RNA干扰下调p53抑制因子iASPP表达对膀胱癌细胞的影响

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.     

RNA干扰下调p53抑制因子iASPP表达对膀胱癌细胞的影响

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.     

电穿孔法介导雄激素受体基因沉默抑制裸鼠膀胱癌移植瘤生长的研究

目的 观察电穿孔法转染靶向雄激素受体(AR)的小干扰RNA(siRNA)对裸鼠人膀胱癌移植瘤生长的影响.方法 建立人膀胱癌细胞124的荷瘤裸鼠模型,于肿瘤直径0.5 cm大小时,瘤体接受AR-siRNA的电转染治疗,转染无效序列siRNA为阴性对照组,瘤体未受电穿击为空白对照组.观察裸鼠肿瘤生长;4周后处死,绘制肿瘤生长曲线;肿瘤组织石蜡包埋,常规切片,TUNEL法检测移植瘤凋亡.结果 电转染AR-siRNA后瘤体组织AR基因的表达显著被抑制,也能明显抑制裸鼠移植瘤的生长;TUNEL检测凋亡率为(13.1±6.9)%,显著高于阴性对照组(P＜0.01).结论 电穿孔法靶向AR的siRNA可有效阻断种植瘤内AR表达,阻断雄激素受体途经信号传导,进而诱导细胞凋亡,能明显抑制人膀胱癌裸鼠皮下移植瘤的生长.

Abstract：

Objective To investigate the antitumor efficacy of small interfering RNA (siRNA)mediated inhibition of androgen receptor (AR) gene expression in T24 bladder tumor xenografts using electroporation. Methods Athymic mouse human bladder cancer transplantation tumor model was established by injecting T24 cells subcutaneously. When tumor diameter was exceeded 0. 5 cm, AR-siRNA was deliered into tumor xenografts by electroporation. The cells with nonspecific siRNA delivery and un-treatment served as control groups. Tumor volumes were measured weekly. The animals were sacrificed after the treatments, and the histological changes of xenografts were observed by Hematoxylin and Eosin ( HE) staining and TUNEL assay. Results The athymic mouse exnograft tumor model was established successfully.After AR-siRNA treatment, tumor growth was inhibited as compared with the controls. The number of TUNEL-positive cells was significantly increased in AR-siRNA group as compared with the nonspecific siRNA group ( P ＜ 0. 01). Conclusion siRNA targeting AR gene could inhibit obviously the growth of athymic mouse human T24 bladder carcinoma transplantation tumor by inducing apoptosis.     

携带4-1BBL基因的胃癌细胞总RNA转染树突状细胞疫苗的研究

目的 观察携带4-1BBL基因的胃癌细胞总RNA转染树突状细胞疫苗,在小鼠体内诱导的抗肿瘤效应以及对小鼠免疫力的影响.方法 用脂质体法把pMKITneo/4-1 BBL质粒导入小鼠胃癌细胞MFC内,再提取此细胞的总RNA并转染至树突状细胞内制备MFC/4-1BBL/DC疫苗;把MFC细胞注射于20只615小鼠皮下建立荷瘤小鼠胃癌模型,并随机分为对照组、DC组、MFC/DC组和MFC/4-1BBL/DC组,每组5只;在MFC细胞接种后第7、14天给予相应DC疫苗治疗,于肿瘤细胞接种后第21天,处死实验动物,测量瘤重、测定肿瘤细胞的凋亡率及外周血的CD4+、CD8+T细胞和NK细胞的含量.结果 MFC/4-1BBL/DC组小鼠平均瘤重(2.06±0.39)g显著低于对照组(3.82±0.57)g、DC组(3.63±0.51)g、MFC/DC组(2.67±0.32)g(P＜0.05);且MFC/4-1BBL/DC组肿瘤细胞凋亡率(28.58±2.84)%明显高于MFC/DC组(25.03±1.88)%、DC组(20.66±2.71)%及对照组(19.09±2.73)%(P＜0.05);MFC/4-1BBI/DC组小鼠外周血CID4+T、NK细胞数明显高于对照组和其他治疗组(P＜0.05).结论 携带4-1BBL基因的胃癌细胞总RNA转染树突状细胞疫苗能够提高荷瘤机体的免疫能力、抑制肿瘤细胞的生长、促进肿瘤细胞的凋亡.

Abstract：

Objective To observe the induced anti-tumor effects and immune state in vivo by the transfection of dendritic cell vaccine into the total RNA of gastric cancer cells carrying 4-1BBL gene.Methods The liposome-mediated pMKITneo/4-1BBL gene was inserted into the MFC gastric cancer cells,and the cell total RNA was extracted and transfected into dendritic cells to make the MFC/4-1BBL/DC vaccine.After MFC cells were injected into the 615 mouse to establish tumor-bearing mouse model,and those 20 mice were randomly divided into control group,DC group,MFC/DC group,MFC/4-1BBL/DC group.The dendritic cell vaccine was subcutaneously injected on the day 7 and 14 after inoculation of tumor cells,and on the day 21 animals were killed and tumor weight was measured,tumor cell apoptosis rate was assayed and the contents of CD4 +,CD8 + T cells and NK cells in peripheral blood were analyzed.Results The mean tumor weight in MFC/4-1BBL/DC group (2.06 ±0.39) g was significantly less than that in control group (3.82 ±0.57) g,DC group (3.63 ±0.51 ) g,MFC/DC group (2.67 ±0.32) g (P ＜0.05).Moreover the apoptosis rate in MFC/4-1BBL/DC group (28.58 ± 2.84 ) % was significantly higher than that in MFC/DC group (25.03 ± 1.88)%,DC group (20.66 ±2.71 )% and control group ( 19.09 ±2.73 )% (P ＜0.05).The percentage of CD4 + T,NK cells in MFC/4-1BBL/DC group was significantly higher than in other groups ( P＜0.05).Conclusion The transfection of dendritic cell vaccine into the total RNA of gastric cancer cells carrying 4-1BBL gene can increase immunity of the bearing tumor mice,inhabit tumor growth and promote tumor cell apoptosis.     

胰腺星状细胞对胰腺癌细胞增殖能力的影响及机制

目的 观察胰腺星状细胞(PSC)对人胰腺癌细胞株增殖能力的影响,探讨其分子机制.方法 用免疫印迹实验检测成纤维细胞生长因子(FGFs)在胰腺癌细胞及PSC中的表达及分泌;噻唑蓝(MTT)比色法检测PSC细胞条件培养基(CDM)对胰腺癌细胞体外增殖的影响,以及除去FGFs之后影响的变化;MTT法检测PSC细胞CDM对Cyclopamine抑制胰腺癌细胞增殖作用的影响;体内实验观察PSC对胰腺癌细胞成瘤能力的影响.结果 PSC主要表达并分泌FGF2,而胰腺癌MIA PaCa-2和PANC-1细胞则主要表达FGF5;PSC细胞CDM刺激48 h后,胰腺癌MIA PaCa-2和PANC-1细胞体外增殖能力分别为对照组的(1.2993±0.1170)倍和(1.2447±0.0123)倍(P＜0.05);中和CDM中FGFs后,FGFs(-)CDM对胰腺癌细胞增殖的刺激作用显著削弱;与单纯注射胰腺癌PANC-1细胞的裸鼠皮下种植瘤比较,PSC与PANC-1细胞混合注射组的平均肿瘤体积、质量均明显增加,分别由380.13 mm3和170 g增加至601.31 mm3和349 g(P＜0.05);PSC细胞CDM处理后,Cyclopamine对胰腺癌MIA PaCa-2和PANC-1细胞的增殖抑制作用显著下降(P＜0.01).结论 PSC在体内、体外均显著刺激胰腺癌细胞的增殖,增强其成瘤能力,其机制与PSC旁分泌FGF2作用于癌细胞相关;PSC显著降低了胰腺癌细胞对Hedgehog信号通路特异性抑制剂Cyclopamine治疗的敏感性.

Abstract：

Objective To assess the effects of pancreatic stellate cells (PSC) on proliferation of pancreatic cancer cells and to reveal its possible mechanism. Methods Expression and secretion of fibroblast growth factor (FGF) ligands in both pancreatic cancer cells and stellate cells were determined by immunoblotting analysis. Mmethyl thiazol tetrazolium (MTT) assay was used to examine the effects of conditioned medium (CDM) of PSC on the proliferation of pancreatic cancer cells. An in vivo tumorigenicity assay was used to evaluate the effect of PSC on xenograft formation in cancer cells. Results FGF2 was predominantly expressed and secreted by PSC. Yet MIA PaCa-2 and PANC-1 pancreatic cancer cells mainly expressed FGF5. CDM of PSC enhanced the proliferation of MIA PaCa-2 and PANC-1 cells for ( 1. 2993 ±0. 1170 ,P ＜0. 05 ) times and ( 1. 2447 ±0. 0123 ,P ＜0. 05 ) times, respectively. Neutralizing the effects of FGFs in the CDM by heparin-sepharose precipitation abolished this effect. The volume and weight of subcutaneous tumors in nude mice by injection of combination of pancreatic cancer cells with PSC were significantly greater than those by injection of PANC-1 cells alone (380. 13 mm3 and 170 g versus 601.31 mm3and 349 g,P ＜0. 05, respectively). The CDM of PSC reduced the antiproliferative effect by cyclopamine on pancreatic cancer cells ( P ＜ 0. 01, respectively). Conclusion We identified in this study a mechanism based on stroma-tumor interactions involving PSC that can contribute to enhance the proliferation of human pancreatic cancer cells.     

慢病毒介导CCL5-siRNA对乳腺癌细胞生物学行为的影响

Objective To study the effect of lentivirus-mediated CCL5-RNAi on the biological behaviors of human breast cancer cells. Methods CCL5-specific siRNA gene was synthesized and cloned into the recombinant lentiviral vector, pGCSIL-GFP. Human high-metastatic breast cancer cells, MDA-MB-231, were infected by CCL5-siRNA recombinant lentivirus, which was set as KD group. Cells infected with CCL5-NC was as NC group, and cells cultured was as CON group. The expression of CCL5 mRNA and protein in MDA-MB-231 cells was detected by RT-PCR and western blot, respectively. Cell growth suppression and cell cycle was observed by MTT assay and fluorescence activated cell sorting (FACS). Colony formation and migration ability were determined by colony-rorming assay and Boyden chamber method. Results After infection of CCL5-siRNA recombinant lentivirus, the expression level of CCL5 mRNA and protein in MDA-MB-231 cells as well as the colony formation and migration ability decreased significantly, but cell's proliferation was not affected obviously. Compared with MDA-MB-231 (0.88± 0.15) and MDA-MB-231/CCL5-NC (1.00±0.07) cells, the expression of CCL5 mRNA in MDA-MB-231/ CCL5-siRNA decreased to 0.18±0.03, P<0.01. Compared with MDA-MB-231/CCL5-NC (1.82±0.18) cells, the expression of CCL5 protein in MDA-MB-231/CCL5-siRNA decreased to 0.33±0.13, P <0.01. Colony-forming assay and Boyden chamber method showed that the colony formation and migration ability of MDA-MB-231/CCL5-siRNA decreased markedly (P<0.05). The clone count in KD group was (0.33± 0.10), which was a significant decrease from (0.97±0.09) (NC group) and (1.04±0.07) (CON group), P<0.05. The number of cells that migrated through the chamber membrane of KD group (38± 15) was less than that of NC group (77±11, P <0.05) and CON group (69±9, P <0.05). However, MTT assay and FACS revealed that the proliferation of MDA-MB-231/CCL5-siRNA was not different from MDA-MB-231/CCL5-NC and MDA-MB-231 (P>0.05), the proliferation index (PI) of group KD, NC and CON were (0.48±0.02), (0.44±0.05) and (0.47±0.02) respectively. The difference was not statistically significant by multiple comparison (P>0.05). Conclusion CCL5-specific siRNA can specifically suppress the colony formation and migration of human high-matastatic breast cancer cells.     

慢病毒介导CCL5-siRNA对乳腺癌细胞生物学行为的影响

Objective To study the effect of lentivirus-mediated CCL5-RNAi on the biological behaviors of human breast cancer cells. Methods CCL5-specific siRNA gene was synthesized and cloned into the recombinant lentiviral vector, pGCSIL-GFP. Human high-metastatic breast cancer cells, MDA-MB-231, were infected by CCL5-siRNA recombinant lentivirus, which was set as KD group. Cells infected with CCL5-NC was as NC group, and cells cultured was as CON group. The expression of CCL5 mRNA and protein in MDA-MB-231 cells was detected by RT-PCR and western blot, respectively. Cell growth suppression and cell cycle was observed by MTT assay and fluorescence activated cell sorting (FACS). Colony formation and migration ability were determined by colony-rorming assay and Boyden chamber method. Results After infection of CCL5-siRNA recombinant lentivirus, the expression level of CCL5 mRNA and protein in MDA-MB-231 cells as well as the colony formation and migration ability decreased significantly, but cell's proliferation was not affected obviously. Compared with MDA-MB-231 (0.88± 0.15) and MDA-MB-231/CCL5-NC (1.00±0.07) cells, the expression of CCL5 mRNA in MDA-MB-231/ CCL5-siRNA decreased to 0.18±0.03, P<0.01. Compared with MDA-MB-231/CCL5-NC (1.82±0.18) cells, the expression of CCL5 protein in MDA-MB-231/CCL5-siRNA decreased to 0.33±0.13, P <0.01. Colony-forming assay and Boyden chamber method showed that the colony formation and migration ability of MDA-MB-231/CCL5-siRNA decreased markedly (P<0.05). The clone count in KD group was (0.33± 0.10), which was a significant decrease from (0.97±0.09) (NC group) and (1.04±0.07) (CON group), P<0.05. The number of cells that migrated through the chamber membrane of KD group (38± 15) was less than that of NC group (77±11, P <0.05) and CON group (69±9, P <0.05). However, MTT assay and FACS revealed that the proliferation of MDA-MB-231/CCL5-siRNA was not different from MDA-MB-231/CCL5-NC and MDA-MB-231 (P>0.05), the proliferation index (PI) of group KD, NC and CON were (0.48±0.02), (0.44±0.05) and (0.47±0.02) respectively. The difference was not statistically significant by multiple comparison (P>0.05). Conclusion CCL5-specific siRNA can specifically suppress the colony formation and migration of human high-matastatic breast cancer cells.     

慢病毒介导CCL5-siRNA对乳腺癌细胞生物学行为的影响

Objective To study the effect of lentivirus-mediated CCL5-RNAi on the biological behaviors of human breast cancer cells. Methods CCL5-specific siRNA gene was synthesized and cloned into the recombinant lentiviral vector, pGCSIL-GFP. Human high-metastatic breast cancer cells, MDA-MB-231, were infected by CCL5-siRNA recombinant lentivirus, which was set as KD group. Cells infected with CCL5-NC was as NC group, and cells cultured was as CON group. The expression of CCL5 mRNA and protein in MDA-MB-231 cells was detected by RT-PCR and western blot, respectively. Cell growth suppression and cell cycle was observed by MTT assay and fluorescence activated cell sorting (FACS). Colony formation and migration ability were determined by colony-rorming assay and Boyden chamber method. Results After infection of CCL5-siRNA recombinant lentivirus, the expression level of CCL5 mRNA and protein in MDA-MB-231 cells as well as the colony formation and migration ability decreased significantly, but cell's proliferation was not affected obviously. Compared with MDA-MB-231 (0.88± 0.15) and MDA-MB-231/CCL5-NC (1.00±0.07) cells, the expression of CCL5 mRNA in MDA-MB-231/ CCL5-siRNA decreased to 0.18±0.03, P<0.01. Compared with MDA-MB-231/CCL5-NC (1.82±0.18) cells, the expression of CCL5 protein in MDA-MB-231/CCL5-siRNA decreased to 0.33±0.13, P <0.01. Colony-forming assay and Boyden chamber method showed that the colony formation and migration ability of MDA-MB-231/CCL5-siRNA decreased markedly (P<0.05). The clone count in KD group was (0.33± 0.10), which was a significant decrease from (0.97±0.09) (NC group) and (1.04±0.07) (CON group), P<0.05. The number of cells that migrated through the chamber membrane of KD group (38± 15) was less than that of NC group (77±11, P <0.05) and CON group (69±9, P <0.05). However, MTT assay and FACS revealed that the proliferation of MDA-MB-231/CCL5-siRNA was not different from MDA-MB-231/CCL5-NC and MDA-MB-231 (P>0.05), the proliferation index (PI) of group KD, NC and CON were (0.48±0.02), (0.44±0.05) and (0.47±0.02) respectively. The difference was not statistically significant by multiple comparison (P>0.05). Conclusion CCL5-specific siRNA can specifically suppress the colony formation and migration of human high-matastatic breast cancer cells.     

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.     

MiR-519d靶向调控OCT4抑制膀胱癌细胞系5637增殖

目的:探讨miR-519d对膀胱癌细胞系5637增殖的影响及分子机制。方法:通过转染miRNA mimics在膀胱癌细胞系5637中过表达miR-519d,分别利用MTS及细胞集落形成试验,检测膀胱癌细胞系5637增殖能力的改变。通过双荧光素酶报道实验和Western Blot方法验证miR-519d对OCT4的靶向调控。利用OCT4特异性干扰RNA证实miR-519d通过靶向调控OCT4抑制膀胱癌细胞系5673增殖。结果:在膀胱癌细胞系过表达miR-519d后,细胞活性、增殖能力明显下降;双荧光素酶报道实验结果表明,相对于各对照组,共转染OCT4 3'UTR载体与miR-519d组荧光素酶相对活性明显降低(P0.05)。过表达miR-519d后5637细胞中OCT4蛋白表达下调。利用siRNA特异性下调OCT4表达后,5637细胞活性及增殖能力同样受到抑制。结论:miR-519d能靶向结合OCT4 3'UTR序列,通过下调OCT4的表达抑制膀胱癌细胞系5637的增殖。     

siRNA沉默高迁移率蛋白A2基因对人膀胱移行细胞癌细胞T24增殖凋亡的影响

目的 观察siRNA靶向沉默HMGA2基因在人膀胱移行细胞癌T24细胞中表达的变化,探讨抑制HMGA2基因对T24细胞增殖、周期和凋亡的影响.方法 针对人HMGA2基因构建siRNA干扰片段,瞬时转染T24细胞后,采用Western-blot方法检测转染siRNA干扰片段48h后T24细胞蛋白表达量的变化,CCK-8法检测T24细胞增殖和流式细胞仪（FCM）检测T24细胞周期和凋亡情况.结果 转染后48h的T24细胞HMGA2蛋白表达水平较空白对照组（CON）和阴性对照组（NC）显著下降,差异有统计学意义（P＜0.05）,转染HMGA2-siRNA的实验组抑制T24细胞增殖,T24细胞S期细胞比例（35.47±0.23）％高于CON组和NC组（P＜0.05）,实验组T24细胞凋亡率为（17.25±0.24）％,明显高于CON组和NC组（P＜0.05）.结论 HMGA2基因的特异性siRNA可以抑制T24细胞增殖,细胞周期阻滞在S期,并促进其凋亡.     

Clusterin对膀胱癌细胞增殖能力机制的研究

摘 要：目的：Cluterin（CLU）又称载脂蛋白J,在生物体中有两种存在的形式：分泌型（sCLU）和核型（nCLU）。sCLU常常与肿瘤的发生发展有密切的关系。越来越多的研究表明,sCLU与膀胱癌耐药有关,高表达常提示预后不良。然而,鲜有针对sCLU调控膀胱癌参与肿瘤发生发展机制的研究。本次实验探讨sCLU调控膀胱癌发生发展的分子机制。方法：我们收集了16对膀胱癌组织样本,通过实时定量聚合酶链反应(RT-PCR)检测sCLU的表达。利用Si-RNA抑制膀胱癌细胞clusterin基因的表达,利用CCK-8增殖实验,平板克隆实验研究sCLU对膀胱癌细胞增殖的影响。同时,我们利用蛋白印迹分析（Western Blot）检测MAPK通路蛋白研究sCLU对膀胱癌细胞的调控机制。结果：16对膀胱癌组织验证表明sCLU在膀胱癌组织中高表达。敲低膀胱癌细胞中sCLU的表达,细胞增殖能力明显受到抑制（P<0.05）,P38/MAPK信号通路相关蛋白表达降低。结论： sCLU可能通过P38/MAPK通路调控膀胱癌细胞的增殖能力。     

TRPM7在膀胱癌细胞增殖与凋亡中的调控作用

目的 探讨薄荷醇受体7（transient receptorpotential melastatin 7,TRPM7）在膀胱癌细胞株T24细胞增殖与凋亡中的调控作用及分子机制.方法 采用RT-PCR及Western blot检测T24细胞株中TRPM7 mRNA及蛋白的表达;分别采用通道阻滞剂及基因沉默的方法阻断TRPM7离子通道的功能,MTT法检测细胞存活率,流式细胞术检测细胞周期分布及细胞凋亡率,Western blot检测Cdk4、Cdk6及Cyto C的表达.结果 RT-PCR及Western blot证实T24细胞株中存在TRPM7 mRNA及蛋白的表达;采用基因沉默及通道阻滞剂阻断TRPM7的功能后,T24细胞存活率分别下降了56.48％和54.87％,处于G0/G1期的细胞随阻滞剂浓度增加而显著增加,细胞凋亡率亦随之增加并呈浓度依赖性,与对照组比较差异均有统计学意义（P＜0.05）.Western blot显示阻断TRPM7后,T24细胞Cdk4、Cdk6的表达减少,而Cyto C的表达增加.结论 TRPM7可促进T24细胞增殖,抑制细胞凋亡.这一过程可能通过调节Cdk4、Cdk6及Cyto C的表达来实现.阻断TRPM7的功能,能够抑制T24细胞增殖,促进细胞凋亡,可能为临床治疗膀胱癌提供新的靶点.     

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.     

Devazepide suppresses cell proliferation and migration,and induces apoptosis in bladder carcinoma

BackgroundThis study aimed to examine the effects of devazepide on the proliferation, migration, and apoptosis of human bladder cancer (BC) 5637 cells, and its mechanism.MethodsA cell counting kit-8 (CCK-8) for cell viability assays, a colony formation assay, and immunofluorescence were applied to detect the effects of devazepide on the proliferation of 5637 cells. Cell cycle assay, cell apoptosis assay and wound healing assay were performed to detect the effects of devazepide on the cell cycle, apoptosis, and migration of 5637 cells. The protein expression of CyclinD1, Bcl-2-associated X protein (Bax), poly ADP-ribose polymerase 1 (PARP1), and Cleaved Caspase-3 in 5637 cells was detected by a western blot assay.ResultsThe proliferation of 5637 cells was significantly inhibited (P<0.001) after incubation with 12, 25, and 50 µM devazepide for 48 and 72 h. A treatment of 25 µM devazepide for 48 h induced G1–S cell cycle arrest and apoptosis (P<0.01), and inhibited cell migration (P<0.05). By western blot assay, we found that devazepide can down-regulate CyclinD1 expression, and up-regulate Bax, PARP1, and Cleaved Caspase-3 expression.ConclusionsDevazepide inhibits the migration and proliferation of human BC 5637 cells by arresting the G1–S cell cycle, and induces cell apoptosis.     

Pseudomonas aeruginosa-mannose–sensitive hemagglutinin inhibits epidermal growth factor receptor signaling pathway activation and induces apoptosis in bladder cancer cells in vitro and in vivo

ObjectivesPseudomonas aeruginosa-mannose–sensitive hemagglutinin (PA-MSHA), a peritrichous P. aeruginosa strain with MSHA fimbriae, has been shown to be a valuable anticancer drug in many kinds of cancers. However, the effect of PA-MSHA on bladder cancer has not been elucidated. In this study, we focused on the antitumor activities and related mechanisms of PA-MSHA on bladder cancer in vitro and in vivo.Materials and methodsSV-40-immortalized normal uroepithelial cells (SV-HUC-1) and human bladder cancer cell lines (T24, 5637, and HT-1376) were treated with PA-MSHA or PA (heat-killed P. aeruginosa). At first, the effect of PA-MSHA on cancer cell proliferation was measured using Cell Counting Assay Kit-8 (CCK-8), whereas the changes of cell morphology were observed by transmission electron microscopy. The early apoptosis induced by PA-MSHA was evaluated by flow cytometry, and the expression level of apoptosis-related molecules was detected using Western blot assay. We then investigated the activation of the epidermal growth factor receptor signaling pathway stimulated by PA-MSHA; the expression and phosphorylation of several key regulators involved in the EGFR signaling pathway were detected. At last, xenograft tumor in nude mice was used to further investigate the antitumor effect of PA-MSHA in vivo.ResultsOur results showed that PA-MSHA could efficiently inhibit proliferation and induce apoptosis in human bladder cancer cell lines. Furthermore, cells stimulated with PA-MSHA exhibited an inactivation of EGFR signaling. In vivo, PA-MSHA treatment significantly suppressed tumor growth and induced apoptosis in xenografts tumor in nude mice.ConclusionsPA-MSHA could efficiently inhibit proliferation and induce apoptosis in human bladder cancer cells in vitro and in vivo, which is associated with the inactivation of EGFR signaling pathway, and it might be used as a potential therapeutic agent for bladder cancer.     

哺乳动物雷帕霉素靶蛋白抑制剂temsirolimus对膀胱癌作用的实验研究

目的 研究哺乳动物雷帕霉素靶蛋白( mTOR)抑制剂temsirolimus对膀胱癌T24、BIU-87细胞株的作用,探讨以mTOR为靶点治疗膀胱癌的应用前景。 方法 膀胱癌T24、BIU-87细胞株经不同浓度temsirolimus作用后,采用噻唑盐法检测temsirolimus对细胞株增殖的影响;流式细胞技术检测temsirolimus对细胞周期、细胞凋亡的影响;细胞迁移实验及transwell细胞侵袭实验检测temsirolimus对肿瘤迁移和浸润的影响;蛋白质印迹法检测mTOR的表达情况;制作裸鼠T24细胞株移植瘤模型,检测temsirolimus对移植瘤生长的作用,免疫组化检测移植瘤Ki-67表达情况。 结果 temsirolimus能够抑制膀胱癌T24、BIU-87细胞株增殖,呈浓度和时间依赖性;temsirolimus能够抑制膀胱癌细胞迁移能力,temsirolimus作用于T24、BIU-87细胞株24 h后,0 nmol/L组划痕修复率分别为(88.9±14.1)％、(83.6±16.3)％,高于5 nmol/L组的(42.7±11.6)％、(36.9±9.7)％,差异有统计学意义(P＜0.05);temsirolimus能够抑制膀胱癌细胞浸润能力,temsirolimus作用于T24、BIU-87细胞株24h后,0 nmol/L组浸润细胞数(26.5±5.8)、(28.2±4.6),高于5 nmol/L组的(19.0±3.8)、(21.3±5.1),差异有统计学意义(P＜0.05);temsirolimus导致膀胱癌细胞周期阻滞于G0/G1期,temsirolimus作用T24、BIU-87细胞株48 h后,5 nmol/L组G0/G1期细胞分别占(77.46±6.11)％、(73.39±4.94)％,高于0 nmol/L组的(65.99±5.01)％、(60.15±3.98)％,差异有统计学意义(P＜0.05);各组细胞凋亡率差异无统计学意义(P＞0.05);temsirolimus能够阻断mTOR的磷酸化,T24、BIU-87细胞株0 nmol/L组p-mTOR/β-actin值分别为0.92±0.09、1.01±0.08,明显高于5 nmol/L组的0.47±0.05、0.04±0.01,差异有统计学意义(P＜0.05);temsirolimus能够抑制裸鼠T24细胞株移植瘤生长,给药21 d后,给药组移植瘤体积为(147.6±74.4) mm3,对照组为(351.1±139.9) mm3,差异有统计学意义(P＜0.05);给药组移植瘤Ki-67表达阳性细胞百分率为(35.5±6.7)％,低于对照组的(67.3±8.4)％,差异有统计学意义(P＜0.05)。 结论 mTOR抑制剂temsirolimus对膀胱癌细胞具有明显的抑制作用,可考虑将mTOR作为膀胱癌治疗的靶点。     

膀胱癌细胞株T24中侧群细胞的分离和功能鉴定

目的 观察膀胱癌细胞株T24中是否存在侧群(SP)细胞及其比例,并鉴定其功能.方法 利用双波长流氏细胞仪(FACS)检测T24中SP细胞的比例,并证实这些SP细胞是否具有癌干细胞的特点.结果 T24中SP细胞占34.7%;与非侧群(NSP)细胞比较,SP细胞有更强的生长增殖能力和克隆形成能力(P＜0.05),表达更高的ATP结合转运蛋白G超家族成员2(ABCG2)和干性基因,对放化疗有更强的抵抗能力,有更多的细胞处于G_0/G_1期(87.4%比63.3%,P＜0.05);分选后的sP和NSP细胞经过约10 d的常规培养,SP细胞中NSP的比例占76.2%,而NSP细胞中SP的比例只占2.6%.结论 膀胱癌细胞株T24中存在很高比例的SP细胞,而且这些SP细胞有癌干细胞的特点.