of vascular endothelial growth factor （VEGF）, VEGF-C and VEGF receptor-3 （VEGFR-3） between prostate cancer （PCa） tissues and adjacent benign tissues, and to explore the correlations among MVD, Jewett-Whitmore staging, Gleason scores and expressions of VEGF, VEGF-C and VEGFR-3 in the progression of PCa. Methods： An immunohistochemical approach was adopted to detect the expressions of CD34, VEGF, VEGF-C and VEGFR-3 in both cancer areas and peripheral benign areas of 71 primary prostatic adenocarcinoma specimens. A statistic analysis was then performed according to the experimental and clinic data. Results： Significantly upregulated expressions of VEGF, VEGF-C and VEGFR-3 were all found in malignant epithelium/cancer cells compared with adjacent benign epithelium （P 〈 0.01）. Patients in stage D had a significantly higher score than patients in stage A, B or C when comparing the expression of VEGF-C or VEGFR-3 in the tumor area （P 〈 0.01）. In addition, significant correlations were observed between Jewett-Whitmore staging and VEGF-C （rs = 0.738, P 〈 0.01）, clinical staging and VEGFR-3 （rs = 0.410, P 〈 0.01）, VEGF-C and Gleason scores （rs = 0.401, P 〈 0.01）, VEGFR-3 and Gleason scores （rs = 0.581, P 〈 0.001） and MVD and VEGF （rs = 0.492, P 〈 0.001）. Conclusion： Increased expressions of VEGF and VEGF-C were closely associ- ated with progression of PCa. The main contribution of increased VEGF expression for PCa progression was to upregulate MVD, which maintained the growth advantage of tumor tissue. However, the chief role of increased expressions of VEGF-C and VEGFR-3 was to enhance lymphangiogenesis and provide a main pathway for cancer cells to disseminate. （Asian J Androl 2006 Mar; 8： 169-175）     

胃良性和恶性病变组织中β-半乳糖凝集素3和接骨木凝集素的表达及其临床病理意义

Objective To study the expressive levels of galectin-3(gal-3) and sambucus nigra agglutinin (SNA) and their clinicopathological significance in the benign and malignant lesions of stomach. Methods EnVisonTM immunohistochemistry for assaying gal-3 expressive level and ABC cytochemistry for determining SNA expressive level were used in conventional paraffin-embedded sections from specimens of gastric cancer(n=49), peritumoral tissues(n=20), metastastic foci of lymph nodes(n=36), and different types of benign lesions(n=80). Results The positive rates of gal-3 and SNA were significantly higher in gastric cancer tissues than those in peritumoral tissues and different types of benign lesions (P<0.05, P<0.01). The positive cases of gal-3 and/or SNA in peritumoral tissues and benign lesions showed mild- to severe-atypical hyperplasia of mucous epithelial cells. No difference was found between the primary foci and metastatic foci in gal-3 and SNA expressions (P>0.05). The positive rates of gal-3 and SNA were significantly lower in histologic grade Ⅱ, infitrating depth T1,T2 and no-metastasis of regional lymph node than those in histologic grade Ⅲ, Ⅳ, infitrating depth T3,T4 and metastasis of lymph node in gastric cancer(P<0.05). The positive rates of gal-3 and SNA were higher in lymphnede metastatic site N1 and no-metastasis of distant organs than those in lymphnede metastatic site N2, N3 and metastasis of distant organs, but no significant difference was found (P>0.05). The consistence was found between the expression of gal-3 and SNA in gastric cancer tissues (χ2=6.59,P<0.05). Conclusions The expressive levels of gal-3 and SNA may be important molecular markers of lectins for reflecting the carcinogenesis, progression and biological behaviors in gastric cancer.     

胃良性和恶性病变组织中β-半乳糖凝集素3和接骨木凝集素的表达及其临床病理意义

Objective To study the expressive levels of galectin-3(gal-3) and sambucus nigra agglutinin (SNA) and their clinicopathological significance in the benign and malignant lesions of stomach. Methods EnVisonTM immunohistochemistry for assaying gal-3 expressive level and ABC cytochemistry for determining SNA expressive level were used in conventional paraffin-embedded sections from specimens of gastric cancer(n=49), peritumoral tissues(n=20), metastastic foci of lymph nodes(n=36), and different types of benign lesions(n=80). Results The positive rates of gal-3 and SNA were significantly higher in gastric cancer tissues than those in peritumoral tissues and different types of benign lesions (P<0.05, P<0.01). The positive cases of gal-3 and/or SNA in peritumoral tissues and benign lesions showed mild- to severe-atypical hyperplasia of mucous epithelial cells. No difference was found between the primary foci and metastatic foci in gal-3 and SNA expressions (P>0.05). The positive rates of gal-3 and SNA were significantly lower in histologic grade Ⅱ, infitrating depth T1,T2 and no-metastasis of regional lymph node than those in histologic grade Ⅲ, Ⅳ, infitrating depth T3,T4 and metastasis of lymph node in gastric cancer(P<0.05). The positive rates of gal-3 and SNA were higher in lymphnede metastatic site N1 and no-metastasis of distant organs than those in lymphnede metastatic site N2, N3 and metastasis of distant organs, but no significant difference was found (P>0.05). The consistence was found between the expression of gal-3 and SNA in gastric cancer tissues (χ2=6.59,P<0.05). Conclusions The expressive levels of gal-3 and SNA may be important molecular markers of lectins for reflecting the carcinogenesis, progression and biological behaviors in gastric cancer.     

RCAS1在胰腺癌诊断中的作用

Objective To evaluate the effect of receptor-binding cancer antigen expressed on SiSO cells(RCASI) as serum tumor marker on the diagnosis of pancreatic cancer. Methods Receiver-operating characteristics(ROC) curve methods were used to assay the serum content of RCAS1 ,CA19-9 and CA242 in 46 patients with pancreatic cancer, 18 patients and 20 normal tissues of chronic pancreatitis detected by enzymelinkod immunosorbent assay (ELISA), and the results were analyzed by statistics methods. The expressions of RCAS1 protein were analyzed by immunohistochemical method in 32 patients with pancreatic cancer, 10 patients with chronic pancreatitis and 6 cases of normal pancreatic specimens. Results The serum levels of RCAS1 ,CA19-9 and CA242 in pancreatic cancer were higher than that in chronic pancreatitis respectively (P ＜ 0.01). The area under curve of RCAS1, CA19-9 and CA242 were 0.826, 0.804 and 0.737 ,respectively. Subgroup analysis indicated that the RCAS1 and CA19-9 levels of pancreatic cancer patients without obstructive jaundice were lower than those for patients with obstructive jaundice (P＜0.01). CA19-9 levels of patients with resectable pancreatic cancer were lower than those with unresectable pancreatic cancer(P ＜ 0.01). Immunohistochemistry showed that the expression rates of RCAS1 in pancreatic cancer and chronic pancreatitis were 87.5% and 40.0% ,respectively (P ＜0. 05). Conclusions In diagnosis of pancreatic cancer,the clinical value of RCAS1 is available. And the combination test of RCAS1 and CA19-9 have clinical value to evaluate if the pancreatic cancer can be resected before operation.