Expression profile of epithelial-mesenchymal transition markers in non–muscle-invasive urothelial carcinoma of the bladder: Correlation with intravesical recurrence following transurethral resection

ObjectivesTo evaluate the expression of molecular markers involved in epithelial-mesenchymal transition (EMT), a key process mediating the progression of malignant tumors, in non–muscle-invasive urothelial carcinoma of the bladder (NMIUCB) to clarify the significance of these markers as predictors of intravesical recurrence in patients treated with transurethral resection (TUR).Materials and methodsExpression levels of 13 EMT markers, including E-cadherin, N-cadherin, β-catenin, γ-catenin, fibronectin, matrix metalloproteinase (MMP)-2, MMP-9, Slug, Snail, TWIST, vimentin, ZEB1, and ZEB2, in TUR specimens obtained from 161 consecutive patients with NMIUCB were measured by immunohistochemical staining.ResultsOf these 13 markers, significant differences in the incidence of intravesical recurrence were noted according to expression levels of E-cadherin, N-cadherin, MMP-2, MMP-9, and TWIST. Univariate analysis also identified expression levels of E-cadherin, N-cadherin, MMP-2, MMP-9 and TWIST, in addition to the tumor size, pathological T category, and concomitant carcinoma in situ, as significant predictors of intravesical recurrence-free survival. Of these significant factors, expression levels of E-cadherin, MMP-9, and TWIST; tumor size; and concomitant carcinoma in situ appeared to be independently associated with intravesical recurrence-free survival on multivariate analysis. Furthermore, there were significant differences in recurrence-free survival according to positive numbers of these 5 independent risk factors (i.e., positive for 0 or 1 factor vs. positive for 2 factors vs. positive for 3 or more factors).ConclusionsConsideration of expression levels of EMT-associated markers in TUR specimens, in addition to conventional prognostic parameters, would contribute to the accurate prediction of intravesical recurrence following TUR for NMIUCB.     

Expression of survivin in bladder cancer cell lines using quantitative real-time polymerase chain reaction

ObjectivePrevious studies have reported that survivin expression is significantly associated with various malignancies including bladder cancer. However, the relationship between the expression of survivin and the tumor stage and grade of bladder cancer still require further study.MethodsTo determine whether survivin plays a role in the differentiation of bladder cancer cells, we conducted a preliminary study to examine the expression of survivin in bladder cancer cell lines.ResultsIn this study, we observed that the gene expression fold changes of survivin ranged from 3.2 to 16.7 in various tumor grades (G1–G4) of bladder cancer cell lines, which were higher than that in normal human urothelial cell line. With the worse differentiation of bladder cancer cell lines, the gene expression fold changes of survivin increased significantly (3.2-fold in RT4, 5.8-fold in 5637, 6.6-fold in T24, and 16.7-fold in HT1197). In addition, we observed different genotypes among various cell lines (C/C in HUC4449, C/G in RT4, C/G in 5637, G/G in T24, and C/C in HT1197). The relationship between survivin ?31 C/G polymorphism and various bladder cancer cell lines was non-significant. However, the overexpression of survivin may be associated with aggressive features of bladder cancer.ConclusionOur findings suggest that survivin could be a potential therapeutic target through the inhibition of cell proliferation in bladder cancer.     

Expression of Med19 in bladder cancer tissues and its role on bladder cancer cell growth

ObjectivesThe human Med19 gene encodes a critical subunit that stabilizes the whole mediator complex. To understand the role of Med19 in bladder cancer, we studied the effects of lentivirus-mediated suppression of Med19 expression on bladder cancer cells in vitro and in vivo.Methods and materialsIn this study, immunohistochemical analysis was used to demonstrate the expression of Med19 in human bladder cancer. The lentivirus vectors containing a small hairpin RNA (shRNA) to target Med19 were constructed. After bladder cancer cells (5637 and T24) were infected, RT-PCR and Western blotting were used to measure Med19 expression. The influence of Med19 on the proliferation of bladder cancer cells were assessed using MTT, BrdU, colony formation and tumorigenicity experiments. Cell cycle was analyzed with flow cytometric assay.ResultsMed19 was up-regulated in human bladder cancers compared with adjacent benign tissues by immunohistochemical analysis, but was strongly inhibited in 5637 and T24 bladder cancer cells infected with lentiviruses delivering shRNA against Med19. The down-regulation of Med19 increased the proportion of cells in G0/G1 phases and attenuated the growth of 5637 and T24 cells in vitro. The tumorigenicity of Med19-suppressed T24 cells was decreased after inoculation into nude mice.ConclusionsOur results suggested that lentiviruses delivering shRNA against Med19 may be a promising tool for bladder cancer therapy.     

Evaluation of thymosin β4 in the regulation of epithelial-mesenchymal transformation in urothelial carcinoma

ObjectivesTo study the underlying alteration in the expression of epithelial markers involved in epithelial-mesenchymal transition (EMT), and elucidate the potential mechanism(s) for Tβ4-induced EMT-like phenotypic changes in bladder cancer cells.Materials and methodsAll tissue samples in this study were obtained from clinical patients of the Union Hospital of Tongji Medical College, and were confirmed by surgery and pathology. Of these, normal bladder tissues (control), primary urothelial carcinoma of different grades (Stage pTa, Stage pT3), bladder paracancerous tissues, accompanied with 2 bladder cancer cell lines (BIU-87 and T24), were divided into 6 groups. Quantitative RT-PCR, Western blotting, and immunohistochemical study of adhesion molecules Tβ4, ILK, E-cadherin, and β-catenin involved in EMT were carried out. A lentiviral gene transferring vector containing the RNA polymerase III-dependent U6 promoter to express short hairpin RNA (shRNA) directed against Tβ4 was also applied. In the present study, all agents were evaluated using commercial kits.ResultsA strong correlation between the expression levels of Tβ4, ILK, E-cadherin, and β-catenin was found in the bladder transitional cell carcinoma (TCC) patients. In the BIU-87 and T24 bladder cancer cells overexpressing Tβ4, which were accompanied by a loss of E-cadherin as well as a cytosolic accumulation of β-catenin, up-regulation of ILK was also revealed. The inhibition of the Tβ4 expression with lentiviral shRNA vector could raise EMT-like phenotypic changes, significantly depressed motility, and subsequent invasiveness of bladder cancer cells.ConclusionsOur results imply that the Tβ4 is likely to play a crucial role in EMT progression, and that inhibition of the Tβ4 expression or interactions with other genes should be novel therapeutic targets for bladder cancers with high invasive and metastatic potential.     

Phospho-Akt pathway activation and inhibition depends on N-cadherin or phospho-EGFR expression in invasive human bladder cancer cell lines

ObjectivesA particular interest in epithelial-mesenchymal transition (EMT), which takes place during embryonic development, provided potential mechanisms involved in the progression of many epithelial tumors, including bladder cancer (BC). The phospho-Akt signaling pathway is supposed to be involved in invasion and progression of human tumors, including BC. Moreover, it has been demonstrated in bladder cancer cell lines that N-cadherin or phospho-epithelial growth factor receptor (EGFR) expression are correlated to tumor progression. Our objectives were to evaluate the potential phospho-Akt pathway involvement in N-cadherin and/or phospho-EGFR positive BC cell lines and to evaluate the prognostic value of E- and N-cadherin expression in patients undergoing cystectomy for invasive BC.Materials and methodsWe screened a panel of invasive and noninvasive BC cell lines for E- and N-cadherin, phospho-EGFR, and phospho-Akt expression using the Western blot technique (WB). The potential role of N-cadherin in invasion was assessed by Matrigel assays with and without the N-cadherin blocking monoclonal antibody GC-4. Then we used the Affymetrix microarray technique to evaluate the prognostic value of E- and N-cadherin expression in 30 patients undergoing a cystectomy for invasive BC.ResultsN-cadherin and phospho-EGFR expression are associated with Akt activation and with invasive behavior modulation. Even if Akt activation is sufficient in promoting invasion, its inactivation by LY294002 (PI-3 kinase inhibitor) is less efficient on invasion than inhibition of N-cadherin and phospho-EGFR by GC-4 (monoclonal antibody) and gefitinib (anti-tyrosine kinase), respectively. N-cadherin and phospho-EGFR inhibition decreased phospho-Akt activation but also caused restoration and reinforcing of E-cadherin expression, respectively, while phospho-Akt inhibition did not have any impact on E-cadherin expression. In a group of high-risk bladder tumors (T₁G₃), N- and E-cadherin expression could be considered as a prognostic marker. In a group of patients with invasive BC (pT₂-T₄) undergoing cystectomy, we showed a shorter overall survival when BC expressed N-cadherin (P = 0.0064) and when E-cadherin expression was down-regulated (P = 0.00165). The N _positive /E _negative profile has the worst prognosis (P = 0.00153).ConclusionsWe confirmed the partial responsibility of p-Akt activation in invasion of some BC cell lines expressing N-cadherin or p-EGFR and also the potential role of N-cadherin and p-EGFR as target in cancer therapy. N/E- cadherin expression profile has a significant prognostic value in invasive BC.     

Roles of HIF-1α in a novel optical orthotopic spontaneous metastatic bladder cancer animal model

Although metastatic disease is lethal in the majority of bladder cancer cases, study on the molecular mechanism(s) of this process suffers from the limited source of distant metastatic tumor tissues and very few suitable animal models. To address this need, we generated an orthotopic animal model by instilling human bladder cancer T24-tumorigenic (T24-t) cells into mouse bladder, and sublines were subsequently derived as primary (T24-parental, T24-P) and lung metastatic (T24-L) sites. Data from invasion, migration, and adhesion assays suggested higher metastatic potential of T24-L cells than T24-P cells in vitro. Using two metastatic models to assess the metastatic ability in vivo, T24-L cells exhibited higher incidence of tumor metastasis. Mechanistically, the up-regulation of MMP-1 and HIF-1α was observed in T24-L cells. Knocking down HIF-1α can significantly down-regulate the expression of MMP-1, accompanied by the decreased invasion ability in vitro. Using immunohistochemical staining, we further observed HIF-1α elevation in the metastatic lymphomatic tissues compared with the primary bladder cancer tissues from the same patients. Taken together, our study provides the evident of the function of HIF-1α/MMP-1 in regulating metastasis of bladder cancer and HIF-1α as a potential target for controlling bladder cancer metastasis.     

Sirolimus Attenuates Calcineurin Inhibitor-Induced Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma

BackgroundCalcineurin inhibitors (CNIs), which are potent immunosuppressants (ISs), increase the risk for hepatocellular carcinoma (HCC) recurrence after liver transplantation (LTx). Epithelial-mesenchymal transition (EMT) is a key process in which epithelial cancer cells lose their polarity, resulting in cancer progression and metastasis. The aim of this study was to evaluate the effect of sirolimus (SRL) individually and in combination with other ISs to reduce EMT.MethodsHCC SK-Hep1 cells were used and various ISs (SRL, tacrolimus, cyclosporine A, or mycophenolate mofetil) were administered at 2 dosages and in combination therapies. Mice were transplanted with SK-Hep1 cells (in the liver) and were monitored after 2 weeks.ResultsThe in vitro treatment with SRL showed a dose-dependent attenuation of cell proliferation and migration in case of the individual and IS combination treatments; further, decreased levels of pro-EMT proteins, namely, N-cadherin, transforming growth factor-β, ZEB1, Slug, and Snail were observed. In contrast, E-cadherin expression was upregulated after both the individual and IS combination treatments. These results were also observed in the samples from mice transplanted with the SK-Hep1 cells.ConclusionThe present study demonstrated that SRL reduced HCC metastasis by inhibiting EMT. Thus, our findings provide a rationale for the use of SRL in combination with ISs in HCC LTx patients.     

RNA干扰下调p53抑制因子iASPP表达对膀胱癌细胞的影响

目的 探讨RNA干扰使p53抑制因子iASPP表达下调对膀胱癌细胞的影响.方法 iASPP siRNA慢病毒感染入膀胱癌细胞株5637和T24,实时定量PCR和蛋白质印迹法检测iASPP的表达;噻唑盐法测定细胞生长;集落形成测定法检测集落形成比率;荧光激活细胞分选术检测细胞周期.结果 iASPP siRNA慢病毒感染后细胞iASPP mRNA为0.60±0.02,低于阴性对照慢病毒组(1.00±0.03,P＜0.05).膀胱癌细胞iASPP蛋白表达降低.iASPP下调能抑制入膀胱癌细胞5637和T24的生长和增殖(P＜0.05),每个集落中细胞数显著减少(P＜0.05),集落数目也低于阴性对照组(P＜0.05).iASPP siRNA慢病毒5637细胞培养中的G1期细胞比率明显高于阴性对照慢病毒组,分别为(51.94±0.98)%和(46.00±0.77)%;T24细胞结果与之类似,分别为(60.04±0.45)%和(53.62±0.69)%;组间比较差异均有统计学意义(P＜0.05).结论 RNA干扰能够使膀胱癌细胞iASPP表达下调,从而抑制膀胱癌细胞的生长和增殖.

Abstract：

Objective To discuss the effects of silencing of iASPP gene on human bladder cancer cells. Methods RNAi silencing of iASPP gene in bladder cancer cell 5637 and T24 cells were used by lentiviral mediated interfering short hairpin RNAs. Cell proliferation was tested by MTT assay, and rate of colony was tested by colony formation assay. Cell cycles were tested by using fluorescence-activated cell sorting. Results Down-regulation of iASPP could inhibit the growth and proliferation of human bladder cancer cells (P＜0.05). iASPP know-down could decrease the colony formation of 5637 and T24 cells (P＜0, 05). Knocking down of iASPP in 5637 and T24 cells showed cell arrested at G1. Conclusions Silencing of iASPP gene could inhibit proliferation and colony formation of bladder cancer, iASPP might be an important target for gene therapy of bladder cancer.     

Prognostic significance of the Ki67 index and programmed death-ligand 1 expression after radical cystectomy in patients with muscle-invasive bladder cancer

ObjectivesTo investigate the association between Ki67 index and programmed death-ligand 1 (PD-L1) expression in muscle-invasive bladder cancer (MIBC) patients after RC.Materials and MethodsWe retrospectively evaluated 262 MIBC patients treated with RC between April 2004 and April 2020. The impact of Ki67 index and PD-L1 expression on prognosis was evaluated by univariate Cox regression analysis. In addition, a pathomolecular risk score, including Ki67 and PD-L1, was developed to predict prognosis and pathological factors. We also evaluated the link between the Ki67 index and PD-L1 under the IL-6 stimulation in the bladder cancer cell lines of T24 and 5637 cells.ResultsThe median age and follow-up period was 69 years and 52 months, respectively. Ki67 index and PD-L1 expression were significantly associated with tumor recurrence. Univariate Cox regression analysis showed that pT3–4, mixed histology, lymphovascular invasion positive (LVI+), pN+, Ki67-high (>17%), and PD-L1+ were significantly associated with recurrence-free survival (RFS). The pathomolecular risk score was developed using resection margin+ (1 point), mixed histology (1 point), LVI+ (1 point), pN+ (1 point), and Ki67-high (1 point). RFS and overall survival were significantly shorter in patients with higher pathomolecular risk scores (>1) than in those with lower risk scores (≤1). Cell proliferation was significantly increased in the T24 and 5637 cells under the IL-6 stimulation, while PD-L1 expression was not.ConclusionsA significant effect of Ki67-high and PD-L1 expression on poor prognosis was observed in patients with MIBC. Further studies are necessary to elucidate the precise mechanisms of cell proliferation and PD-L1 expression in patients with MIBC.     

LIN28在膀胱癌和膀胱癌细胞系中的表达及意义

目的：探讨LIN28在膀胱癌组织和细胞系中表达情况,以及与mieroRNA初级Let-7g（pri—Lev7g）之间关系,推测其可能临床意义及对肿瘤进展的影响。方法：采用常规RT-PCR、miRNA转录、免疫荧光和免疫组化方法,检测LIN28mRNA和pri-Let-7g表达,以及LIN28蛋白表达定位。结果：2例膀胱癌细胞系均表达LIN28mRNA,T24表达较强,免疫荧光显示这两个细胞系均表达LIN28蛋白,阳性部位位于细胞胞质,T24荧光强度强于5637。所选10例膀胱癌和相应癌旁组织均表达LIN28mRNA,二者并无明显不同,与临床分级也无明确关系。免疫组化显示癌组织LIN28表达阳性并定位于胞质,而癌旁正常组织LIN28表达为阴性。此外,两个细胞系pri—Let-7g表达较强,而癌和癌旁组织的pri—Let-7g表达强度无明显差异,需进一步检测其成熟Let-7g在这些组织中是否存在不同,以明确这些miRNA是否发生生物合成的转录后阻断。结论：明确T24和5637两个膀胱癌细胞系均可作为研究LIN28、Let-7与其相应靶基因关系的体外实验模型。尽管并不确定膀胱癌和癌旁组织LIN28、Let-7g表达强度与临床分级是否相关,但至少明确LIN28／LIN28在膀胱癌中表达,为探讨LIN28和Let-7在泌尿系统来源的其他恶性肿瘤中的作用提供借鉴和实验依据。     

A novel role of Id-1 in regulation of epithelial-to-mesenchymal transition in bladder cancer

Background and objectiveInhibitor of differentiation or DNA binding -1 (Id-1) has been shown to be increased in several types of advanced cancer, and to be associated with aggressive and metastatic abilities of cancer cells. Recently, more and more evidence indicates that epithelial-to-mesenchymal transition (EMT) is an important mechanism taking place during tumor invasion and metastasis, but the molecular pathways underlying EMT have not been clearly established. This study was to investigate the expression of Id-1 in bladder cancer and its association with EMT.Materials and methodsA total of 169 tissues, consisting of 147 primary bladder cancers and 22 adjacent normal tissues were included in this study. Id-1, E-cadherin, and β-catenin were examined immunohistochemically in paraffin sections. The pBabe-Id-1 expression retroviral vector and retroviral vectors containing an Id-1-specific small interfering RNA oligonucleotides (si-Id-1) were transfected into 2 bladder cancer cell lines respectively. Then, we used Western blotting and immunofluorescent staining to detect the cellular expression of epithelial markers and mesenchymal markers. The invasion and migration ability of bladder cancer cells were identified by type I collagen invasion assay and wound closure assay.ResultsWe demonstrated that increased Id-1 expression was associated with advanced tumor stage and grade. In addition, the increased Id-1 expression in bladder tumors was also correlated with decreased membranous E-cadherin and β-catenin expression. In vitro, studies showed that inactivation of the Id-1 gene conferred morphologic transition of bladder cancer cells from a fibroblastic to epithelial appearance, and overexpression of Id-1 could lead to acquisition of a fibroblastic spindle cell phenotype accompanied by loss of cell-to-cell contacts. By Western blotting and immunofluorescent staining, we showed that the expression level of Id-1 was correlated with the expression of mesenchymal markers but was inversely correlated with the expression of epithelial markers. Moreover, results of collagen invasion and wound closure assays showed ectopic Id-1 expression led to increased ability of invasion and migration.ConclusionsOur results suggest that Id-1 may play roles in tumor progression and EMT activation in bladder cancer.     

The role of tumour microenvironment-driven miRNAs in the chemoresistance of muscle-invasive bladder cancer–a review

Successful treatment for muscle-invasive bladder cancer is challenged by the ability of cancer cells to resist chemotherapy. While enormous progress has been made toward understanding the divergent molecular mechanisms underlying chemoresistance, the heterogenous interplay between the bladder tumour and its microenvironment presents significant challenges in comprehending the occurrence of chemoresistance. The last decade has seen exponential interest in the exploration of microRNA (miRNA) as a tool in the management of chemoresistance. In this review, we highlight the miRNAs involved in the tumour microenvironment crosstalk that contributes to the chemoresistance in bladder cancer. Decrypting the role of miRNAs in the interplay beholds scope for future clinical translational application in managing the long-standing concerns of chemoresistance in muscle-invasive bladder cancer.     

胃癌原发灶中上皮间质转化相关因子和CD133的表达及其与临床病理特征和预后的关系

目的研究上皮间质转化（EMT）相关因子Snail、E-cadherin、N-cadherin与胃癌患者临床病理特征、预后及胃癌肿瘤起始细胞表面标志物CD133表达的关系。方法利用Western blot方法检测50例胃癌及癌旁正常胃黏膜组织中EMT相关因子及CD133蛋白的定位及定量表达,分析EMT相关因子及CD133蛋白表达与胃癌患者的临床病理学指标的关系,Spearman等级相关分析EMT相关因子和CD133表达的关系,Kaplan-Meier方法分析EMT相关因子及CD133表达与胃癌患者生存的关系。结果①胃癌组织中Snail、N-cadherin及CD133蛋白表达相对灰度值明显高于其在癌旁正常胃黏膜组织中的表达（Snail：0.599±0.114比0.259±0.108,P=0.020;N-cadherin：0.754±0.154比0.329±0.134,P=0.001;CD133：0.635±0.119比0.485±0.116,P=0.029）,E-cadherin蛋白表达相对灰度值明显低于其在癌旁正常胃黏膜组织中的表达（0.378±0.123比0.752±0.156,P=0.003）。②Snail蛋白、N-cadherin蛋白表达平均相对灰度值在有血管浸润、淋巴管浸润、N3淋巴结转移及肿瘤直径≥5 cm和Ⅲ＋Ⅳ期胃癌患者中的表达明显高于无血管浸润、淋巴管浸润、N0～N2淋巴结转移及肿瘤直径〈5 cm和Ⅰ＋Ⅱ期的胃癌患者（P〈0.05）,而E-cadherin蛋白表达平均相对灰度值在有血管浸润、淋巴管浸润、N3淋巴结转移及Ⅲ＋Ⅳ期胃癌患者中的表达显著低于无血管浸润、淋巴管浸润、N0～N2淋巴结转移及Ⅰ＋Ⅱ期的胃癌患者（P〈0.05）,CD133蛋白表达平均相对灰度值在有淋巴管浸润、N3淋巴结转移、肿瘤直径≥5 cm和Ⅲ＋Ⅳ期胃癌患者中的表达显著高于无淋巴管浸润、N0～N2淋巴结转移、肿瘤直径〈5 cm和Ⅰ＋Ⅱ期的胃癌患者（P〈0.05）。③Snail、N-cadherin蛋白表达与CD133蛋白表达分别均呈正相关（rs=0.278,P=0.048;rs=0.406,P=0.003）,而E-cadherin蛋白表达与CD133蛋白表达呈负相关（rs=-0.504,P=0.000）。④Snail、N-cadherin及CD133蛋白低表达组的生存时间明显长于其高表达者（P〈0.05）,联合EMT相关因子和CD133蛋白表达能够最有效预测患者生存。结论EMT与胃癌肿瘤起始细胞特性之间存在明显相关,并且两者与胃癌的高侵袭的临床病理特征相关,联合EMT相关因子Snail、E-cadherin、N-cadherin与CD133能够最有效预测胃癌患者的预后。     

两种不同前列腺癌上皮细胞间质转化模型转录调控因子的比较及意义

目的:初步筛选、对比决定两种不同前列腺癌上皮细胞间质转化(EMT)模型(LNCaP/HIF1α细胞模型、ARCaP细胞模型)表型的转录因子,为利用这两个模型深入研究前列腺癌转移机制奠定基础。方法:分别体外培养LNCaP/HIF1α细胞模型中的LNCaP细胞、稳定高表达缺氧诱导因子HIF1α的LNCaP细胞和ARCaP细胞模型中的IF11细胞、IA8细胞,提取总RNA行RT-PCR,检测目前发现的决定EMT表型的5种转录因子(Snail、Slug、ZEB1、SIP1、Twist1),对比两种模型的差异。结果:LNCaP/HIF1α细胞模型中的两种细胞均不同程度地表达5种转录因子,其中Slug、Twist1有差异,表型转化后Slug表达升高,而Twist1表达降低(P<0.05);ARCaP细胞模型中的两种细胞均不同程度地表达Snail、Slug、ZEB1、SIP1,不表达Twist1,其中ZEB1、Slug有差异,表型转化后ZEB1、Slug表达升高(P<0.05)。结论:决定不同来源的前列腺癌EMT模型表型转化的转录调控机制各不相同,Slug可能是决定HIF1α诱导LNCaP细胞EMT表型转化的关键转录因子,而ZEB1、Slug则可能在ARCaP细胞EMT表型转化中起决定作用。     

Interferon-α inhibits cyclooxygenase-1 and stimulates cyclooxygenase-2 expression in bladder cancer cells in vitro

The enzymes cyclooxygenase-1 (Cox-1) and cyclooxygenase-2 (Cox-2) catalyze the initial step in the formation of prostaglandins (PGs). PGs are known to be involved in numerous processes, for example inflammation, immune responses, carcinogenesis, and tumor angiogenesis. The formation of PGs is stimulated in various cancers since the expression of Cox-2 is upregulated. Interferon (IFN)-α is used in the treatment of bladder cancer, although not all of the effects of such treatment are thoroughly known. Therefore, we investigated the expression of cyclooxygenases in two bladder cancer cell lines, 5637 and T24, under basal conditions and in the presence of human recombinant IFN-α (100, 1,000, and 10,000 U/ml). The mRNA of Cox-1 and Cox-2 was expressed in both cultured bladder carcinoma cell lines. The level of Cox-1 expression was low in 5637 cells and higher in T24 cells. In contrast, Cox-2 expression was prominent in 5637 cells and low in T24 cancer cells. The highest IFN-α concentration (10,000 U/ml) decreased the expression of Cox-1 to 47 and 28% of the control levels in 5637 and T24 cells, respectively. In contrast, Cox-2 expression increased in both cell lines. In 5,637 cells, Cox-2 expression increased 1.3-fold with 10,000 U/ml of IFN-α. In T24 cells, the maximum effect was achieved by 1,000 U/ml of IFN-α, which increased the expression of Cox-2 up to 2.4-fold. These findings may have relevance in the outcome of patients treated with IFN-α because upregulated Cox-2 expression may suppress the cell-mediated defense system. On the other hand, the inhibition of Cox-1 could be beneficial because Cox-1 is known to stimulate angiogenesis. Received: 5 August 1999 / Accepted: 8 September 2000     

LIN28A和LAMP1在膀胱癌细胞系中的表达及意义

目的:探讨LIN28A和LAMP1在膀胱癌细胞系中表达情况,以及两者之间的关系,推测其可能临床意义及对肿瘤进展的影响。方法:采用RT-PCR检测膀胱癌细胞系LIN28A、LIN28B和LAMP1表达,免疫荧光检测LIN28A和LAMP1二者蛋白表达定位;LIN28A敲减后通过qRT-PCR检测LAMP1的mRNA表达变化。结果:5个癌细胞系T24、UM-UC3、J82、5637和SW780和正常移行上皮细胞系SV-HUC-1均表达LIN28A,其中J82也表达LIN28B;5个癌细胞系均表达LAMP1,SV-HUC-1不表达LAMP1;LIN28A和LAMP1蛋白均定位在胞浆;LIN28A敲减后对LAMP1的mRNA表达变化无明显影响,相应蛋白变化需要进一步验证。结论:4个膀胱癌细胞系T24、5637、UM-UC3和SW780可以用于LIN28A与肿瘤相关的机制研究,而J82可用于LIN28B的机制研究。LIN28A对肿瘤细胞和干细胞的调控方面可能具有相似性,敲减后对其靶点mRNA表达量无明显影响,LAMP1蛋白可能对肿瘤细胞侵袭转移具有抑制作用。     

LncRNA DANCR regulates lymphatic metastasis of bladder cancer via the miR-335/VEGF-C axis

BackgroundSubstantial evidence indicate that long non-coding RNA (lncRNA) and microRNA (miRNA) act as key role in bladder cancer. Differentiation antagonistic ncRNA (DANCR) could be used as a biomarker in the occurrence and development of cancer. This study aims to explore the mechanism of DANCR/miR-335/VEGF-C axis affecting lymphatic metastasis of bladder cancer.MethodsqRT-PCR detects the expression of DANCR in bladder cancer cell lines (SW780, 5637, T24, UM-UC-3) and normal bladder cell lines (SV-HUC-1), and selects T24 cell lines for subsequent experiments. The expression levels of DANCR, miR-335 and VEGF were measured by qRT-PCR, and the dual luciferase reporter gene verified the targeted regulation of DANCR on miR-335 and miR-335 on VEGF. CCK-8, Transwell and Wound healing assay detect the proliferation, invasion and migration ability of bladder cancer cells, Endothelial cell adhesion assay and Western blot further prove the lymphatic metastasis of bladder cancer.ResultsIn this study, DANCR was highly expressed in bladder cancer cell lines. Transfection of si-DANCR significantly inhibits the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells. Dual luciferase assay confirmed that DANCR targets miR-335/VEGF-C. Transfection of miR-335 mimic promotes the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells, overexpression of DANCR eliminates the promotion of miR-335 mimic on bladder cancer cells. Further experiments proved that inhibition of miR-335 and overexpression of VEGF-C can reverse the inhibitory effect of silencing DANCR on bladder cancer cells.ConclusionsIn bladder cancer, DARCR plays an important role, which regulates the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells through the miR-335/VEGF-C molecular axis.