CONDITIONED MEDIUM OF HUMAN NASOPHARYNGEAL CARCINOMA EPITHELIOID CELL LINE CNE_(1) CONTAINED THE ACTIVITIES OF TRANSFORMED GROWTH-INHIBITING FACTORS

The hormone defined serum free conditioned medium (SFCM) of human nasopharyngeal carcinoma epithelioid cell line (CNE1) was assayed by both the 3H-thymidine incorporation test and the soft agar test. It was found that the SFCM stimulated the growth of long-term serum-free cultured CNE4 cells in ac-cordence with the fact that the growth rate of long-term serum-free cultured CNE1 cells was directly proportional to the plating density. Alternatively 5% SFCM inhibited the growth of short-term serum-free cultured CNE4 cells by 51% in which the indicator cell remained the responsiveness state of growing in the serum-supplemented medium to the effector of interest. Furthermore, SFCM resulted in the inhibition of anchorage-independent growth of CNE4 cells and A431 cells. Also in soft agar test. SFCM reduced the colony formation of NRK(?),9F cells in the presence of EGF or EGF plus TGF-β. These finding suggested that CNE4 secreted autocrine growth stimulating factor(s) and growth inhibiting factor(s) in the serum-free medium, the latter strongly reverse malignant phenotypes of CNE4 and A431 cells in serum-supplemented surrounding.

T cells expressing a LMP1-specific chimeric antigen receptor mediate antitumor effects against LMP1-positive nasopharyngeal carcinoma cells in vitro and in vivo

T cells modified with chimeric antigen receptor are an attractive strategy to treat Epstein-Barr virus（EBV） associated malignancies.The EBV latent membrane protein 1（LMP1） is a 66-KD integral membrane protein encoded by EBV that consists of transmembrane-spanning loops.Previously,we have identified a functional signal chain variable fragment（scFv） that specifically recognizes LMP1 through phage library screening.Here,we constructed a LMP1 specific chimeric antigen receptor containing anti-LMP1 scFv,the CD28 signalling domain,and the CD3ζchain（HELA/CAR）.We tested its functional ability to target LMP1 positive nasopharyngeal carcinoma cells.HELA/CAR cells were efficiently generated using lentivirus vector encoding the LMP1-specific chimeric antigen receptor to infect activated human CD3＋ T cells.The HELA/CAR T cells displayed LMP1 specific cytolytic action and produced IFN-γ and IL-2 in response to nasopharyngeal carcinoma cells overexpressing LMP1.To demonstrate in vivo anti-tumor activity,we tested the HELA/CAR T cells in a xenograft model using an LMP1 overexpressing tumor.Intratumoral injection of anti-LMP1 HELA/CAR-T cells significantly reduced tumor growth in vivo.These results show that targeting LMP1 using HELA/CAR cells could represent an alternative therapeutic approach for patients with EBV-positive cancers.

Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells

In order to study the mechanism of the effect of heparin on apoptosis in carcinoma cells, the nasopharyngeal carcinoma cell line CNE2 was used to identify the effect of heparin on apoptosis associated with the expression of c-myc, bax, bcl-2 proteins by use of Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), agarose gel electrophoresis, and flow cytometry, as well as Western blot analysis. The results showed that heparin induced apoptosis of CNE2 cells including the morphologic changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the 'ladder pattern' revealed by agarose gel electrophoresis of DNA in a concentration-dependent manner. The number of TUNEL-positive cells was dramatically increased to 33.6+/-1.2% from 2.8+/-0.3% by treatment with heparin in different concentrations (10 to approximately 40 kU/L). The apoptotic index was increased to 32.5% from 3.5% by detecting SubG1 peaks on flow cytometry. Western blot analysis showed that levels of bcl-2, bax and c-myc were significantly overexpressed by treatment with the increase of heparin concentrations. These results suggest that heparin induces apoptosis of CNE2 cells, which may be regulated by differential expression of apoptosis-related genes.

Inhibition of Pim-1 attenuates the proliferation and migration in nasopharyngeal carcinoma cells

Objective:To explore the role of proto-oncogene Pim-1 in the proliferation and migration of nasopharyngeal carcinoma(NPC) cells.Methods:Pim-1 expressions in NPC cell lines CNE1,CNE1-GL,CNE-2Z and C666-1 were examined by KT-PCR,western blotting and immunoflucesence,respectively.After CNE1,CNE1-GL and C666-1 cells were treated with different concentrations of Pim-1 special inhibitor,quercelagetin,the cell viability,colony formation rate and migration ability were analyzed.Results:Pim-1 expression was negative in well-differentiated CNE1 cells,whereas expressed weakly positive in poor-differentiated CNE-2Z cells and strongly positive in undifferentiated C666-1 cells.Interestingly,CNE1-GL cells that derived from CNE1 transfected with an Epstein Barr virus latent membrane protein-1 over-expression plasmid displayed stronger expression of Pim-1.Treatment of CNE1-GL and C666-1 cells with quercelagetin significantly decreased the cell viability,colony formation rate and migration ability but not the CNE1 cells.Conclusions:These findings suggest that Pim-1 overexpression contributes to NPC proliferation and migration,and targeting Pim-1 may be a potential treatment for anti-Pim-1-expressed NPCs.

EFFECTS OF EB VIRUS ENCODED LMP1 ON DIFFERENTIATION OF NASOPHARYNGEAL CARCINOMA CELLS

Objective: To investigate the effects of Epstein- Barr virus (EBV) encoded latent membrane protein 1 (LMP1) expression on tumor cell differentiation in early-stage nasopharyngeal carcinoma (NPC). Methods: Thirty-one biopsies of early-stage NPC were collected from the Cancer Center, Sun Yat-sen University. All 31 NPCs were of non-keratinizing carcinoma in histological type. The Epstein-Barr virus early RNAs (EBERs) were detected by use of DAKO PNA Probe (Y5200) and PNA ISH Detection Kit (K5201). The LMP1, a-catenin, b-catenin, g-catenin, high- molecular and low-molecular weight cytokeratins were detected by immunohistochemistry. Results: All 31 carcinoma biopsies studied showed a considerable number of EBERs-positive tumor cells, and 19 out of 31 cases (61.29%) expressed LMP1. The mean percentage of g-catenin expression in LMP1 positive group (53.25±34.12% ) was significantly lower than that (80.42±15.77%) in LMP1 negative group, (P<0.01). The g-catenin expression was positively correlated with the expression of low molecular weight cytokeratin (r=0.440, P<0.05). There were positive correlations for the expression of a-catenin, b-catenin and g-catenin in between. Conclusion: The LMP1 could down-regulate the expression of g-catenin and thus inhibit the tumor cell differentiation in early-stage NPC.

MiR-210 regulates cell cycle in nasopharyngealcarcinoma cell line (CNE-1) under hypoxic condition by reducing the expression of cyclin D1

Objective： The aim of our study was to determine the underlying mechanism of miR-210 on regulation of the cell cycle in nasopharyngeal carcinoma cell line CNE-1, particularly through regulation of cyclin D1, under hypoxic conditions. Methods： The CNE-1 cell line was induced with hypoxia, and the expression levels of endogenic miR-210 and cyclin D1 were detected by real-time PCR and Western blotting. Next, the luciferase assay was used to confirm that cyclin D1 is a target gene for miR-210. Cell cycle and cell proliferation were detected in CNE-1 cells that were cultured under hypoxic conditions with either overexpression or knockout of miR-210 using flow cytometry and MTT assay, respectively. Results： Hypoxia induced the expression of miR-210, resulting in reduced mRNA and protein levels of cyclin D1 and repression of cyclin D1 in CNE-1 cells. Further analysis indicated that miR-210 directly binded to the 3＇UTR of the cyclin D1 gene, thus regulated the expression of cyclin DI. The flow cytometry assay showed that, under hypoxic conditions, miR-210 blocked CNE-1 cells in the G1 phase, and miR-210 also inhibited the proliferation of CNE-1 cells. Conclusion： Under hypoxic conditions, miR-210 directly reduced the expression of cyclin D1, leading to CNE-1 cells blocked in G1 phase.

Expression of E-selectin, integrinβ_1 and immunoglobulin superfamily member in human gastric carcinoma cells and its clinicopathologic significance

AIM： To study the expression levels of E-selectin, integrin β1 and immunoglobulin supperfamily memberintercellular adhesion molecule-1 （ICAM-1） in human gastric carcinoma cells, and to explore the relationship between these three kinds of cell adhesion molecules and gastric carcinoma. METHODS： The serum contents of E-selectin, integrin β1 and ICAM-1 were detected by enzyme-linked immunosorbent assay （ELISA）, in 47 healthy individuals （control group） and in 57 patients with gastric carcinoma （gastric carcinoma group） respectively prior to operation and 7 d after operation. RESULTS： The serum E-selectin, ECAM-1 and integrin β1 were found to be expressed in both control and gastric carcinoma groups. However, they were highly expressed in patients with gastric carcinoma patients before operation or with unresectable tumours. The expression levels of ICAM-1 and integrin β1 were significantly higher in gastric carcinoma patients than in controls （P 〈 0.01）. A comparison of the E-selectin levels between the two groups showed statistically insignificant differnce （P = 0.64）. In addition, the expression levels were all decreased substantially in the postoperative patients subjected to radical resection of the tumours, indicating that the high level expressions of these compounds might be the important factor for predicting the prognosis of these patients. CONCLUSION： Serum E-selectin, ICAM-1 and integrin β1 expression levels are probably related to the metastasis and relapse of gastric cancer.

Effect of artesunate on human endometrial carcinoma HEC-1B cells

Objective： To observe the effect of the artesunate （ART） on cellular proliferation in vitro, to search for the possible anti-tumor mechanism of ART on endometrial carcinoma at the molecular level and to provide the experimental and theoretical foundations for the clinical applications of ART. Methods： The cell proliferation was observed by microscope; MTT was used to examine the effects of ART on proliferation of HEC-1B cells, and flow cytometric analysis was used to detect cell cycle and apoptosis. The human endometrial carcinoma HEC-1B cells were conventionally cultured; ART was administered with a concentration of 40 μg/ml before the total RNA were extracted, mRNA expression of Survivin, Caspase-3, N-Cadherin, E-Cadherin, Fibronectinl and Cox-2 were detected using RT-PCR. Results： ART reduced proliferation in human endometrial carcinoma cell line HEC-1B in a dose- and time-dependent effect. The cells of G0/G1 stage were significantly increased （P〈0.05）, but the cells of G2/M stages were significantly decreased （P〈0.05）, so it has shown that the cell cycle was probably blocked in G0/G1 stage. After intervention with ART at 20 and 80 μg/ml for 48 h, cellular apoptosis rate respectively was （36.42±0.77）% and （11.77±0.58）%, and the difference was statistically significant compared with the control （[6.64±0.191%, P〈0.01）. The expression of Cox-2 mRNA in the ART group was lower than those of control group, yet the expression of Caspase-3 and E-Cadherin mRNA in the ART group was higher than those of control group. Conclusion： ART can inhibit HEC-1B cell growth and proliferation in a dose- and time-dependent manner. Furthermore, ART can induce apoptosis in a dose-dependent manner. ART is able to downregulate Cox-2 mRNA expression and to upregulate E-Cadherin and Caspase-3 mRNA expression. So we can conclude that ART could induce the endometrial carcinoma HEC-1B cell apoptosis and inhibit tumor cell proliferation.

EFFECT OF MATRINE ON EXPRESSION OF HCCR1 AND HCCR2 PROTEINS IN CULTURAL HUMAN HEPATOCELLULAR CARCINOMAS CELLS

Objective： To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas （HCC） cells at the level of gene and protein. Methods： Three methods, representational difference analysis （RDA） of cDNA, microarrays and fluorescence in situ hybridization （FISH） were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results： Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion： Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.

LMP2A, AnnexinA2, Rad52 and Gli1 expression in nasopharyngeal carcinoma and their correlation with tumor malignancy

Objective:To study the LMP2A, AnnexinA2, Rad52 and Gli1 expression in nasopharyngeal carcinoma and their correlation with tumor malignancy.Methods:Nasopharyngeal cancer tissue confirmed by fibreoptic nasopharyngoscopic biopsy and mild chronic rhinitis mucosa inflammation tissue in Renmin Hospital of Wuhan University between May 2014 and February 2017 were selected to extract the RNA, and then fluorescence quantitative PCR kits were used to determine the expression of LMP2A, AnnexinA2, Rad52, Gli1, epithelial-mesenchymal transition genes and cell cycle genes.Results: LMP2A, AnnexinA2, Rad52 and Gli1 mRNA expression in nasopharyngeal cancer lesions were significantly higher than those in rhinitis mucosa inflammation lesions, and the higher the clinical stage, the higher the LMP2A, AnnexinA2, Rad52 and Gli1 mRNA expression in nasopharyngeal cancer lesions;E-cadherin mRNA expression in nasopharyngeal cancer lesions was significantly lower than that in rhinitis mucosa inflammation lesions and negatively correlated with LMP2A and Gli1 while N-cadherin, Vimentin and ZEB2 mRNA expression were significantly higher than those in rhinitis mucosa inflammation lesions and positively correlated with LMP2A and Gli1;CyclinD1, CyclinE and PCNA mRNA expression in nasopharyngeal cancer lesions were significantly higher than those in rhinitis mucosa inflammation lesions and positively correlated with with AnnexinA2 and Rad52.Conclusion:The high expression of LMP2A, AnnexinA2, Rad52 and Gli1 in nasopharyngeal carcinoma can promote epithelial-mesenchymal transition and cell cycle process in cancer cells.

循环肿瘤细胞PD-L1表达对鼻咽癌患者的预后价值评估

目的探讨循环肿瘤细胞(CTC)上细胞程序性死亡配体1(PD-L1)表达对鼻咽癌患者预后的意义。方法收集2021年6月10日—2023年7月10日经病理学确诊的鼻咽癌患者59例,检测患者外周血CTC和PD-L1的表达情况。分析CTC和PD-L1+CTC与鼻咽癌患者临床病理特征的相关性;评估CTC和PD-L1+CTC检测对鼻咽癌患者预后的临床诊断价值。结果59例患者的CTC检出率为76.3%(45/59),检出数量为(1.4±1.1)个。不同M分期,CTC检出数量的差别有统计学意义(P=0.026)。PD-L1+CTC的检出率为35.6%(21/59),检出数量为(0.4±0.7)个。鼻咽癌患者的2 a疾病进展率为8.5%(5/59),PD-L1+CTC患者相较于PD-L1-CTC患者具有更低的无进展生存率(PFS)和总生存率(OS),差别均有统计学意义(P=0.016,P=0.012)。Cox回归分析显示,PD-L1+CTC是鼻咽癌患者PFS(HR=9.244,95%CI:1.030~82.991,P=0.047)和OS(HR=190.642,95%CI:1.389~341.562,P=0.023)的预后影响因素。结论PD-L1+CTC是鼻咽癌患者PFS和OS的预后影响因素,CTC和PD-L1检测可能辅助鼻咽癌患者的预后评估。

Hugl-1 induces apoptosis in esophageal carcinoma cells both in vitro and in vivo

AIM: To determine whether the human giant larvae homolog 1 gene (Hugl-1/Llg1/Lgl1) exerts tumor suppressor effects in esophageal cancer. METHODS: We constructed a Hugl-1 expression plasmid, pEZ-M29-Hugl1, for gene transfection. We transfected the pEZ-M29-Hugl1 plasmid into Eca109 esophageal cancer cell lines with Lipofectamine 2000 to overexpress Hugl-1. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the effects of the plasmid on Hugl-1 expression. In vitro cell proliferation and apoptosis were examined separately by cell counting Kit-8 (CCK-8) assay, flow cytometry, and Western blotting before and after the transfection of the plasmid into Eca109 cells. Cell cycle distribution was assessed with flow cytometry. The effect of Hugl-1 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice. Expression of Hugl-1 in xenograft tumor was analyzed by immunohistochemistry.The transferase-mediated dUTP nick end-labeling (TUNEL) technique was performed to detect and quantitate apoptotic cell. RESULTS: The transfection efficiency was confirmed with real-time RT-PCR and Western blotting. Our results show that compared with control groups the mRNA levels and protein levels of Hugl-1 in pEZ-M29-Hugl1-treated group were remarkably increased (P < 0.05). The CCK-8 assay demonstrated that the growth of cells overexpressing Hugl-1 was significantly lower than control cells. Cell cycle distribution showed there was a G 0 /G 1 cell cycle arrest in cells overexpressing Hugl-1 (64.09% ± 3.14% vs 50.32% ± 4.60%, 64.09% ± 3.14% vs 49.13% ± 2.24%). Annexin V-fluorescein isothiocyanate revealed that apoptosis was significantly increased in cells overexpressing Hugl-1 compared with control group (17.33% ± 4.76% vs 6.90% ± 1.61%, 17.33% ± 4.76% vs 6.27% ± 0.38%). Moreover, we found that Hugl-1 changes the level of the anti-apoptotic protein Bcl-2 and the proapoptotic protein Bax and the activation of both caspase-3 and caspase-9. With a TUNEL assay, we found that Hugl-1 markedly increased the apoptosis rate of Eca109 cells in vivo (60.50% ± 9.11% vs 25.00% ± 12.25%). It was shown that Hugl-1 represents a significantly more effective tumor suppressor gene alone in a xenograft tumor mouse model. This data suggest that Hugl-1 inhibited tumor growth and induced cell apoptosis in vivo . CONCLUSION: These results suggest that Hugl-1 induces growth suppression and apoptosis in a human esophageal squamous cell carcinoma cell line both in vitro and in vivo .

Transcription factor EGR-1 inhibits growth of hepatocellular carcinoma and esophageal carcinoma cell lines

AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. CONCLUSION: The decreased expression of egr-1 might play a role in the dysregulation of normal growth in the cancerous process of HCC and EC. Egr-1 gene of transfected HHCC and ECa109 cells showed obvious suppression of the cell growth and malignant phenotypes, but no suppression in SMMC7721 (HCC cell line) cells.

GSTM1,GSTT1,GSTP1 and CYP1A1 genetic polymorphisms and susceptibility to esophageal cancer in a French population:Different pattern of squamous cell carcinoma and adenocarcinoma

AIM:To evaluate the association between CYP1A1 and GSTs genetic polymorphisms and susceptibility to esophageal squamous cell carcinoma(SCC)and esophageal adenocarcinoma(ADC)in a high risk area of northwest of France. METHODS:A case-control study was conducted to investigate the genetic polymorphisms of these enzymes (CYPIAI*2C and GSTP1 exon 7 Val alleles,GSTMI*2/*2 and GSTTl *2/*2 null genotypes).A total of 79 esophageal cancer cases and 130 controls were recruited. RESULTS:GSTMI*2/*2 and CYPIAI*IA/*2C genotype frequencies were higher among squamous cell carcinomas at a level dose to statistical significance(OR =1.83,95% CI 0.88-3.83,P=0.11;OR=3.03,95% CI 0.93-9.90,P=0.07, respectively).For GSTP1 polymorphism,no difference was found between controls and cases,whatever their histological status.Lower frequency of GSTT1 deletion was observed in ADC group compared to controls with a statistically significant difference(OR=13.31,95% CI 1.66-106.92,P<0.01). CONCLUSION:In SCC,our results are consistent with the strong association of this kind of tumour with tobacco exposure.In ADC,our results suggest 3 distinct hypotheses: (1)activation of exogenous procarcinogens,such as small halogenated compounds by GSTT1;(2)contribution of GSTT1 to the inflammatory response of esophageal mucosa,which is known to be a strong risk factor for ADC, possibly through leukotriene synthesis;(3)higher sensitivity to the inflammatory process associated with intracellular depletion of glutathione.

顺铂诱导TNF-α自分泌引发头颈部鳞状癌细胞RIP1/RIP3/MLKL的坏死性凋亡

目的探讨顺铂是否能够诱导头颈部鳞状癌细胞产生TNF-α,进而激活RIP1/RIP3/MLKL依赖性的坏死性凋亡通路,抑制鳞癌细胞的增殖,并探讨其分子机制。方法选取头颈部鳞状癌细胞系HN4和SCC4作为实验对象,分为对照组、顺铂组、caspases抑制组、坏死性凋亡抑制组。利用CCK-8法检测顺铂刺激后24 h的细胞存活率。随后采用Western blotting检测caspase-8以及坏死性凋亡通路蛋白(RIP1/RIP3/MLKL)和NF-κB(p65)、TNF-α表达情况;结合细胞划痕实验和Western blotting检测上皮间充质转化相关蛋白(N-cadherin、Vimentin、E-cadherin)的表达情况,评估坏死性凋亡对头颈部鳞状癌细胞迁移能力的影响。结果顺铂对HN4、SCC4细胞毒性IC_(50)分别约为10μg/mL、15μg/mL。在顺铂刺激下,与坏死性凋亡抑制剂组相比,caspase-8表达降低(P<0.05),N-cadherin、Vimentin表达降低(P<0.05)、E-cadherin表达升高(P<0.05),坏死性凋亡通路蛋白(RIP1/RIP3/MLKL)表达升高(P<0.05),TNF-α和NF-κB(p65)蛋白表达均升高(P<0.05)。在顺铂组中,细胞愈合率显著降低于对照组和坏死性凋亡抑制剂组。结论顺铂作用可激活头颈部鳞状癌细胞NF-κB信号通路,并促进TNF-α自分泌,从而引发鳞癌细胞经由RIP1/RIP3/MLKL通路依赖性的坏死性凋亡反应,并抑制肿瘤细胞性增殖。

Role of JNK signaling pathway in sensitivity to radiotherapy of nasopharyngeal carcinoma

Objective:The aim of the study was to investigate the effect of c-Jun N-terminal protein kinase(JNK) signaling pathway on influencing the sensitivity to radiotherapy of human nasopharyngeal carcinoma CNE cells.Methods:Human nasopharyngeal carcinoma CNE multicellular spheroids(MCS) were constructed with three dimensional cell culture methods.Western blot was employed to analyze the activity of JNK signaling pathway in MCS after X-ray irradiation,and the expression of caspase-3 protein before and after using SP600125(a special inhibitor of JNK).X-ray induced cell apoptosis in MCS before and after treated with SP600125 were detected by TUNEL.Results:The level of JNK phosphorylation in MCS was a dynamic course after radiation,and there was a phosphorylation peaks at 2 h later,the apoptotic rate of MCS(P < 0.05) and the expression of caspase-3 protein(P < 0.05) were significantly increased after treated with SP600125.Conclusion:The transient activation of JNK played a important role in sensitivity to radiotherapy of CNE MCS via mediating survival signals,blocking this pathway accelerate cell apoptosis,which may be related to the increased expression of caspase-3.

Expression of PinX1 and hTERT in basal cell carcinoma and their implications

Objective This study aimed to investigate the expression and significance of PIN2/TERF1 interacting, telomerase inhibitor 1(Pin X1) and human telomerase reverse transcriptase(h TERT) in basal cell carcinoma(BCC). Methods Real-time polymerase chain reaction and immunohistochemistry were performed to quantify the m RNA expressions and integrated optical density(IOD), respectively, of Pin X1 and h TERT in BCC specimens(n = 30), as well as in normal skin specimens(n = 15). Results The m RNA expression level and IOD of Pin X1 in the BCC samples were both significantly lower than those in the control specimens(P < 0.05). Conversely, the m RNA expression level and IOD of h TERT in BCC were both significantly higher than that in the control samples(P < 0.05). The correlation between the expression levels of Pin X1 and h TERT showed no statistical significance(P > 0.05). Conclusion Downregulation of Pin X1 and upregulation of h TERT expression may be associated with the activation and maintenance of telomerases in the induction of BCC.

LMP-1、Bcl-2表达与鼻咽癌侵袭转移的相关性

目的分析鼻咽癌组织中潜伏膜蛋白(LMP)-1、B淋巴细胞瘤(Bcl)-2基因与其侵袭转移的关系。方法选择120例NPC患者进行研究,使用鼻内镜采集病灶组织,采用免疫组织化学法检测组织中LMP-1、Bcl-2的表达,根据TNM分期评估NPC肿瘤侵袭和转移程度,分析LMP-1、Bcl-2与NPC侵袭和转移的关系。结果120例NPC患者中LMP-1阳性共86例,占71.67%,Bcl-2阳性共90例,占75.00%;LMP-1、Bcl-2阳性患者中EBV-DNA阳性占比居高,与LMP-1、Bcl-2阴性者相比,差异有统计学意义(P<0.05)。TNM不同分期患者LMP-1、Bcl-2阳性表达率相比较,差异有统计学意义(P<0.05)。采用卡方Phi和Cramer's V系数检验发现,NPC组织中LMP-1、Bcl-2表达与肿瘤侵袭和转移均显著相关(P<0.05)。结论LMP-1、Bcl-2在NPC组织中阳性表达率普遍较高,且LMP-1、Bcl-2阳性表达与肿瘤侵袭和转移有关。

Effects of biological clock gene BMAL1 and hypoxia-inducible factor HIF-1αon proliferation,migration and radiotherapy sensitivity of nasopharyngeal carcinoma cells HONE1

Objective To understand the effects of clock gene BMAL1 and HIF-1α(Hypoxia inducible factor-1α)on proliferation,migration and sensitivity to radiotherapy of nasopharyngeal carcinoma cells HONE1.At the same time,whether the biological clock gene BMAL1 can affect the expression of HIF-1αprotein was investigated.It will lay the foundation for further study on the correlation between clock gene BMAL1 and HIF pathway.Methods BMAL1 gene overexpression and interference lentivirus and HIF-1αgene interference lentivirus were constructed respectively,and were transfected into nasopharyngeal carcinoma cells HONE1.Western blot was used to verify the establishment of overexpressed and knockdown BMAL1 cell lines and HIF-1αgene knockdown cell line,and to investigate the expression of HIF-1αprotein in overexpressed and knockdown BMAL1 cell lines.CCK-8 cell proliferation test and scratch test were used to analyze the proliferation and migration ability of cells.Cell apoptosis after radiotherapy was analyzed by flow cytometry.The effects of BMAL1 and HIF-1αon the sensitivity of HONE1 radiotherapy in nasopharyngeal carcinoma cells after X-ray irradiation at different doses(0Gy,2Gy,4Gy,6Gy)were detected by clone formation assay.Results The overexpression of BMAL1 gene and lentivirus interference were constructed to effectively up regulate and down regulate the expression of BMAL1 protein in nasopharyngeal carcinoma cells HONE1.Meanwhile,HIF-1αgene interference lentivirus was constructed to effectively down-regulate the expression of HIF-1αprotein in nasopharyngeal carcinoma cell line HONE1,and successfully screen out stable nasopharyngeal carcinoma cell lines.Western blot results showed that overexpression of BMAL1 gene could inhibit the expression of HIF-1αprotein in HONE1 of nasopharyngeal carcinoma cells,while knockdown of BMAL1 gene promoted the expression of HIF-1αprotein in HONE1 of nasopharyngeal carcinoma cells(P<0.05).CCK-8 cell proliferation and scratch test showed that overexpression of BMAL1 gene or knockdown of HIF-1αgene could inhibit the proliferation and migration of HONE1 cells(P<0.05).Flow cytometry results showed that after 8Gy irradiation for 72 h,the apoptosis rate of BMALl gene overexpression group was higher than that of the overexpression control group,similarly,the apoptosis rate of HIF-1αgene knockdown group was higher than that of the knockdown control group(P<0.05).After X-ray irradiation at different doses(0Gy,2Gy,4Gy,6Gy),clon-formation experiment showed that the clon-formation rate and cell survival fraction of BMALl overexpression group or HIF-1αknockdown group were lower than those of negative control group(P<0.05).Sigmaplot analysis showed that the D0,Dq and SF2 of the BMAL1 overexpression group or HIF-1αknockdown group were lower than those of the negative control group,and the radiosensitization ratios were 1.381 and 1.063,respectively.Conclusion Overexpression of BMAL1 gene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1,increase apoptosis after radiotherapy and improve radiosensitivity.Knock down HIF-1αGene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1,increase apoptosis after radiotherapy and improve radiosensitivity.In nasopharyngeal carcinoma cells HONE1,overexpression of BMAL1 gene can inhibit the expression of HIF-1αprotein while knockdown of BMAL1 gene can promote the expression of HIF-1αprotein.

lncRNA MSC-AS1通过调节miR-429/RhoA轴对人鼻咽癌细胞增殖、凋亡、迁移和侵袭的影响

目的探索长链非编码RNA(lncRNA)MSC-AS1通过调节微小RNA(miR)-429/Ras同源基因家族成员A(RhoA)轴对人鼻咽癌(NPC)细胞增殖、凋亡、侵袭和迁移的影响。方法实时荧光定量PCR法(qPCR)检测NPC细胞系(CNE-1、HNE2、HONE-1细胞)以及人鼻咽上皮细胞系NP69细胞中lncRNA MSC-AS1、miR-429、RhoA mRNA的表达水平。以CNE-1细胞为研究对象,将si-MSC-AS1、miR-429 inhibitor、miR-429 mimics及相应阴性对照分别转染或共转染CNE-1细胞,记为对照组(未转染)、si-NC组、si-MSC-AS1组、mimics NC组、miR-429 mimics组、si-MSC-AS1+inhibitor NC组、si-MSC-AS1+miR-429 inhibitor组。MTT法、流式细胞术、Transwell分别检测细胞增殖、凋亡、迁移和侵袭能力;双荧光素酶实验分别验证miR-429与MSC-AS1、RhoA的靶向关系;免疫印迹法检测RhoA、B淋巴细胞因子相关x蛋白(Bax)、上皮细胞钙黏蛋白(E-cadherin)、细胞周期素D1(CyclinD1)、神经钙黏蛋白(N-cadherin)的表达水平。结果miR-429分别与MSC-AS1、RhoA存在靶向关系。与NP69细胞相比,CNE-1、HNE2、HONE-1细胞中MSC-AS1、RhoA mRNA的表达水平显著增加,miR-429表达水平显著降低,差异有统计学意义(P<0.05)。与对照组、si-NC组相比,si-MSC-AS1组细胞增殖率、迁移和侵袭数、MSC-AS1、N-cadherin、RhoA、CyclinD1比较表达水平显著下降,细胞凋亡率、E-cadherin、Bax显著增加,差异有统计学意义(P<0.05);与对照组、mimics NC组相比,miR-429 mimics组细胞增殖率、迁移和侵袭数、N-cadherin、RhoA、CyclinD1表达水平显著下降,细胞凋亡率、miR-429、E-cadherin、Bax显著增加,差异有统计学意义(P<0.05);抑制miR-429表达水平可减弱干扰MSC-AS1对细胞恶性生物学行为的影响(P<0.05)。结论干扰MSC-AS1可以抑制NPC细胞增殖、侵袭及迁移,促进其凋亡,可能与miR-429/RhoA轴有关。