Over-expression of LRIG3 suppresses growth and invasion of bladder cancer cells

The purpose of this study was to investigate the impact of leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) on the biological features of bladder cancer cell lines. The plasmids of over-expressed LRIG3 and the blank plasmid serving as control were transfected into the bladder cancer cell lines, T24, EJ and BIU-87, and the expression levels of LRIG3 mRNA and protein were detected by using real-time PCR and Western blotting. The changes in the cell cycle and apoptosis were examined by using flow cytometry. The invasive ability was measured by Transwell assay, and CCK-8 assays were used to measure the proliferation of cells. As compared with the control group, the LRIG3 mRNA and protein expression levels in LRIG3 cDNA-transfected group were raised significantly (P<0.05). The average number of cells with up-regulated LRIG3 passing through the inserted filter was decreased significantly as compared with the control group (P<0.05). Up-regulation of LRIG3 also could inhibit proliferation and induce apoptosis of T24, EJ and BIU-87 cells. Except BIU-87, the T24 and EJ cells transfected with LIRG3 cDNA were arrested in G 0 /G 1 phase compared to the control group (P<0.05). In conclusion, the over-expression of LRIG3 could influence the cell cycle and invasion, inhibit proliferation and induce apoptosis in the three bladder cancer cell lines.     

Expression and biological function of N-myc down-regulated gene 1 in human cervical cancer

The expression of N-myc down-regulated gene 1 (NDRG1) has previously been reported to be involved in the proliferation,differentiation,invasion and metastasis of cancer cells,but its role in cervical cancer is still unclear.This study aimed to investigate the expression of NDRG1gene in human cervical cancer and its effect on aggressive tumor behaviors.The NDRG1 expression in cervical tissues and cells was detected by RT-PCR.Specific expression plasmid pEGFP-N1-NDRG1-GFP was used to enhance the expression of NDRG1 in human cervical cancer cell lines.The mRNA and protein level of NDRG1 was assessed by RT-PCR and Western blotting,respectively.Its effects on cell proliferation,migration,invasion,cell cycle and apoptosis were detected by MTT,transwell migration assay and flow cytometry (FCM),respectively.The results showed that the expression of NDRG1 in cervical cancer tissues and cells was significantly lower than in normal cervical tissues (P<0.001).After transfection with pEGFP-N1-NDRG1-GFP,the mRNA and protein expression of NDRG1 was up-regulated in Siha cells,which suppressed cell proliferation (P<0.001),induced cell cycle arrest (P<0.05),reduced invasion and migration of Siha cells (P<0.05),but caused no cell apoptosis.Moreover,vascular endothelial growth factor (VEGF),a tumor-induced angiogenesis factor,was markedly reduced and E-cadherin,a cell adhesion molecule,was increased in the cells transfected with pEGFP-N1-NDRG1-GFP.It was concluded that up-regulated NDRG1 may play a role in the suppression of malignant cell growth,invasion and metastasis of human cervical cancer.     

Effect of Smac on TRAIL-induced apoptosis of prostate cancer cell line PC-3 and the molecular mechanism

The effect of Smac gene on the TRAIL-induced apoptosis of the prostate cancer cell line PC-3 and the molecular mechanism were investigated. The Smac gene was transfected into PC-3 cells under the induction of liposome. The intrinsic Smac gene expression was detected by Western blotting. After treatment with TRAIL as an apoptosis inducer, in vitro cell growth activity was as-sayed by MTT colorimetry. The apoptosis rate of PC-3 cells was determined by annexin Ⅴ-FITC and propidium iodide staining flow cytometry. The expression of cellular XIAP and caspase-3 genes was examined by Western blotting. Smac-transfected cells (PC-3/Smac group) had significantly in-creased Smac protein level as compared with PC-3 controls (P<0.01). After induction with 100-200 ng/mL TRAIL for 12-36 h, cellular proliferation rate in PC-3/Smac group was significantly lower than in PC-3 controls (P<0.05). After induction with 100 ng/mL TRAIL for 24 h, the apoptosis rate in PC-3/Smac group was significantly enhanced as compared with that of PC-3 controls (P<0.05). Ac-cordingly, the XIAP expression level was down-regulated significantly (P<0.05) and caspase-3 sub-unit P20 was up-regulated significantly (P<0.05). It is suggested that the over-expression of cellular Smac can inhibit inhibitor of apoptosis proteins (IAPs), enhance caspases activity and the apoptosis rate of PC-3 cells induced by TRAIL, which may provide a useful experimental basis for prostate cancer therapy.     

Isolation and identification of cancer stem-like cells from side population of human prostate cancer cells

It has been widely verified by various sorting methods that cancer stem cells (CSCs) exist in different types of tumor cells or tissues.However,due to lack of specific stem cell surface markers,CSCs are very difficult to be separated from some cancer cells,which becomes the key barrier of functional studies of CSCs.The sorting method by side population cells (SP) lays a solid foundation for in-depth and comprehensive study of CSCs.To identify the existence of SP in prostate cancer cell lines,we applied flow cytometry sorting by SP to cultures of prostate cancer cell lines (TSU,LnCap,and PC-3),and the cancer stem-like characteristics of SP were verified through experiments in vitro and in vivo.The proportion of SP in TSU cells was calculated to be 1.60%±0.40% (±s),and that in PC-3 and LnCap cells was calculated to be 0.80%±0.05% and 0.60%±0.20%,respectively.The colony formation assay demonstrated that the colony formation rate of SP to non-SP sorted from TSU via flow cytometry was 0.495±0.038 to 0.177±0.029 in 500 cells,0.505±0.026 to 0.169±0.024 in 250 cells,and 0.088±0.016 to 0.043±0.012 in 125 cells respectively.In the in vivo experiments,tumors were observed in all the mice on the 10th day after injecting 50 000 cells subcutaneously in SP group,whereas when 5×106 cells were injected in non-SP group,tumors were developed in only 4 out of 8 mice until the 3rd week before the end of the experiment.Our results revealed that prostate cancer cells contain a small subset of cells,called SP,possessing much greater capacity of colony formation and tumorigenic potential than non-SP.These suggest that SP in prostate cancer cells may play a key role in the self-renewal and proliferation,and have the characteristics of cancer stem-like cells.Dissecting these features will provide a new understanding of the function of prostate CSCs in tumorigenicity and transformation.     

Down-regulation of p110β expression increases chemosensitivity of colon cancer cell lines to oxaliplatin

This study examined the synergetic effect of class ?A Phosphoinositide 3-kinases cata-lytic subunit p110β knockdown in conjunction with oxaliplatin treatment on colon cancer cells. Down-regulation of p110β by siRNA interference and oxaliplatin treatment were applied in colon cancer cell lines HT29, SW620 and HCT116. MTT assay was used to measure the inhibitory effect of p110β knockdown on the proliferation of colon cancer cell lines. SubG1 assay and Annexin-Ⅴ FITC/PI double-labeling cytometry were applied to detect cell apoptosis. And cell cycle was evalu-ated by using PI staining and flow cytometry. The expression of caspase 3, cleaved PARP, p-Akt, T-Akt and p110β was determined by western blotting. The results suggested that down-regulation of p110β expression by siRNA obviously reduced cell number via accumulation in G0-G1 phase of the cell cycle in the absence of notablely increased apoptosis in colon cancer cell lines HT29 and SW620 (S phase arrest in HCT116). Moreover, inhibition of p110β expression increased oxaliplatin-induced cell apoptosis and cell cycle arrest in HT29, HCT116 and SW620 cell lines. In addition, increases of cleaved caspase-3 and cleaved PARP induced by oxaliplatin treatment were determined by im-munoblotting in p110β knockdown group compared with normal control group and wild-type group. It is concluded that down-regulated expression of p110β could inhibit colon cancer cells proliferation and result in increased chemosensitivity of colorectal cancer cells to oxaliplatin through augmentation of oxaliplatin-induced cell apoptosis and cell cycle arrest.     

Silencing MTA1 by RNAi Reverses Adhesion,Migration and Invasiveness of Cervical Cancer Cells （SiHa） via Altered Expression of p53, and E-cadherin/β-catenin Complex

It has been reported that metastasis-associated gene 1 (Mta1) is overexpressed in many malignant tumors with high metastatic potential. In addition, some studies indicated that MTA1 participated in invasion, metastasis, and survival of cancer cells by regulating cell migration, adhesion and proliferation. But the role of MTA1 is unclear in vitro in the development of cervical cancer cells. This study investigated whether and how MTA1 mediated cell proliferation, migration, invasion and adhesion in cervical cancer. MTA1 expression level was detected by Western blot in two cervical cancer cell lines of different invasion potentials. The effects of MTA1 expression on SiHa cell apoptosis, cycle, proliferation, migration, invasion and adhesion were tested by flow cytometry, MTT, wound-healing assay, Transwell assay and adhesion assay, respectively. The expression levels of p53, E-cadherin, and β-catenin activity were evaluated in untreated and treated cells. The results showed that MTA1 protein expression was significantly higher in SiHa than in HeLa, which was correlated well with the potential of migration and invasion in both cell lines. Furthermore, the cell invasion, migration and adhesion capabilities were decreased after inhibition of MTA1 expression mediated by Mta1-siRNA transfection in SiHa. However, no significant differences were found in cell apoptosis, cycle, and proliferation. In addition, E-cadherin and p53 protein levels were significantly up-regulated, while β-catenin was significantly down-regulated in SiHa transfected with the siRNA. These results demonstrated that MTA1 played an important role in the migration and invasion of cervical cancer cells. It was speculated that the decreased migration and invasion capability by inhibiting the MTA1 expression in the SiHa cell line may be mediated through the altered expression of p53, and E-cadherin/β-catenin complex. MTA1 could serve as a potential therapeutic target in cervical cancer.     

In vitro and in vivo evidence of metallopanstimulin-1 in gastric cancer progression and tumorigenicity

Purpose:The metallopanstimulin-1(MPS-1)gene is a growth factor-inducible gene,which is highly expressed in many human cancers and may be involved in the progression towards tumor malignancy.However,it is unclear whether MPS-1 plays any role in gastric cancer development or progression.Our studies were designed to clarify the MPS-1 expression pattern and to explore its potential role in gastric cancer.Experimental Design:The expression pattern of MPS-1 was determined in primary gastric cancer specimens and gastric cancer cell lines via immunohistochemistry and Western blotting.To investigate the functional significance of MPS-1 expression,three small interfering RNA(siRNA)expression plasmids were constructed and transfected into gastric cancer cell line SGC7901.The stable cell lines transfected with the siRNA targeting MPS-1 mRNA plasmids were selected and the biological features of these cells were examined.Results:MPS-1 was overexpressed in 86% of the gastric cancer tissues and all gastric cancer cells.In addition,MPS-1 expression was significantly increased and corresponded with the tumor-node-metastasis clinical stage,and was significantly higher in the late stage(P<0.01).The MPS-1 expression level was significantly decreased in the transfected cells with MPS-1-specific siRNA expression plasmid pRNAT-133.Furthermore,the stable transfected cancer cells exhibited an increase in the incidence of spontaneous apoptosis and a decrease in growth ability and tumorigenicity in nude mice.Conclusions:These results provide strong evidence that MPS-1 plays an important role in gastric cancer cell proliferation and development,and suggests that MPS-1 is a promising target for gastric cancer treatment.     

胃癌组织和SGC-7901细胞中nucleostemin基因表达的实验研究

目的检测nucleostemin(NS)基因在胃癌组织和胃癌SGC-7901细胞中的表达情况。方法21例胃癌手术标本,提取胃癌组织和胃癌SGC-7901细胞株总RNA,用半定量RT-PCR方法检测胃癌组织和SGC-7901中NS基因的表达情况。用脂质体法将PCDNA4/C-NS-silencer质粒及空载体PCDNA4/C-vector质粒分别转染SGC-7901细胞,分别命名为silencer组和vector组,未转染的SGC-7901细胞记为normal组,RT-PCR方法检测NS基因表达量。用MTT法检测3组细胞的生长增殖情况。结果胃癌组织和SGC-7901细胞中皆有NS基因表达。定性分析显示,与vector组和normal组比较,silencer组细胞趋于分化,NS基因表达量下降。细胞培养24 h、48 h、72 h后,si-lencer组和vector组细胞增殖抑制率间差别均有统计学意义(P<0.01)。结论胃癌组织和SGC-7901细胞中有NS基因表达;NS基因特异性RNA干扰使NS基因表达量下降,抑制SGC-7901细胞株体外增殖。     

黄芩素对人前列腺癌 PC －3细胞增殖、侵袭的抑制作用及机制研究

目的：探讨黄芩素对人前列腺癌PC－3细胞增殖、侵袭的影响及其相关机制。方法培养人前列腺癌PC－3细胞,分别予20μL的培养液（对照组）、无水乙醇（溶媒组）、黄芩素溶液（100、200μmol／L黄芩素组）处理细胞48 h后,通过四甲基偶氮唑蓝（ MTT）法分析PC－3细胞的增殖情况,Transwell侵袭实验检测PC－3细胞的侵袭能力,荧光实时定量PCR检测PC－3细胞中Ezrin mRNA表达,Western blot测定PC－3细胞中Ezrin、Bcl－2、Bax蛋白表达,原位末端标记法（ TUNEL）检测PC－3细胞凋亡率。结果100、200μmol／L黄芩素作用PC－3细胞48 h后,细胞增殖抑制率、凋亡率以及Bax蛋白表达水平增加,平均穿膜细胞数以及Ezrin、Bcl－2蛋白表达水平降低,且具有剂量依赖性（ P＜0．01）,然而溶媒组上述指标与对照组比较,差异无统计学意义（ P＞0．05）。结论黄芩素能够降低人前列腺癌PC－3细胞的增殖、侵袭性,其机制可能与抑制Ezrin蛋白表达及促进肿瘤细胞凋亡有关。     

Nucleostemin特异性RNA干扰联合化疗药物对肿瘤细胞增殖抑制作用的体外实验研究

目的：研究Nucleostemin特异性RNA干扰（RNAi）联合4种化疗药物对人食管癌细胞株Eca-109体外增殖的影响。方法：1.用NS特异性RNAi表达载体转染Eca-109细胞。2.实验分组：分为silencer组（S组）、normal组（N组）、单纯用药组和联合用药组。S组为转染NS特异性siRNA表达载体的Eca-109细胞;N组为正常Eca-109细胞;单纯用药组为单纯应用4种化疗药物作用于Eca-109细胞;联合用药组为转染NS特异性RNAi表达载体分别联合4种化疗药物作用于Eca-109细胞。3.利用细胞计数法、镜下细胞形态学观察和MTT法检测各组细胞的增殖情况。结果：联合用药各组增殖速率明显低于单纯用药各组,而增殖抑制率明显高于单纯用药各组,差异具有显著性（p〈0.05）。结论：4种化疗药物与NS特异性RNAi联合对Eca-109细胞都具有一定的协同抑制作用。     

E2F陷阱DNA对前列腺癌细胞增殖的影响

目的 观察转录因子E2F陷阱DNA策略对雄激素非依赖性前列腺癌细胞PC-3M增殖的影响。方法 采用脂质体Lipofectarnine将E2F decoy DNA、ARE decoy DNA和Control decoy DNA分别转染PC-3M细胞，倒置相差显微镜观察细胞形态变化，MTT检测其对细胞增生活性的影响。结果 E2F decoy DNA转染后的PC-3M细胞的生长受到明显抑制，转染后的细胞形态变化。结论 E2F decoy DNA明显抑制雄激素非依赖性前列腺癌PG3M细胞增殖。     

低氧微环境下siRNA靶向沉默HIF 2α基因对前列腺癌PC 3细胞增殖和凋亡的影响

目的 观察体外低氧条件下缺氧诱导因子-2α(HIF-2α)在人前列腺癌细胞PC-3中的表达情况,并研究siRNA沉默HIF-2α基因对前列腺癌PC-3细胞生长及凋亡的影响.方法 在体外培养的PC-3细胞中加入化学诱导剂CoCl2建立低氧模型,采用逆转录PCR(RT-PCR)和Western印迹法检测CoCl2诱导HIF-2αmRNA和蛋白表达的时-效和量-效关系.合成HIF-2αsiRNA片段并转染前列腺癌PC-3细胞,采用RT-PCR及Western印迹法检测HIF-2αsiRNA对HIF-2α基因表达的影响,采用MTT法检测转染HIF-2αsiRNA后对PC-3细胞生长的抑制情况,采用流式细胞仪检测RNA干扰对PC-3细胞凋亡的影响.结果 (1)低氧可诱导PC-3细胞的HIF-2αmRNA表达,与常氧组比较,低氧12,24及48 h组的HIF-2αmRNA和蛋白表达量逐渐升高(P<0.05).100μmol/L的CoCl2作用48 h可作为最佳前列腺癌细胞PC-3低氧模型.(2)低氧+HIF-2αsiRNA干扰组的HIF-2αmRNA和蛋白表达水平明显低于低氧+阴性对照组和低氧组(P<0.05),而低氧+阴性对照组与低氧组的HIF-2αmRNA和蛋白表达水平无显著性差异(P>0.05).低氧+HIF-2αsiRNA干扰组细胞增殖受抑制、细胞凋亡增加(P<0.05),而常氧组、低氧组、低氧+阴性对照组细胞增殖活力和凋亡差别均无统计学意义(P>0.05).     

雄激素受体对IgG蛋白表达及前列腺癌细胞增殖和迁移的影响

目的 研究前列腺癌细胞中雄激素受体(AR)对免疫球蛋白lgG表达的影响,及对前列腺癌细胞增殖、迁移的作用.方法 (1)Western blot检测雄激素依赖性前列腺癌细胞株LNCap与去势抵抗性前列腺癌细胞株PC-3中AR、IgG蛋白的表达;(2)AR基因(pCDNA3.1+)转染PC-3构建AR过表达的PC-3-AR细胞,沉默LNCap中AR基因表达构建LNCap-siAR细胞;Westernblot检测AR及IgG表达量;Q-PCR检测细胞株中IgG mRNA表达量;四氮甲基唑氮、细胞划痕方法检测细胞的增殖及迁移能力.结果 LNCap细胞与PC-3细胞相比较,AR高表达,IgG低表达(P＜0.05).PC-3-AR细胞lgG降低表达(P＜0.01),细胞增殖及迁移能力减弱;LNCap-siAR细胞IgG增高表达(P＜0.01),细胞增殖及迁移能力增强.结论 AR调控IgG蛋白表达呈负相关并与前列腺癌细胞增殖、迁移能力相关.     

沉默液泡型ATP酶c亚基ATP6V0C抑制人前列腺癌细胞侵袭的分子机制

目的： 采用RNA干涉技术沉默液泡型ATP酶c亚基ATP6V0C的表达,探索ATP6V0C在前列腺癌侵袭中的分子作用机制,并研究其与LASS2/TMSG1的关系。方法与结果： ATP6V0C在高转移潜能人前列腺癌细胞系（PC-3M-1E8和PC-3M）中的表达明显高于低转移潜能人前列腺癌细胞系（PC-3M-2B4和PC-3）, 选择ATP6V0C表达量最高的PC-3M-1E8细胞进行基因沉默实验,发现ATP6V0C的siRNA转染PC-3M-1E8后,其基质金属蛋白酶2（matrix metalloprotein-2,MMP-2）和基质金属蛋白酶9(MMP-9)蛋白的表达量及MMP-2的分泌量均无明显变化,但上清液中MMP-9的活性明显减弱,与空白对照组及阴性对照组相比,差异有统计学意义（P<0.05);干扰组细胞外氢离子浓度及液泡型ATPase的活性明显降低（P<0.05);细胞划痕修复实验及transwell体外侵袭实验结果显示ATP6V0C siRNA干扰组细胞的体外迁移及侵袭能力明显减弱（P<0.05)。PC-3M-1E8细胞转染ATP6V0C siRNA后,LASS2/TMSG1的表达明显减弱（P<0.05)。免疫荧光双重染色结果显示ATP6V0C蛋白与LASS2/TMSG1共定位于胞浆和胞膜,干扰组共定位信号与对照组相比明显减弱。结论： 特异性siRNA沉默ATP6V0C能抑制人前列腺癌细胞的迁移和侵袭能力,其机制与ATP6V0C沉默后抑制V-ATPase的活性,使细胞外氢离子浓度降低,抑制MMP-9的激活,从而影响细胞外基质的降解和重塑有关。靶向siRNA干扰ATP6V0C的表达后,可能通过反馈调节机制抑制LASS2/TMSG1的表达,但其具体分子机制尚待进一步研究。     

岩藻糖基转移酶II对前列腺癌细胞生物学功能的影响及机制

目的：探讨岩藻糖基转移酶II（FUT2）对前列腺癌细胞生物学功能的影响及其潜在机制。方法：通过免疫组化技术检测FUT2 在前列腺癌组织中的表达情况。利用脂质体转染技术构建低表达FUT2的前列腺癌细胞株及其对照细胞株,并通过MTT实验、平板克隆实验、Transwell实验检测干扰FUT2表达对前列腺癌细胞生物学功能的影响。采用Western blot检测β-catenin、Cyclin D1和ICAM-1等蛋白表达水平。结果：FUT2在前列腺癌组织中的表达水平明显升高（P ＜0.05）。干扰FUT2的表达后,前列腺癌细胞的增殖、迁移和侵袭能力下降,Cyclin D1表达下调,ICAM-1表达上调（P ＜0.05）。结论：FUT2促进前列腺癌细胞的增殖、迁移和侵袭能力。     

RNA干扰Aurora-A基因对前列腺癌细胞株PC-3影响的研究

目的采用RNA干扰技术特异性阻断前列腺癌细胞PC-3中Aurora-A的表达,观察其对肿瘤细胞生长的抑制作用。方法采用免疫组化方法检测Aurora-A在前列腺癌细胞中的表达。体外化学合成特异针对Aurora-A基因的小干扰RNA(small interfering RNA,siRNA),以脂质体转染前列腺癌细胞系PC-3,荧光显微镜及流式细胞仪确定最佳转染效率。MTT法检测细胞增殖抑制率,流式细胞仪检测细胞凋亡率。RT-PCR和Western-blot检测Aurora-A mRNA和蛋白的表达。结果 Aurora-A蛋白主要表达于PC-3细胞胞浆中;脂质体的最佳转染效率高于90%;siR-NA1组24h、48h、72h细胞增殖抑制率分别为(0.377±0.035)%、(0.597±0.065)%、(0.749±0.061)%,与各组比较均有显著性差异;siRNA1组细胞凋亡率为(50.593±1.208)%,与各组比较均有显著性差异;RT-PCR检测siRNA1组Aurora-A mRNA相对值为(1.354±0.087),与各组比较均有显著性差异;Western-blot检测显示siRNA 1组蛋白表达量明显低于对照组。结论应用RNAi技术靶向沉默Aurora-A基因可以下调Aurora-A mRNA及蛋白的表达,抑制细胞增殖,促进细胞凋亡。