RNA干扰PAR基因表达对PC3细胞生长的影响

背景与目的：PAR（prostate androgen regulated）基因在雄激素非依赖性的前列腺癌中特异性高表达,可能与细胞的恶性转化有关。本研究采用RNA干扰技术下调前列腺癌PC3细胞中PAR基因的表达,并探讨其对PC3细胞恶性表型的影响及作用机制。方法：构建针对PAR基因的短发夹RNA（short hairpin RNA,shRNA）表达质粒psiRNA-PAR1、psiRNA-PAR2和psiRNA-PAR3,转染PC3细胞;采用RT-PCR法检测其对PAR基因的表达抑制作用;细胞计数、软琼脂克隆形成实验、流式细胞术检测PAR基因表达下调对PC3细胞生长的抑制作用及其作用机制。结果：3组shRNA表达质粒均可抑制PC3细胞中PAR基因的表达;PAR基因的下调可使细胞生长速率明显减慢,增殖受到抑制;克隆形成能力降低,其中以psiRNA-PAR1的抑制效应最明显,并在转染48h后对PAR基因表达的抑制率达最高峰,为（81.18±1.68）%;流式细胞术检测结果表明,细胞周期阻滞于G2/M期,凋亡增加。结论：PAR基因表达下调,明显地抑制PC3细胞的生长,其作用机制主要是通过诱导细胞G2/M期阻滞和凋亡实现的;PAR基因可能是一个新的与雄激素非依赖性前列腺癌细胞恶性表型相关的癌基因,有望作为基因及药物治疗雄激素非依赖性前列腺癌的一个新靶点。     

辐射介导下HeLa细胞中Cdc25B与IER5蛋白表达的关系

目的：探索辐射介导下HeLa细胞中Cdc25B与IER5蛋白表达的关系。方法：用4 Gy γ射线分别照射正常HeLa细胞和IER5-siRNA-HeLa细胞,收集照射后0、0.5、2、4、8、12、16、24、36 h的细胞样本,并设未照射细胞为对照组。采用qPCR法检测照射后Cdc25B mRNA的表达;Western blot检测细胞内IER5和Cdc25B蛋白的表达;流式细胞术检测照射后G2期细胞周期阻滞情况。结果：qPCR结果显示,照射后0.5~24 h内,与对照组相比,正常HeLa细胞内Cdc25B mRNA在0.5 h先下降2 h再上升之后再下降,差异均有统计学意义（P均 < 0.05）;在IER5-siRNA-HeLa细胞内Cdc25B mRNA在0.5 h先下降后再上升,差异均有统计学意义（P均 < 0.05,2 h组除外）。Western blot结果显示,照射后0.5~24 h内,与对照组相比,正常HeLa细胞内Cdc25B蛋白从2 h开始上升8 h开始下降,差异有统计学意义（P均 < 0.01）;IER5-siRNA-HeLa细胞内Cdc25B蛋白从0.5 h开始下降8 h开始逐渐上升,差异有统计学意义（P均 < 0.01,24 h除外）。照射后0~36 h内,与对照组相比,正常HeLa细胞与IER5-siRNA-HeLa细胞的IER5蛋白表达水平分别在照后8 h与0 h后开始出现明显上升,差异均具有统计学意义（P均 < 0.05）;而Cdc25B蛋白表达水分别在照射后8 h与0 h后开始出现下降,差异均具有统计学意义（P均 < 0.05）,正常HeLa细胞与IER5-siRNA-HeLa细胞中Cdc25B蛋白与IER5蛋白的表达分别从8 h与0 h后开始呈现负相关关系（r分别为-0.740和-0.669,P均 < 0.01）,当两种细胞中Cdc25B蛋白表达量下降时,G2期细胞周期阻滞比例升高（P < 0.05）。结论：辐射介导下HeLa细胞中Cdc25B蛋白表达变化是由Cdc25B mRNA变化引起的,并且Cdc25B与IER5蛋白表达呈负相关关系。     

细胞分裂周期蛋白25同源蛋白C与肿瘤放疗敏感性

细胞分裂周期蛋白25同源蛋白C（Cdc25C）在真核细胞的有丝分裂中起重要调节作用。真核细胞中的G2-M进程主要由细胞周期蛋白依赖性激酶1（CDK1）-细胞周期蛋白B（cyclinB）复合物调控。CDK1-cyclinB复合物由Cdc25 C激活促进细胞从G2期进入M期,Cdc25 C活性是细胞周期进入M期的关键之一。提高Cdc25 C活性可促进G2-M期转变,去除电离辐射诱导的G2-M期阻滞,使损伤的DNA在未得到修复的情况下进入细胞分裂期,可导致细胞的增殖性死亡,而提高放疗敏感性。     

Cdc25C cyclin B1在二烯丙基二硫化物诱导人胃癌BGC823细胞G2/M期阻滞中的作用

目的：研究二烯丙基二硫化物（DADS）对人胃癌BGC823细胞的增殖及细胞周期的影响以及Cdc25C、cyclin B1的作用。方法：采用MTT法检测BGC823细胞的生长活性；流式细胞术分析DADS对细胞周期分布的影响；Western blot观察DADS对Cdc25C蛋白、cyclin B1蛋白表达的影响。结果：MTT比色实验结果显示，DADS对人胃癌细胞具有明显的生长抑制作用，且呈显著的剂量-效应依赖关系（P〈0．05）。流式细胞分析发现，DADS作用12h时，G2/M期细胞增加到30．1％；24h时增加到58．1％；36h达50．2％（P〈0．05）。Westernblot结果表明，DADS呈时间依赖性抑制人胃癌BGC823细胞周期Cdc25C磷酸酶的表达，cyclin B1表达在DADS处理24h后开始下降（P〈0．05）。结论：DADS可抑制人胃癌BGC823细胞增殖，其增殖抑制与细胞周期G2/M期阻滞有关；DADS可能是通过抑制Cdc25C、cyclin B1表达使部分BGC823细胞停滞在G2／M期。     

RNA干扰沉默信息调节因子2同源蛋白1阻滞前列腺癌PC3细胞周期

  目的  观察沉默信息调节因子2同源蛋白1（SIRT1）小干扰RNA（siRNA）对前列腺癌PC3细胞生长增殖、细胞周期和P21、P27细胞周期调节蛋白及视网膜母细胞瘤（retinoblastoma,Rb）蛋白表达变化影响,探讨SIRT1在前列腺癌发生中的可能作用机制。  方法  体外培养PC3细胞,分空白对照（Mock）组、发夹siRNA对照（scramble siRNA）组和SIRT1 siRNA转染组。RT-PCR和Western blot方法检测SIRT1 siRNA的转染效率;MTT方法测定细胞生长抑制率;流式细胞术检测细胞周期;Western blot方法检测P21、P27蛋白表达和Rb蛋白的磷酸化状态。  结果  与Mock组和scramble siRNA组比较,SIRT1 siRNA组SIRT1 mRNA和蛋白表达降低,明显抑制PC3细胞增殖,阻滞在G1期。P21和P27蛋白表达增加,Rb蛋白磷酸化受到抑制。  结论  SIRT1 siRNA可抑制前列腺癌PC3细胞增殖、阻滞细胞周期,其机制可能与改变P21、P27细胞周期蛋白表达和Rb蛋白的磷酸化状态相关。      

外源性IL-18基因对C6胶质瘤细胞周期及相关基因表达的影响

目的:本研究拟从分子水平探讨IL-18基因转染对C6胶质瘤细胞周期及其相关基因表达的影响。方法:用流式细胞仪检测C6/IL-18细胞周期、细胞增殖指数的变化;以MTT法检测细胞的增殖活性及细胞抑制率,用RT-PCR、Western blot分析C6/IL-18细胞PCNAc、yclin D1c、yclin B1、p21 mRNA及蛋白的表达。结果:与C6/LXSN细胞及亲代C6细胞相比,C6/IL-18细胞周期有所改变,表现为G0/G1期细胞增多、G2/M期细胞减少,细胞增殖指数(PI)与细胞增殖活性降低。C6/IL-18细胞的PCNAc、yclin D1c、yclin B1 mRNA及蛋白表达降低,p21 mRNA及蛋白表达增高。结论:外源性IL-18基因表达可降低C6细胞的增殖活性,下调PCNA,cyclin D1,cyclin B1表达,上调p21基因表达,其作用机制有待进一步研究。     

SPOCK2基因表达上调对前列腺癌细胞增殖的影响

目的:探讨SPOCK2基因表达上调对前列腺癌细胞增殖的影响。方法:通过Real-time PCR及Western Blot检测前列腺癌组织及细胞中SPOCK2基因的表达。进一步通过基因重组技术上调前列腺癌细胞系DU145及LNCaP中SPOCK2基因表达,通过CCK8检测细胞增殖情况变化,通过流式细胞仪检测细胞周期及凋亡情况。结果:前列腺癌组织中SPOCK2表达显著低于正常对照组（P<0.05）,应用基因重组片段有效上调SPOCK2基因表达后,转染组细胞增殖率均显著低于阴性对照组及空白对照组,差异有显著统计学意义（P<0.05）。转染组G0/G1期细胞百分比显著高于阴性对照组及空白对照组,差异有显著统计学意义（P<0.05）。转染组S期显著低于阴性对照组及空白对照组,差异有显著统计学意义（P<0.05）。DU145细胞转染组凋亡率显著高于阴性对照组及空白对照组,差异有显著统计学意义（P<0.05）。结论:SPOCK2上调可以抑制细胞增殖,促进细胞凋亡,使细胞停滞于G0/G1期,表明该基因可能通过影响细胞增殖,作为抑癌基因在前列腺癌的发生发展中发挥重要作用。     

用RNAi沉默survivin基因表达对前列腺癌细胞系PC3的影响

目的：运用RNA干扰（RNA interference，RNAi）技术阻断前列腺癌细胞系PC3中survivin基因的表达，并研究survivin基因沉默后对PC3细胞产生的影响。方法：用真核转录载体pSilencer^TM3．1-H1 neo构建针对survivin基因的重组真核转录载体pSilencer3．1-SVVl、pSilencer3．1-SVV2和pSilencer3．1-SVV3，并用脂质体法转染前列腺癌细胞系PC3，通过RT—PCR、蛋白印迹实验检测survivin的表达变化，并用流式细胞术、MTT法检测转染后PC3细胞的凋亡、细胞周期、细胞生长速度、对顺铂的敏感性等方面的变化。结果：重组载体pSilencer3．1-SVV2和pSilencer3．1-SVV3有效地阻断了PC3细胞中survivin基因在mRNA和蛋白水平上的表达（P〈0．01）。重组载体转染PC3细胞后，与对照组相比，细胞的凋亡增加了10％～15％；细胞生长速度明显变慢，其细胞数在84h时与对照组相比减少约30％；G1期细胞增加了10％。G2期和S期细胞减少了5％以上。加入顺铂后，pSilencer3．1-SVV2、pSilencer3．1-SVV3重组质粒转染的PC3细胞的存活数下降了35％～45％，细胞凋亡则增加了10％-14％。结论：初步证明了survivin基因在前列腺癌细胞分化增殖、抗凋亡等方面所扮演的重要角色，为进一步阐明survivin基因与前列腺癌的关系以及以survivin基因为靶点的前列腺癌基因治疗研究奠定了基础。     

低剂量照射对细胞周期和修复基因表达的影响

目的观察低剂量照射对A549(肺癌细胞)和2BS细胞(人胚胎肺成纤维细胞)细胞周期及修复基因表达的影响.探讨肿瘤细胞与正常细胞在诱导修复基因表达方面的差异,并结合细胞周期变化,进一步阐明适应性反应形成机制.方法(1)应用流式细胞技术,对不同剂量照射后A549和2BS细胞周期进行分析;(2)应用Northern-blot检测不同照射剂量DNA修复基因hR24L、bRAD6和bRAD52转录水平的变化.结果(1)2BS细胞经75mGy X线照射组,及低剂量加高剂量照射组于照后30分钟即出现明显的G2期阻滞,且细胞周期于24小时内恢复.A549细胞在75mGy X线照射后,细胞周期未发生变化;(2)2BS细胞在75mGy X照射下,hR24L、hRAD6表达增强,bRAD52无变化.A549细胞低剂量照射修复基因表达未见显著变化.结论低剂量照射后2BS细胞与A549细胞周期改变存在差异,且细胞周期的调控与DNA修复基因的表达有着密切的关系.     

宫颈鳞状上皮FoxM1及Cdc25B细胞核内蛋白水平与高危型HPV感染的相关性研究

背景与目的：宫颈癌是妇科最常见的恶性肿瘤,人乳头状瘤病毒（human papilloma virus,HPV）与宫颈癌/癌前病变的发生关系密切。宫颈组织活检RNA标本及宫颈癌细胞系中发现HPV 16 E7基因与FoxM1基因表达具有相关性。为发掘辅助诊断宫颈鳞癌及其癌前病变的免疫组织化学（immunohistochemistry,IHC）标志物,分析宫颈组织中高危型HPV感染（high-risk HPV,hrHPV）和转录因子FoxM1及其下游蛋白Cdc25B的细胞核内水平之间的相关性。方法：宫颈活检、锥切及子宫切除组织学标本来自复旦大学附属肿瘤医院病理科资料库（2007—2009年）,用于细胞核FoxM1和Cdc25B蛋白IHC检测,并与23种基因型HPV DNA检测以及P16INK4a（P16）和Ki-67之IHC结果比较。结果：包括22例正常、28例CIN1、50例CIN2/3和40例鳞癌在内共计140例入组。CIN2+中hrHPV感染率100%（90/90）,FoxM1、Cdc25B、P16和Ki-67阳性率分别为100.00%（90/90）、94.44%（85/90）、85.56%（77/90）和97.78%（88/90）,且上述标志物阳性率均随宫颈病变严重程度加剧而上升（Jonckheere-Terpstra检验,P均＜0.000 1）。FoxM1和Cdc25B表达与hrHPV感染、P16和Ki-67阳性率相关（Spearman相关检验,P均<0.000 1）。FoxM1和Cdc25B诊断CIN2+效果佳,曲线下面积（area under curve,AUC）值分别为0.850和0.822。结论：人宫颈鳞状上皮中细胞核FoxM1和Cdc25B蛋白水平与hrHPV感染相关,有望成为诊断与鉴别诊断CIN2+的潜在辅助指标。     

2-Methoxyestradiol inhibits hepatocellular carcinoma cell growth by inhibiting Cdc25 and inducing cell cycle arrest and apoptosis

PURPOSE: 2-Methoxyestradiol (2-ME) is a physiological metabolite of estrogen, which can inhibit growth of many types of tumor cells, including hepatocellular carcinoma, both in vitro and in vivo. The exact mechanisms of its action are still unclear. We have studied the mechanisms of growth inhibition of several of human and rat hepatoma and normal liver cells by 2-ME. METHODS: Human (Hep3B, HepG2, PLC/PRF5) and rat (McA-RH7777, JM-1) hepatoma and normal rat (CRL-1439) and human (CRL-11233) liver cell lines were cultured in vitro, in presence of 2-ME, and its IC50s were determined. Cell cycle arrest, Cdc25 phosphatase inhibition and apoptosis induction were studied. Finally, the effect of 2-ME on the growth of JM-1 rat hepatoma cells in rat liver was determined in vivo. RESULTS: The IC50 range for growth inhibition of hepatoma cells was found to be between 0.5 and 3 muM. In contrast, normal rat hepatocytes and liver cell lines were resistant to 2-ME up to 20 muM. JM-1 cells were arrested in the G2/M phase of the cell cycle. Cdc25A and Cdc25B, cell cycle controlling phosphatases, activities were inhibited in vitro and 2-ME was found to likely bind to their catalytic site cysteines. As a consequence, their cellular substrates Cdk1 and Cdk2 were tyrosine phosphorylated. Caspase-3 was cleaved suggesting apoptotic cell death. Moreover, growth of JM-1 tumors, which were transplanted into rat liver, was also inhibited by treatment with 2-ME in vivo. CONCLUSIONS: 2-Methoxyestradiol is a selective, potent and relatively non-toxic hepatoma growth inhibitor both in vitro and in vivo. Cell cycle arrest of hepatoma cells was likely mediated by binding and inactivation of the Cdc25 phosphatases and induction of apoptosis.     

Paclitaxel induced apoptosis in breast cancer cells requires cell cycle transit but not Cdc2 activity

Purpose Paclitaxel (PTX) is a widely used chemotherapy agent and may cause cell death by apoptosis subsequent to microtubule (MT) disruption. In this paper, we have investigated whether cell cycle transit and or Cdc2 (Cdk1) activity is required for the apoptosis induced by PTX.Methods Cell cycle was analyzed by flow cytometry, Cdc2 was assayed bio chemically. Cdc2 activity was decreased by siRNA and dominant negative (dn) Cdc2 expression. Cells were arrested by chemical or biological inhibitors in a G₁or S phase. Apoptosis was measured by DNA fragmentation and examination of nuclei by microscopy. JNK and AKT activations were assessed as well.Results Cell cycle inhibition was highly effective in decreasing PTX induced apoptosis. MT morphology was not altered by these inhibitors. PTX induced JNK activity or AKT mediated BAD phosphorylation was unaffected by cell cycle inhibitors. Abrogation of Cdc 2 activity was without effect on PTX induced apoptosis.Conclusions While cell cycle transit is required for PTX induced apoptosis; Cdc2 activity is not required.     

三氧化二砷诱导卵巢癌OVCAR-3细胞周期阻滞及凋亡

目的探讨As2O3对卵巢癌细胞增殖的抑制作用及对细胞周期和凋亡的影响.方法采用MTT法及集落形成实验测定细胞的增殖活力,通过细胞形态学观察细胞分裂及凋亡,应用流式细胞仪进行细胞周期解析、凋亡及bcl-2蛋白表达的检测.结果As2O3呈剂量依赖性抑制OVCAR-3细胞增殖,抑制50%细胞生长的药物浓度(IC50)为2μmol/L.0.5～5μmol/L的As2O3作用7天,集落抑制率均在40%以上(P＜0.01).2μmol/L与5μmol/L的As2O3作用12 h后,细胞周期出现了G2/M期阻滞及亚二倍体凋亡峰,随着作用时间的延长,凋亡细胞随G2/M期细胞减少而逐渐增多.细胞形态学观察可见分裂期细胞及凋亡细胞明显增加.0.5～5μmol/L的As2O3均能下调bcl-2蛋白的表达.结论As2O3能够抑制卵巢癌OVCAR-3细胞的增殖,诱导M期阻滞及细胞凋亡.     

5'-nitro-indirubinoxime induces G2/M cell cycle arrest and apoptosis in human KB oral carcinoma cells

Our previous study demonstrated that the novel indirubin derivative, 5'-nitro-indirubinoxime (5'-NIO), effectively arrested the tumor growth through the inhibition of cell proliferation and the induction of apoptosis. However, the precise molecular mechanisms underlying 5'-NIO-induced antitumor activity remain unclear. Here, we report that 5'-NIO inhibits the proliferation of human KB oral carcinoma cells via the cell cycle arrest in G2/M phase. 5'-NIO reduced the activity of Cdc2/cyclin B complex through the inhibition of the PLK1 expression. Partially, 5'-NIO also arrested cell cycle in G1/S phase via the reduction of CDK4 and cyclin D1/D3 levels by p16 and induction of the level of p21waf1. Using flow cytometry analysis, we showed that 5'-NIO-induced cell cycle arrest is followed by apoptosis. We determined further that 5'-NIO-induced apoptosis is accomplished by the mitochondria-dependent activation of the caspase cascade. Overall, these observations suggest the potential value of 5'-NIO as a candidate for a therapeutic modality for the treatment of oral cancer.     

Targeting mixed lineage kinases in ER-positive breast cancer cells leads to G2/M cell cycle arrest and apoptosis

Estrogen receptor (ER)-positive tumors represent the most common type of breast cancer, and ER-targeted therapies such as antiestrogens and aromatase inhibitors have therefore been widely used in breast cancer treatment. While many patients have benefited from these therapies, both innate and acquired resistance continue to be causes of treatment failure. Novel targeted therapeutics that could be used alone or in combination with endocrine agents to treat resistant tumors or to prevent their development are therefore needed. In this report, we examined the effects of inhibiting mixed-lineage kinase (MLK) activity on ER-positive breast cancer cells and non-tumorigenic mammary epithelial cells. Inhibition of MLK activity with the pan-MLK inhibitor CEP-1347 blocked cell cycle progression in G2 and early M phase, and induced apoptosis in three ER-positive breast cancer cell lines, including one with acquired antiestrogen resistance. In contrast, it had no effect on the cell cycle or apoptosis in two non-tumorigenic mammary epithelial cell lines. CEP-1347 treatment did not decrease the level of active ERK or p38 in any of the cell lines tested. However, it resulted in decreased JNK and NF-κB activity in the breast cancer cell lines. A JNK inhibitor mimicked the effects of CEP-1347 in breast cancer cells, and overexpression of c-Jun rescued CEP-1347-induced Bax expression. These results indicate that proliferation and survival of ER-positive breast cancer cells are highly dependent on MLK activity, and suggest that MLK inhibitors may have therapeutic efficacy for ER-positive breast tumors, including ones that are resistant to current endocrine therapies.     

Rab25基因siRNA诱导人卵巢癌细胞A2780……

 目的 观察Rab25基因的小分子干扰RNA（siRNA）对肿瘤细胞的诱导凋亡作用。方法 成功构建Rab25基因的siRNA，稳定转染靶细胞，MTT法检测细胞增生率；流式细胞仪（FCM）检测细胞生长周期和细胞凋亡率；DNA琼脂糖凝胶电泳法（DNA ladder）确定凋亡。结果 细胞生长明显受到抑制，增生能力下降；阻断细胞生长于G1期（转染pSUPER／Rab25 siRNA组细胞G1期达到88.6 %）；细胞凋亡增加，转染pSUPER／Rab25 siRNA组细胞凋亡率为12.3 %，与A2780组细胞及转染空载体组细胞相比，差异有统计学意义（P＜0.01）；DNA凝胶电泳可观察到细胞凋亡特异的DNA梯形带。结论 Rab25基因的siRNA可抑制对肿瘤细胞的生长，诱导其凋亡，可为卵巢癌基因治疗开辟新途径     

Induction of G1 arrest and apoptosis in androgen-dependent human prostate cancer by Kuguacin J,a triterpenoid from Momordica charantia leaf

In this study, we focused on the effects of a bitter melon (Momordica charantia) leaf extract (BMLE) and a purified component, Kuguacin J (KuJ), on androgen-dependent LNCaP human prostate cancer cells. Both treatments exerted growth inhibition through G1 arrest and induction of apoptosis. In addition, KuJ markedly decreased the levels of cyclins (D1 and E), cyclin-dependent kinases (Cdk2 and Cdk4) and proliferating cell nuclear antigen, and caused an increase in p21 and p27 levels. Its induction of apoptosis was accompanied by an increase in cleavage of caspase-3 and poly (ADP-ribose) polymerase, attributable to augment of Bax/Bcl-2 and Bad/Bcl-xL and reduction of survivin levels. BMLE and KuJ also reduced the expression of androgen receptor (AR), prostate-specific antigen (PSA) while induced P53 protein level. Down-regulation of p53 by RNA interference indicated that BMLE and KuJ inhibited cell growth partly through p53-dependent cell cycle arrest and apoptotic pathways. Both BMLE and KuJ caused less toxicity in a normal prostate cell line, PNT1A. Our results suggest that BMLE and a purified component, KuJ, from its diethyl ether fraction could be promising candidate new antineoplastic and chemopreventive agents for androgen-dependent prostate cancer and carcinogenesis.     

Silencing the Metallothionein-2A gene inhibits cell cycle progression from G1- to S-phase involving ATM and cdc25A signaling in breast cancer cells

Metallothioneins (MTs) are a group of metal-binding proteins involved in cell proliferation, differentiation and apoptosis. The MT-2A isoform is generally the most abundant isoform among the 10 known functional MT genes. In the present study, we observed that down-regulation of the MT-2A gene in MCF-7 cells via siRNA-mediated silencing inhibited cell growth by inducing cell cycle arrest in G1-phase (G1-arrest) and a marginal increase in cells in sub-G1-phase. Scanning electron microscopic examination of the cells with silenced expression of MT-2A (siMT-2A cells) revealed essentially normal cell morphology with presence of scattered apoptotic cells. To elucidate the underlying molecular mechanism, we examined the expression of cell cycle related genes in MT-2A-silenced cells and found a higher expression of the ataxia telangiectasia mutated (ATM) gene concomitant with a lower expression of the cdc25A gene. These data suggest that MT-2A could plausibly modulate cell cycle progression from G1- to S-phase via the ATM/Chk2/cdc25A pathway.