High expression of Axl promotes clinical progression of nasopharyngeal carcinoma

AIM: To explore the expression and significance of receptor tyrosine kinase anexelekto (Axl) in nasopharyngeal carcinoma (NPC). METHODS: Immunohistochemistry was used to detect the Axl protein expression of 78 patients with NPC and 32 patients with nasopharyngeal chronic inflammation (NPI). The correlations between the Axl protein levels and the clinical parameters of NPC patients were analyzed. NPC cells were cultured in vitro, and the expression of Axl in well differentiated CNE1 cells, poorly-differentiated CNE2Z cells and undifferentiated C666-1 cells was detected by immunofluorescence staining. After treatment of the CNE1and C666-1 cells with Axl specific inhibitor TP-0903, CCK-8 assay was used to detect cell viability, flow cytometry was adopted to analyze the cell cycle distribution, qPCR was used to examine the mRNA levels of Axl and proliferating cell nuclear antigen (PCNA), and Western blot was used to examine the protein expression of Axl and p-Axl. RESULTS: Axl protein was localized in the cell membrane and cytoplasm. The rate of high expression of Axl in NPC was significantly higher than that in NPI (P<0.01). High Axl expression showed no correlations with NPC patients' age, gender and M stage, while positively correlated with the clinical stage, T stage and N stage (P<0.05). Axl protein showed a low level in the CNE1 cells, but showed a high level in CNE2Z and C666-1 cells. TP-0903 inhibited cell viability in concentration and time dependent manners. TP-0903 at 2 nmol/L showed significant inhibitory effects, as evidenced by arresting the cell cycle at G₀ phase and reducing Axl activity and PCNA expression. CONCLUSION: High expression of Axl promotes the clinical progress of NPC.TP-0903 significantly inhibits the viability of NPC cells, suggesting that Axl may be a valuable target in the NPC treatment.     

Expression of glypican-3 in hepatocellular carcinoma and its clinical significance

AIM: To study the expression of glypican-3 in hepatocellular carcinoma (HCC) tissues and to clarify its clinical significance. METHODS: The expression of GPC3 was detected in 59 cases of HCC and their para-cancerous tissues, 10 cases of intrahepatic cholangiocellular carcinoma (ICC), 11 cases of cirrhotic tissues and 14 cases of normal liver tissues (around haemangioma) by RT-PCR and immunohistochemical staining. The survival curves were constructed using Kaplan-Meier method and evaluated using the log-rank test. In addition, the Cox proportional hazards regression model was established to identify the factors that were independently associated with disease-free survival (DFS). RESULTS: The mRNA expression of GPC3 in the HCC tissues was significantly higher than that in the para-cancerous tissues (83.1% vs 35.6%, χ²=27.53, P<0.01). The protein expression of GPC3 in the HCC tissue was also higher than that in the para-cancerous tissues (78.0% vs 33.2%, χ²=24.97, P<0.01). The expression of GPC3 in ICC tissues, liver cirrhosis tissues and normal liver tissues was undetectable. Kaplan-Meier survival analysis indicated that the GPC3(+)HCC patients had worse 1-year DFS than that of GPC3(-) patients (33.6% vs 72.7%, P<0.05). The HCC patients with para-cancerous GPC3(+) also had worse 1-year DFS than that of the para-cancerous GPC3(-) patients (23.5% vs 40.1%, P<0.05). The DFS rate decreased significantly as the expression intensity of GPC3 increased. The Cox regression model analysis indicated that AFP(+) (odd ratio=0.372, 95% confidence interval: 0.140-0.900, P<0.05), tumor size (odd ratio=5.215, 95% confidence interval: 1.737-15.656, P<0.01), para-cancerous tissue GPC3(+) (odd ratio=0.226, 95% confidence interval: 0.085-0.599, P<0.01) and the intensity of GPC3 expression in HCC tissue (odd ratio=1.946, 95% confidence interval: 1.080-3.507, P<0.05) were the independent risk factors linked to DFS of patients. CONCLUSION: GPC3 protein is highly expressed in the HCC tissues,but not in ICC, cirrhotic liver and normal liver tissues. The expression of GPC3 in para-cancerous tissues and the intensity of GPC3 expression in HCC tissues are the important independent risk factors linked to DFS of patients.     

Expression and clinical significance of SCNM1 in hepatitis B-related hepatocellular carcinoma

AIM: To investigate the expression of sodium channel modifier 1 (SCNM1) in hepatitis B-related hepatocellular carcinoma (HCC) and its relationship with clinicopathological features and prognosis.METHODS: The specimens were collected from 108 patients with hepatitis B-related HCC who were treated in the Third Affiliated Hospital of Sun Yat-sen University from January 2013 to December 2015. All patients signed the informed consent and met the requirements of medical ethics. The mRNA expression level of SCNM1 in hepatitis B-related HCC tissues and tumor-adjacent tissues was detected by RT-qPCR, and the relationship between the mRNA expression of SCNM1 and the clinicopathological characteristics of hepatocellular carcinoma was analyzed. The relationship between SCNM1 expression and the prognosis of the patients was analyzed by Kaplan-Meier plotter.RESULTS: The data from TCGA database, Human Protein Atlas database and Oncomine database showed that the expression of SCNM1 in hepatocellular carcinoma tissues was significantly higher than that in the normal liver tissues (P<0.01). SCNM1 was mainly distributed in the nucleus. The results of RT-qPCR showed that the median mRNA expression of SCNM1 in hepatitis B-related HCC tissues was significantly higher than that in the matched tumor-adjacent tissues (t=8.082, P<0.01). The mRNA expression of SCNM1 was correlated with cirrhosis, alanine aminotransferase and tumor size (P<0.05), but not with sex, age and tumor envelope. The total survi-val time of the HCC patients with high expression of SCNM1 was shorter than that of the patients with low expression of SCNM1 (HR=1.53, P=0.016), and that of the patients with hepatitis B-related HCC was even shorter (HR=2.41, P=0.015).CONCLUSION: SCNM1 is highly expressed in hepatitis B-related HCC and may play an important role in the development of hepatitis B-related HCC.     

Expression of HDAC6 protein in cervical carcinoma tissues and its clinical significance

AIM:To investigate the protein expression of histone deacetylase 6(HDAC6) in cervical carcinoma tissues and its clinical value. METHODS:The method of immunohistochemistry was used to detect the protein expression of HDAC6 in 63 cases of cervical carcinoma tissues, 38 cases of cervical intraepithelial neoplasia(CIN) tissues and 63 cases of normal cervical epithelial tissues. The relationships between the protein expression of HDAC6 and clinical pathological features were analyzed. The protein expression of HDAC6 in randomly selected 4 cases of cervical carcinoma tissues and paired normal cervical epithelial tissues was detected by Western blotting. RESULTS:Positive rates of HDAC6 protein expression in cervical carcinoma tissues were significantly higher than that in CIN tissues or normal cervical epithelial tissues, and there were obvious differences among the 3 groups(P<0.05). The protein expression of HDAC6 was not related to age and histological differentiation(P>0.05), but closely associated with clinical stages, invasive depth and lymph node metastasis(P<0.01 or P<0.05). Furthermore, the result of Western blotting demonstrated that the protein level of HDAC6 in cervical carcinoma tissues was markedly higher than that in normal cervical epithelial tissues. CONCLUSION:HDAC6 may be an important molecular marker for evaluating malignant degree and prognosis of cervical carcinoma.     

Latent membrane protein 1 regulates expression of p27 and RPLP0 in nasopharyngeal epithelial and nasopharyngeal carcinoma tissues

AIM: To observe the protein expression of p27 and ribosomal phosphoprotein large P0(RPLP0) regulated by latent membrane protein 1 (LMP1) in nasopharyngeal epithelial and nasopharyngeal carcinoma tissues. METHODS: The protein levels of p27 and RPLP0 and the relationship with LMP1 were analyzed by Western blotting. The protein expression of LMP1, p27 and RPLP0 was also detected by the method of immunohistochemistry in 30 nasopharyngeal epithelial and 60 nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues. Meanwhile, the significance of clinical pathology was evaluated. RESULTS: The positive rate of LMP1 protein was 73.3% and 90.0% in nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues, respectively. Compared with the LMP1-negative tissues, the protein levels of RPLP0 were low in the nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues with LMP1-positive expression, but the levels of RPLP0 protein were overexpressed. The protein expression of RPLP0 and RPLP0 was related to the age of nasopharyngeal carcinoma patients, the protein level of LMP1, the metastasis of lymph nodes and the TNM classification. The positive expression of p27 protein at high level was usually observed in the patients with young age, or had the characteristics of LMP1 (-), non-metastasis of lymph nodes, and in I or II stage of TNM classification. However, the protein expression of RPLP0 was low (P<0.05). CONCLUSION: LMP1 down-regulates p27 and up-regulates RPLP0 in nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues.     

Mortalin expression in cervical squamous-cell carcinoma and its effect on tumor metastasis

AIM: To investigate the significance of mortalin expression in clinical pathology of cervical squamous-cell carcinoma. METHODS: Immunofluorescence staining was used to detect the location of mortalin in human cervical squamous-cell carcinoma SiHa cells. The protein expression of mortalin was detected in 59 cases of normal cervical epithelial tissues and 93 cases of cervical squamous-cell carcinoma tissues by immunohistochemical staining, and its correlation with clinicopathological features of cervical squamous-cell carcinoma was also analyzed. MTT assay was used to evaluate the optimal concentration and dosing time of mortalin inhibitor MKT-077. After the protein expression of mortalin in SiHa cells was inhibited, wound-healing and migration assays were performed. The protein expression of epithelial-mesenchymal transition (EMT)-related molecules was determined by Western blot. RESULTS: Immunofluorescence staining showed that mortalin was located in the cytoplasm of SiHa cells. The positive rate and strongly positive rate of mortalin in the cervical squamous-cell carcinoma patients were 88.7% (55/62) and 61.3% (38/62), respectively, and they were significantly higher than those in normal cervical epithelial tissues (23.7% and 5.1%, P < 0.01). Additionally, mortalin expression was statistically correlated with the histological grade, clinical stage and lymph node metastasis. After inhibiting the expression of mortalin in the SiHa cells by MKT-077, the results of wound-healing and migration assays showed that the migration ability of SiHa cells was down-regulated. The protein expression of E-cadherin was up-regulated, and vimentin and Snail were significantly down-regulated. CONCLUSION: Mortalin over-expression is an effective biomarker for prediction of malignant potential and poor prognosis of cervical squamous-cell carcinoma.     

《中国蔬菜品种志》

本书由中国农业科学院蔬菜花卉研究所主编,已于2002年9月出版发行。全书分上、下卷,1～6章为上卷,包括根菜类、白菜类、芥菜类、甘蓝类、绿叶菜类及葱蒜类,计2263个品种,1347页;7～12章为下卷,包括瓜类、茄果类、豆类、薯芋类、水生蔬菜类和多年生蔬菜类,计2550个品种,1177页。入志的品种中,地方品种占     

Protein expression of EGFR and c-Kit is associated with primary tumor progression in nasopharyngeal carcinoma

AIM: To investigate the protein expression of 2 receptor tyrosine kinases,epidermal growth factor receptor(EGFR) and c-Kit, in non-keratinizing nasopharyngeal carcinoma (NPC) and to analyze the relationship of that with the clinicopathological parameters. METHODS: Ninety-five samples of stage II and III non-keratinizing NPC biopsies were collected from the Department of Pathology, Sun Yat-sen University Cancer Center. The 10% formalin-fixed paraffin-embedded biopsy blocks were re-sectioned. Besides HE routine staining, immunohistochemistry was performed for detecting the expression of EGFR, c-Kit, latent membrane protein 1(LMP1) and Ki-67. RESULTS: The protein expression rates of EGFR and c-Kit were 70.53% (67/95) and 63.16% (60/95), respectively. The expression score of EGFR was positively correlated with that of c-Kit protein (P<0.05). They were both correlated with T staging (EGFR,P<0.05; c-Kit, P<0.01). Furthermore, the staining intensities of EGFR and c-Kit proteins were also correlated with T staging(EGFR, P<0.05; c-Kit,P<0.01). CONCLUSION: The proteins of EGFR and c-Kit are usually expressed in non-keratinizing NPC cells. The immunoreactive scores of these 2 receptor tyrosine kinases are positively correlated with each other. Either the expression rate or the immunoreactive intensity of EGFR and c-Kit proteins is correlated with primary tumor progression. Immunohistochemical staining of EGFR or c-Kit protein in NPC biopsies could be recognized as an insight into further gene analysis for target therapy.     

Detection and clinical significance of circulating tumor cells in peripheral blood of nasopharyngeal cancer patients with modified immunomagnetic enrichment and fluorescent immunocytochemistry

AIM: To investigate the clinical significance of the new method of modified immunomagnetic enrichment of tumor cells in combination with fluorescent immunocytochemistry to detect circulating tumor cells (CTCs) in peripheral blood samples of patients with nasopharyngeal cancer (NPC). METHODS: Peripheral blood samples were obtained from 76 histology-proven patients with NPC before the initial therapy. After isolation of the mononuclear cells, the CTCs expressing cytokeratin (CK)8/18 in the blood samples were detected by the method of immunomagnetic enrichment in combination with fluorescent immunocytochemistry. The magnetic beads covalent binding with epithelial cell adhesion molecule (EpCAM) antibody were used to enrich the tumor cells which expressed EpCAM. After median following-up for 25 months, the effects of CTCs and other prognostic factors on patient prognosis were thoroughly investigated. RESULTS: None of the positive CK8/18 cells was detected in 20 normal blood samples. The CTCs were detected in 82.9% of the patients (P<0.01). Relapse patients had significantly higher number of median CK8/18⁺ CTCs than the patients without relapse (P<0.01). No association of viral capsid antigen (VCA)-IgA (P>0.05) was observed between the patients with and without relapse. Relapse-free survival rates were lower when the number of peripheral blood CK8/18⁺ CTCs was more than 3. The 2-year relapse-free survival rates were 100%, 100%, 100%, 94.1%, 71.4%, 53.3% and 44.4%(P<0.01) when the numbers of peripheral blood CK8/18⁺ CTCs were 0, 1, 2, 3, 4, 5, 6 before treatment, respectively. Overall survival rates were lower when the number of peripheral blood CK8/18⁺ CTCs was more than 5, but the difference was not significant. The 2-year overall survival rates were 100%, 100%, 100%, 100%, 100%, 100%, 80% and 77.8% (P>0.05) when the numbers of peripheral blood CK8/18⁺ CTCs were 0, 1, 2, 3, 4, 5, 6 before treatment,respectively. The VCA-IgA titer could not predict survivals. Cox multivariate analysis also showed the same results. CONCLUSION: Peripheral blood CK8/18⁺ CTCs are a prognostic factor for initial treatment of NPC.     

The expression of 53BP1 and 53BP2 gene in nasopharyngeal carcinoma

AIM:To investigate the expression map of two p53 binding proteins 53BP1 and 53BP2 in nasopharyngeal carcinoma (NPC) tissue. METHODS:The expression of 53BP1 and 53BP2 mRNA in NPC biopsy and control group are tested by RT-PCR. The expression of two mRNA in NPC paraffin section are examined by in situ hybridization. RESULTS:No expression of 53BP1 mRNA was found in NPC tissue and control group. However, expression of 53BP2 was detected in NPC biopsy and control group by RT-PCR, specific expressoin found cancerous nest in NPC paraffin section by in situ hybridization. CONCLUSION:The high expression of 53BP2 may be related to the development of NPC.     

Expression and clinical significance of Bmi-1 in gastric carcinoma

AIM: To investigate the relationship between expression of Bmi-1 (B cell-specific MLV integration site-1) in gastric cancer and its clinicopathologic significance.METHODS: 146 surgical patients with gastric carcinoma were followed up at least 2 years.Expression of Bmi-1 protein was examined by immunohistochemistry in their archival paraffin embedded tissue specimens.RESULTS: The intensive positive rate of Bmi-1 expression in gastric cancer was 67.8% (99/146).Expression of Bmi-1 was highly correlated with tumor size,clinical stage,lymph node metastasis and T classification (P<0.05),but not with sex,age,tumor differentiation,etc.(P>0.05).The survival rate in the patients with Bmi-1 expression was much lower than that in those patients without Bmi-1 expression (P<0.01).Multivariate analysis indicated that Bmi-1 expression,T classification,lymph node metastasis,distant metastasis,tumor size and postoperative chemotherapy were all significantly prognostic factors of gastric carcinoma.CONCLUSION: Overexpression of Bmi-1 in patients with gastric carcinoma enhances the possibility of invasion and metastasis,implying a poor prognosis.Bmi-1 may serve as fairly a good prognostic factor to indicate biologic behavior and prognosis in gastric carcinoma.     

Inhibitory effect of JIP on AP-1 activity induced by LMP1 in nasopharyngeal carcinoma cells and its mechanism

AIM: To investigate the mechanism of the AP-1 signal transduction pathway inhibited by JIP in nasopharyngeal carcinoma cells. METHODS: AP-1 activity was triggered by Dox-induced LMP1 expression in Tet-on-LMP1-HNE₂ cells (L7). The retention of phospho-JNK in the cytoplasm caused by JIP was examined with immunofluroscence assay. RESULTS: 24 h after transfection of L7 cells with the JIP expression plasmid, the translocation of activated JNK was inhibited, which resulted in the retention of phospho-JNK in the cytoplasm and down-regulation of the AP-1 activity. CONCLUSION: JIP down-regulates the activity of AP-1 through the inhibition of the translocation of JNK.     

Differences of genome-wide methylation level and methylated genes between nasopharyngeal carcinoma cells in same genetic background but different radiation resistance

AIM: To compare the differences of the genome-wide methylation levels and methylated regions between nasopharyngeal carcinoma (NPC) cells in the same genetic background but different radiation resistance (CNE-2 cells and CNE-2R cells). METHODS: Using the method which was developed by Doctor Zhao Cun-you, based on using methyl-sensitive restriction enzyme to measure the genome-wide methylation levels. In addition, MeDIP-Seq was used to analyze the methylated regions in 6 gene functional elements, including the upstream 2k sequence, 5'UTR, coding sequence, intron, 3'UTR and downstream 2k sequence, between CNE-2 cells and CNE-2R cells. RESULTS: The genome-wide methylation level was approximately 30% lower in CNE-2R cells than that in CNE-2 cells. No obvious difference on the amount of genes and the coverage of the peak in the 6 gene functional elements was observed. However, the methylation pattern of plentiful genes had altered in the gene function elements. CONCLUSION: The genome-wide methylation levels and methylated regions between NPC cells in the same genetic background but different radiation resistance were quite different, indicating that the DNA methylation may be associated with NPC radioresistance.     

Correlation between Mnk2 protein expression and prognosis of patients with esophageal squamous cell carcinoma

AIM: To investigate the protein expression of mitogen-activated protein kinase-interacting kinase-2 (Mnk2) and its prognostic effect in the patients with resected esophageal squamous cell carcinoma (ESCC). METHODS: A total of 86 informative patients with surgically resected ESCC and 54 normal esophageal tissues were enrolled. Western blot and immunohistochemistry (IHC) were utilized to assess the protein expression of Mnk2, and its correlation with prognosis was statistically analyzed by the methods of Kaplan-Meier curve and Cox proportional hazard mode. RESULTS: The protein expression of Mnk2 was elevated in most of tumor tissues compared with the adjacent tissues. Clinicopathologic analysis showed that Mnk2 expression was significantly correlated with the TNM stage (P<0.05). Both disease-free survival (DFS) and overall survival (OS) of Mnk2 over-expression patients were shorter than those in Mnk2 negative expression group. Multivariate analysis confirmed that Mnk2 expression, as an independent and significant factor for both DFS and OS, predicted a poor prognosis of the patients with resected ESCC (P<0.05). CONCLUSION: The expression of Mnk2 was significantly related to the TNM stages, and might be a novel predictor for prognosis in ESCC.     

TMT-tagged quantitative proteomics analysis of differentially expressed proteins and enrichment of signaling pathways in nasopharyngeal carcinoma cells with or without SATB1 over-expression

AIM: To analyze the effects of special AT-rich sequence binding protein 1 (SATB1) expression on the protein expression profiles in human nasopharyngeal carcinoma (NPC) cells, and to enrich the differential signaling pathways through bioinformatics analysis. METHODS: SATB1 over-expressing lentivirus and negative control lentivirus were used to infect the CNE1 cells, and then the cell lines were obtained by puromycin stressed method. The total proteins of the 2 cells were extracted, and the differentially expressed proteins were screened by TMT-labeled protein quantification technique and tandem mass spectrometry. The mRNA levels of the differential protein-coding genes were verified by RT-qPCR. GO analysis was used to annotate and enrich the differentially expressed proteins, and the KEGG database was used to enrich and analyze the signaling pathways of differential proteins. RESULTS: SATB1 over-expressing CNE1 cells were established through infected with associated lentivirus. Compared with the control group, 278 differentially expressed proteins were identified in SATB1 over-expressing CNE1 cells, in which 115 were up-regulated and 163 were down-regulated. 10 representative differential protein-coding genes were verified by RT-qPCR, which showed the consistence with the proteomic results. GO analysis indicated differentially expressed proteins were mainly involved in cellular processes, single-organism processes, biological regulation, metabolic processes, protein binding and catalysis. Cell components of differentially expressed proteins mainly existed in cell part, cells and organelles. KEGG analysis showed that differentially expressed proteins were involved in signaling pathways closely related to tumors, includeing MAPK, PI3K-Akt, AMPK, JAK-STAT, p53, PPAR, Hippo and HIF-1 signaling pathways. CONCLUSION: Over-expression of SATB1 significantly alters the protein expression profiles in the NPC cells and affects multiple signaling pathways closely related to tumors. Proteomics also provides a possible macro approach to the screening of molecular mechanisms, therapeutic and prognostic targets for NPC.