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.

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.

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 treat ment with heparin in different concentrations (10～40 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.

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, integrinβ1 and immunoglobulin supperfamily member-intercellular 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, integrinβ1 and ICAM-1 were detected by enzyme-linked immuno-sorbent 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 integrinβ1 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 integrinβ1 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 pi expression levels are probably related to the metastasis and relapse of gastric cancer.

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

瞄准：抄写因素 EGR-1 (早生长反应 gene-1 ) 在房间生长，区别和开发起一个重要作用。它鉴别了 EGR-1 在一些瘤举办重要转变抑制活动，例如纤维肉瘤，胸癌。这个实验被设计在 hepatocellular 癌(HCC ) 和食道的癌(EC ) 的癌的过程调查 egr-1 的角色，然后在这些肿瘤细胞的生长上估价 EGR-1 的效果。方法：第一，在 HCC 和 EC 的 egr-1 的抄写和表达式，帕拉癌的纸巾和他们的正常对应物部分被检测由在 situ 杂交和免疫组织化学，与正常的人的胸和是的鼠标大脑纸巾积极控制。Egr-1 基因当时是进 HCC 的 transfected (HHCC， SMMC7721 ) 并且没有 egr-1 抄写和表示是在场的 EC (ECa109 ) 房间在线。在裸体老鼠的房间生长速度， FCM 房间周期，板克隆形成和 tumorigenicity 被观察，控制仅仅是有向量的房间线 transfected。结果：很少或没有 egr-1 抄写和表示在 HCC， EC 和正常的肝纸巾被检测。egr-1 的表示在 hepatocellular 帕拉被发现更高癌的织物(抄写水平 P=0.000；表示水平 P=0.143，可能因为在盒子的数字的少数) 并且食道的癌症的 dysplastic 织物(抄写水平 P=0.000；表示水平 P=0.001 ) 。egr-1-transfected HHCC (HCC 细胞线) 的生长率细胞和 ECa109 (EC 细胞线) 细胞比控制的慢得多。S 阶段房间，克隆形成和 tumorigenicity 的比例控制的是比这些显著地低的(减少 45.5% 在 HHCC 房间并且 34.1% 在 ECa109 房间；46.6% 和 41.8% ；80.4% 和 72.6% 分别地) 。关于上述项目 SMMC7721 (HCC ) egr-1-transfected 房间和控制之间没有明显的差别。结论：egr-1 的减少的表示可能在 HCC 和 EC 的癌的过程在正常生长的 dysregulation 起一个作用。transfected HHCC 和 ECa109 细胞的 Egr-1 基因显示出细胞生长和在 SMMC7721 (HCC 细胞线) 的恶意的显型，而是没有抑制细胞的明显的抑制。

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.

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。

基于Met/PI3K/Akt信号通路研究五味子甲素对人鼻咽癌细胞HONE-1增殖、迁移和侵袭的影响

目的:研究五味子甲素对人鼻咽癌细胞HONE-1增殖、迁移和侵袭的影响及其可能机制。方法:以HONE-1细胞为研究模型,使用不同浓度[0(空白对照)、10、20、40μmol/L]的五味子甲素处理后,分别采用CCK-8试验、划痕试验和Transwell小室试验检测细胞的增殖、迁移和侵袭能力变化;通过计算机分子对接分析五味子甲素与酪氨酸蛋白激酶(Met)蛋白的结合能力;采用Western blotting法检测细胞中磷酸化酪氨酸蛋白激酶(p-Met)、磷酸化磷脂酰肌醇-3-激酶(p-PI3K)、磷酸化蛋白激酶B(p-Akt)、B淋巴细胞瘤2(Bcl-2)和N-钙黏蛋白(N-cadherin)的相对表达量。结果:与空白对照比较,10、20、40μmol/L五味子甲素处理后细胞的增殖、迁移和侵袭能力均显著减弱(P<0.05);分子对接结果显示,五味子甲素能与Met蛋白的活性口袋稳定结合;Western blotting试验结果显示,与空白对照比较,10、20、40μmol/L五味子甲素处理后细胞中p-Met、p-PI3K、p-Akt、Bcl-2和N-cadherin蛋白的相对表达量均显著降低(P<0.05)。结论:五味子甲素可通过抑制Met/PI3K/Akt信号通路的活化来抑制HONE-1细胞的增殖、迁移和侵袭。

Clinical significance of HBME-1,Galectin-3,and CK19 expression and the status of BRAF mutation in papillary thyroid carcinoma

Objective The aim of this study was to explore the clinical significance of the expression of proteins human bone marrow endothelial cell markers(HBME-1), Galectin-3, and cytokeratin19(CK19), as well as the status of v-raf murine sarcoma viral oncogene homolog B1(BRAF) mutation in papillary thyroid carcinoma(PTC). Methods Immunohistochemical staining was performed in 82 specimens each of PTC and papillary benign lesions to detect the expression of HBME-1, Galectin-3, and CK19. Polymerase chain reaction(PCR) and gene sequencing were performed on 60 specimens each of PTC and papillary benign lesions to detect the status of BRAF mutation. Results The positive expression ratios of HBME-1, Galectin-3, and CK19 in PTC were 98.8%, 97.6% and 100% respectively, which were significantly higher than the expressions in papillary benign lesions(P < 0.05). No significant relationship was observed between the expression of these makers and the clinicopathological features of PTC. The sensitivity of co-expression of HBME-1 and CK19 or HBME-1 and Galectin-3 as diagnostic criteria of PTC was 99.9%, with a specificity of 95.4%. BRAF mutation was detected in 40 of 60 PTC(66.7%) specimens. There was a statistical difference in BRAF mutations between PTC and papillary benign lesions(P < 0.05); there were no associations between BRAF mutation and the clinicopathological features of PTC. Conclusion Combined immunohistochemical staining of HBME-1, Galectin-3, and CK19 can further improve the sensitivity and specificity of differential diagnosis of PTC. BRAF mutation is a significant genetic event, which may have diagnostic value for PTC.

Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated cytotoxicity

A major problem which is poorly understood in the management of bladder cancer is low sensitivity to chemotherapy and high recurrence after transurethral resection. Insulin-like growth factor 1 receptor (IGF-1R) signaling plays a very important role in progression, invasion and metastasis of bladder cancer cells. In this study, we investigated whether IGF-1R was involved in the growth stimulating activity and drug resistance of bladder cancer cells. The results showed: The mRNAs of IGF-1, IGF-2 and IGF-1R were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace leve1s; T24 cell responded far better to growth stimulation by IGF-1 than did normal urothelial cells; blockage of IGF1R by antisense oligodeoxynucleotide (ODN) significantly inhibited the growth of T24 cell and enhanced sensitivity and apoptosis of T24 cells to mitomycin (MMC). These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy.

血清SCC-Ag、VEGF、UHRF1水平联合预测食管鳞癌根治术后复发的价值

目的探讨血清人鳞状细胞癌相关抗原(SCC-Ag)、血管内皮生长因子(VEGF)、环指域1(UHRF1)水平联合预测食管鳞癌根治术后复发的价值。方法回顾性分析2017年5月至2019年6月因食管鳞癌于郑州大学第一附属医院接受根治术治疗的116例患者的临床资料。术前均检测血清SCC-Ag、VEGF、UHRF1水平,比较不同临床病理特征患者各血清指标。术后均随访3 a,根据是否复发将患者分为复发组(56例)和未复发组(60例),并比较两组血清SCC-Ag、VEGF、UHRF1水平和临床病理特征。采用Cox回归模型分析食管鳞癌根治术后复发的危险因素,并绘制受试者工作特征(ROC)曲线分析血清SCC-Ag、VEGF、UHRF1水平联合对食管鳞癌根治术后复发的预测价值。结果最大肿瘤直径>5 cm、有淋巴结转移、TNM分期Ⅲ期、低分化程度患者血清SCC-Ag、VEGF、UHRF1水平均高于最大肿瘤直径≤5 cm、无淋巴结转移、TNM分期Ⅰ~Ⅱ期、高/中分化程度患者(P<0.05);复发组血清SCC-Ag、VEGF、UHRF1水平和最大肿瘤直径>5 cm、有淋巴结转移、TNM分期Ⅲ期、低分化程度占比均高于未复发组(P<0.05);Cox回归模型分析结果显示,在未校正因素时,血清SCC-Ag、VEGF、UHRF1水平均是食管鳞癌患者根治术后3 a内复发的危险因素(RR=2.978、3.031、3.126,P<0.05),校正性别、年龄、肿瘤部位、最大肿瘤直径、淋巴结转移、TNM分期、分化程度后仍是术后3 a内复发的危险因素(RR=1.939、2.112、2.235,P<0.05);ROC曲线分析结果显示,血清SCC-Ag、VEGF、UHRF1水平联合预测食管鳞癌根治术后复发的灵敏度和曲线下面积(AUC)分别为94.64%、0.919,均高于各指标单独预测(P<0.05),其特异度与各指标单独预测比较差异无统计学意义(P>0.05)。结论血清SCC-Ag、VEGF、UHRF1水平与食管鳞癌患者临床病理特征和术后复发均具有一定的相关性,对术后复发均具有预测价值,但三者联合预测价值更高。

杨梅素调节JAK-STAT-IRF1信号通路对鼻咽癌细胞免疫逃逸的影响

目的探讨杨梅素对鼻咽癌细胞免疫逃逸的影响及对JAK-STAT-IRF1信号通路的调节作用。方法体外实验:培养人鼻咽癌CNE1细胞并分为对照组、杨梅素L组、杨梅素M组、杨梅素H组和杨梅素H+Broussonin E组,MTT法检测细胞增殖能力;Hoechst法检细胞凋亡;蛋白免疫印迹(Western blot)技术验证细胞叉头样转录因子3(Foxp3)、维甲酸相关孤核受体γt(RORγt)、磷酸化(p)-Janus激酶(JAK)1、JAK1、p-JAK2、JAK2、p-信号转导与转录激活子(STAT)1、STAT1、干扰素调节因子1(IRF1)蛋白表达。体内实验:建立BALB/c裸鼠鼻咽癌模型,随机分为模型组、杨梅素低剂量组、杨梅素中剂量组、杨梅素高剂量组和紫杉醇组,取瘤体并称重,流式细胞术检测巨噬细胞程序性死亡受体1配体(PD-L1)表达,Western blot法检测瘤体Foxp3、RORγt、p-JAK1、JAK1、p-JAK2、JAK2、p-STAT1、STAT1、IRF1蛋白表达。结果与对照组相比,杨梅素L、M、H组CNE1细胞增殖能力以及RORγt、p-JAK1/JAK1、p-JAK2/JAK2、p-STAT1/STAT1、IRF1蛋白表达降低,细胞凋亡率和Foxp3蛋白表达增加(P<0.05);与杨梅素H组相比,杨梅素H+Broussonin E组细胞增殖能力以及RORγt、p-JAK1/JAK1、p-JAK2/JAK2、p-STAT1/STAT1、IRF1蛋白表达升高,细胞凋亡率和Foxp3蛋白表达减少(P<0.05);与模型组对比,杨梅素低、中、高剂量组Foxp3蛋白表达增加,瘤体质量以及PD-L1、RORγt、p-JAK1/JAK1、p-JAK2/JAK2、p-STAT1/STAT1、IRF1蛋白表达减少(P<0.05)。结论杨梅素可能通过抑制JAK-STAT-IRF1信号通路的激活抑制鼻咽癌细胞免疫逃逸。

丝切蛋白-1、磷酸化丝切蛋白-1、CD11c在鼻咽癌组织中的表达及临床意义

目的探讨丝切蛋白1(Cofilin-1)、磷酸化丝切蛋白1(p-Cofilin-1)及树突状细胞(DCs)亚群在鼻咽癌组织中的表达及与预后的关系。方法回顾性分析2011年1月至2015年12月97例鼻咽癌患者的临床资料,采用免疫组化法检测鼻咽癌组织中Cofilin-1、p-Cofilin-1及CD11c+DCs的表达情况。分析三者表达与鼻咽癌临床病理特征和预后的关系。结果97例鼻咽癌患者中,Cofilin-1低表达率为26.8%(26/97),高表达率为73.2%(71/97);p-Cofilin-1低表达率为27.8%(27/97),高表达率为72.2%(70/97)。CD11c低表达率为80.4%(78/97),高表达率为19.6%(19/97)。M分期、临床分期、治疗后转移、治疗前骨转移和治疗后出现骨转移与Cofilin-1的表达有关(P<0.05);治疗后转移和治疗后出现骨转移与p-Cofilin-1的表达有关(P<0.05);CD11c表达与鼻咽癌临床病理参数均无关(P>0.05)。Cofilin-1低表达组和高表达组1、3、5年生存率分别为84.6%、73.1%、73.1%和93.0%、61.7%、46.7%(P=0.094);低表达组和高表达组1、3、5年无远处转移生存率分别为100.0%、100.0%、100.0%和87.2%、73.3%、68.6%(P=0.002)。p-Cofilin-1低表达组和高表达组1、3、5年生存率分别为85.2%、51.9%、40.4%和92.9%、72.7%、59.3%(P=0.075);低表达组和高表达组1、3、5年无远处转移生存率分别为83.5%、53.7%、53.7%和95.1%、89.9%、85.5%(P=0.001)。CD11c低表达组和高表达组1、3、5年生存率分别为89.7%、61.3%、46.5%和94.7%、89.5%、89.5%(P=0.007);低表达组和高表达组1、3、5年无远处转移生存率分别为88.8%、80.2%、76.1%和100.0%、93.8%、87.1%(P=0.175)。结论Cofilin-1表达水平越高,肿瘤转移风险越高,无远处转移生存时间越短;p-Cofilin-1表达水平越高,肿瘤发生转移的风险越低,无远处转移生存时间越长;CD11c表达水平越高,总生存时间越长。Cofilin-1、p-Cofilin-1、CD11有望成为鼻咽癌靶向治疗和预后预测的标志物。

敲低lncRNA LINC01296调节PD-L1抑制鼻咽癌细胞免疫逃逸的机制研究

目的研究敲低长链非编码RNA(lncRNA)LINC01296调节程序性死亡配体-1(PD-L1)抑制鼻咽癌细胞免疫逃逸的机制。方法分离培养人外周血淋巴细胞后与人鼻咽癌细胞CNE-2Z共培养,同时取C57BL/6小鼠皮下注射CNE-2Z细胞构建鼻咽癌移植瘤模型24只,均随机分为对照组、lncRNA LINC01296敲低组[转染lncRNA LINC01296小干扰RNA(siRNA)]、阴性对照组(转染lncRNA LINC01296 siRNA阴性对照和空载质粒)、lncRNA LINC01296敲低+PD-L1过表达组(转染lncRNA LINC01296 siRNA和PD-L1过表达质粒),分组转染后,流式细胞术检测人外周血淋巴细胞中活化CD8^(+)T细胞比例;细胞计数试剂盒-8法检测人外周血淋巴细胞对CNE-2Z细胞杀伤率;测量移植瘤小鼠肿瘤体积;免疫荧光染色检测移植瘤小鼠肿瘤组织CD8和PD-L1阳性表达;实时荧光PCR实验检测CNE-2Z细胞和肿瘤组织lncRNA LINC01296、PD-L1信使RNA(mRNA)表达;蛋白印迹实验检测CNE-2Z细胞和肿瘤组织PD-L1蛋白表达。结果与对照组相比,lncRNA LINC01296敲低组人外周血淋巴细胞中活化CD8^(+)T细胞比例及对CNE-2Z细胞杀伤率、肿瘤组织CD8阳性比例升高(P<0.05),肿瘤体积、CNE-2Z细胞和肿瘤组织PD-L1阳性比例、CD8和PD-L1双阳性比例、lncRNA LINC01296、PD-L1 mRNA和蛋白表达降低(P<0.05);阴性对照组各指标无明显变化(P>0.05);过表达PD-L1可减弱敲低lncRNA LINC01296对CNE-2Z细胞和小鼠各指标的影响。结论敲低ln⁃cRNA LINC01296可通过下调PD-L1而促进CD8^(+)T细胞活化及浸润,减弱鼻咽癌细胞免疫逃逸,增强CD8^(+)T细胞对其杀伤力。