Small G-protein RhoE is underexpressed in prostate cancer and induces cell cycle arrest and apoptosis

BACKGROUND: RhoE/Rnd3, a recently described novel member of the Rho GTPases family, was discussed as a possible antagonist of the RhoA protein that stimulates cell cycle progression and is overexpressed and/or overactivated in prostate cancer. We investigated the expression of RhoE and its role in cell cycle regulation and apoptosis in the human prostate. METHODS: RhoE expression in cell lines and tissue specimens was assessed by immunoblot analysis, real-time PCR (RT-PCR), and immunohistochemistry. To elucidate RhoE effects on the prostate, RhoE was cloned and overexpressed in DU-145 prostate cancer. Cell cycle modulation and apoptosis was investigated by immunoblot and FACS analysis. RESULTS: Immunoblot analysis showed a strong RhoE signal in both, benign epithelial and stromal cells. In contrast, almost no protein was detected in various prostate cancer cells. On RT-PCR and microarray analysis, RhoE mRNA expression was significantly reduced in malignant tissue when compared to benign samples. RhoE immunostaining was strong in benign tissue, especially in prostate epithelial cells, whereas it was minimal or absent in malignant tissue. Forced RhoE overexpression in a prostate cancer cell line inhibits the expression of two proteins essential for G2/M transition, namely CDC2 and cyclin B1, and induces G2/M arrest. In addition, apoptotic cell death as measured by a cleavage product of caspase 3 is significantly increased in RhoE-overexpressing cells. CONCLUSION: In conclusion, our findings suggest RhoE as a tumor suppressor gene that is downregulated early in the development of prostate cancer.     

RhoE对cd44基因启动子的转录活性及大肠癌细胞系恶性生物学行为的影响

目的研究RhoE对cd44基因启动子的转录调控,并探讨RhoE对裸鼠体内成瘤性的影响。方法采用PCR技术从人胚肾HEK293细胞中扩增出cd44启动子,并将其插入荧光素酶报告基因载体pGL3-Basic中,经测序确定所扩增的DNA序列,并将其转染入SW480及LoVo细胞中监测其活性。将pcDNA3.1-RhoE和pcDNA3.1分别与pGL3-CD44 promoter共转染大肠癌LoVo细胞和SW480细胞,双荧光素报告基因检测活性。另外将pcDNA3.1-RhoE和对照组分别稳定转染LoVo细胞和SW480细胞,并将筛选的稳定表达的细胞分别接种裸鼠,观察肿瘤的生长;用免疫组化的方法检测RhoE对肿瘤组织细胞形态及CD44分子表达的影响。结果测序结果表明,扩增的cd44启动子序列正确,双报告基因实验检测荧光素酶活力表明构建的报告基因具有启动子活性。含cd44启动子序列的报告基因在pcDNA3.1-RhoE阳性的LoVo细胞中表达受到抑制;HE染色显示pcDNA3.1-RhoE转染组较对照组肿瘤细胞明显变小,且大小和形态呈现一致性,已没有瘤巨细胞,相应的肿瘤细胞核的体积也变小。结论 RhoE通过抑制cd44...     

RhoE regulates actin cytoskeleton organization in human periodontal ligament cells under mechanical stress

ObjectivesRhoE and regulator of G-proteins signalling (RGS) 2 were identified as the up-regulated genes in human periodontal ligament (PDL) cells under compression. RhoE belongs to the Rho GTPase family, and RGS2, a novel family of GTPase-activating proteins, turns off the G-protein signalling. Rho family proteins have recently been known to regulate actin cytoskeleton dynamics in various cell types. In this study, we investigated the involvement of RhoE and RGS2 in the regulation of actin filament organization in the PDL cells under mechanical stress.MethodsHuman PDL cells were cultured and subjected to a static compressive force (3.0 g/cm²) for 48 h. To observe changes in the actin cytoskeleton and the expression of RhoE and RGS2 in response to mechanical stress, immunofluorescence analysis was performed. To examine the role of RhoE and RGS2 in actin filament organization, cells were transfected with antisense S-oligonucleotides (ODNs) to RhoE and RGS2.ResultsCompressive force caused a loss and disassembly of actin stress fibres leading to cell spreading. Immunocytochemical study revealed that RhoE and RGS2 expressions were induced by mechanical stress and localized in the perinuclear and in the cell membrane, respectively. The impaired formation of stress fibres caused by compressive forces was recovered by treatment with antisense S-ODN to RhoE to the control levels. However, addition of antisense S-ODN to RGS2 did not affect the stress fibre formation.ConclusionsThese results indicate that the loss and disassembly of stress fibres due to mechanical stress are mediating RhoE signalling, without the exertion of RGS2.     

RhoE在乳腺癌中的表达及与KiSS-1的相关性

目的 研究分析RhoE 在乳腺癌中的表达与KiSS-1 和临床病理参数的关系, 探讨乳腺癌生长、侵袭和转移的分子机制.方法 采用免疫组织化学方法检测106 例乳腺癌组织中RhoE 和KiSS-1 的表达情况,分析RhoE 和KiSS-1 与乳腺癌临床病理特征的关系及二者的相关性.结果 RhoE 和KiSS-1 在乳腺癌组织中的表达与有无淋巴结转移和临床分期有关(P ＜0.05),与患者年龄、绝经期、肿瘤T 分期、M 分期无关(P ＞0.05).伴随着淋巴结转移和临床分期的增加,癌组织中RhoE和KiSS-1 的表达显著下调或缺失,差异有统计学意义(P ＜0.05).随着乳腺癌组织分化程度的降低,RhoE 的表达下调或缺失,差异有统计学意义(P ＜0.05);但与KiSS-1 的表达则无关,差异无统计学意义(P ＞0.05).乳腺癌组织中RhoE 和KiSS-1的表达呈正相关(r ＝0.682,P ＜0.05).结论 RhoE和KiSS-1 的表达与乳腺癌临床分期和淋巴结的转移相关,RhoE 同时还和乳腺癌的组织分化程度相关.其相互作用在乳腺癌细胞的增殖、侵袭和转移中起重要作用,RhoE 可能作为一种肿瘤调节蛋白发挥重要调节作用.     

RhoE在胶质瘤组织中的表达及意义

目的探讨RhoE蛋白在胶质瘤恶性进展中的作用以及与临床预后的关系。方法应用免疫组化法检测2例正常脑组织和37例不同病理级别人脑胶质瘤瘤组织中RhoE蛋白表达情况,评估RhoE蛋白表达与胶质瘤的病理分级及临床预后的关系。结果 RhoE蛋白在正常脑组织和胶质瘤中的免疫染色评分分别为7.00±1.00和3.32±0.372。RhoE蛋白表达与胶质瘤恶性程度呈负相关,相关系数r=-0.733。RhoE蛋白免疫染色评分小于4的胶质瘤患者生存期较短(P<0.05)。结论 RhoE在胶质瘤的发生进展中呈负调控作用,RhoE的低表达与胶质瘤临床预后较差相关,其表达情况对判断胶质瘤患者的预后有一定的参考价值。     

RhoE/Rnd3及CyclinB1蛋白在人乳腺癌中的表达及相关性

目的：探讨RhoE/Rnd3及CyclinB1蛋白在乳腺癌中的表达情况及其相关性。方法：采用免疫组化方法检测60例乳腺浸润性导管癌、30例正常乳腺组织中RhoE/Rnd3及CyclinB1蛋白的表达情况。结果：RhoE/Rnd3及Cyclin B1蛋白在正常乳腺中的表达率分别为73.3%和43.3%,二者的表达差异具有显著性意义（P〈0.05）;在乳腺癌中的表达率分别为40.0%和70.0%,二者的表达差异具有显著性意义（P〈0.05）。RhoE/Rnd3及CyclinB1蛋白表达呈负相关（P〈0.05）。RhoE/Rnd3的表达与患者年龄和淋巴结转移呈负相关,与生育史呈正相关;CyclinB1表达与患者年龄和ER状态呈正相关。结论：RhoE/Rnd3在乳腺癌的发生中呈负调控作用,CyclinB1在乳腺癌发生中呈正调控作用,RhoE/Rnd3可能通过抑制CyclinB1来抑制乳腺癌的发生;RhoE/Rnd3过表达与乳腺癌预后较好有关,CyclinB1过表达与乳腺癌预后不良有关。     

RhoE表达上调对U87细胞NF-κB活性的影响

目的探讨RhoE对转录因子NF-κB活性水平的影响。方法将包含RhoE基因的质粒转染胶质瘤U87细胞;免疫印迹实验分析NF-κB亚单位p65在胞浆和胞核中的蛋白表达水平;细胞免疫化学实验检测p65在细胞中的蛋白表达水平和亚细胞定位;免疫印迹实验检测Ikkβ和IκBα蛋白表达水平。结果将pC MVFlA G-RhoE质粒成功转染U87细胞后,免疫印迹实验显示,胞浆和胞核中p65的蛋白表达水平均较对照组下降;细胞免疫化学结果亦表明,p65在细胞核中的定位量明显减少。免疫印迹实验进一步分析显示,NF-κB上游调控分子IκBα表达增加,而Ikkβ表达减少。结论上调RhoE表达水平能够抑制U87细胞转录因子NF-κB活性水平。NF-κB活性的降低可能与RhoE调控Ikk/IκBα机制相关。     

RhoE/Rnd3蛋白在乳腺癌组织中的表达及意义

目的：探讨RhoE/Rnd3蛋白在乳腺癌组织中的表达情况及意义。方法：采用免疫组化方法检测60例乳腺浸润性导管癌、30例正常乳腺组织中RhoE/Rnd3蛋白的表达情况。结果：RhoE/Rnd3蛋白在正常乳腺组织和乳腺癌中的表达率分别为73.3%和40.0%,二者的表达差异具有显著性意义（P〈0.05）RhoE/Rnd3蛋白在乳腺癌中表达与恶性程度呈负相关（P〈0.05）。RhoE/Rnd3的表达与与患者年龄和淋巴结转移呈负相关,与生育史呈正相关。结论：RhoE/Rnd3在乳腺癌的发生中呈负调控作用,RhoE/Rnd3可能通过抑制Cy-clinB1来抑制乳腺癌的发生;RhoE/Rnd3过表达与乳腺癌预后较好有关。     

RhoE/Rnd3蛋白在子宫腺肌病中的表达及意义

目的探讨RhoE/Rnd3蛋白在子宫腺肌病组织中的表达及其潜在的临床意义。方法应用免疫组织化学方法检测32例正常子宫(对照组)内膜、平滑肌组织及38例子宫腺肌病组织(实验组)中在位内膜(A组)、异位内膜(B组)、平滑肌组织中RhoE/Rnd3蛋白的组织定位和表达程度,分析对比其差异。结果①腺上皮细胞:RhoE/Rnd3蛋白在对照组腺体细胞中的表达均高于实验组在位内膜腺体细胞(t=11.3,P<0.05)及异位内膜腺体细胞(t=9.33,P<0.05);在子宫腺肌病在位内膜腺体细胞与异位内膜腺体细胞中的表达差异无统计学意义(t=1.63,P>0.05);②间质细胞及平滑肌:RhoE/Rnd3蛋白在3组内膜间质细胞及对照组-实验组平滑肌细胞中表达差异均无统计学意义(P>0.05);③子宫腺肌病内膜腺体细胞中RhoE/Rnd3蛋白的表达水平与患者的病程有关,病程超过2年的患者组织中,RhoE/Rnd3蛋白的表达水平明显低于病程不足2年的患者(P<0.05)。结论RhoE/Rnd3蛋白在子宫腺肌病的发生发展中发挥重要作用,有可能是一个重要的肿瘤抑制基因。