MSH2 is required for cell proliferation, cell cycle control and cell invasiveness in colorectal cancer cells

We have investigated the role of MSH2,a mismatch repair gene in cell proliferation,cell cycle control and cell invasiveness in the SW480 human colorectal cancer cell line.RNAi-mediated inhibition of MSH2 expression was achieved using MSH2 shRNA lentiviral expression vectors.Effective knockdown of endogenous MSH2 expression was determined by real-time PCR analysis.The most efficient MSH2 knockdown vector was selected for subsequent studies using SW480 cells.Endogenous MSH2 mRNA levels decreased after lentiviral delivery of the MSH2-RNAi,indicating efficient silencing of MSH2 expression in SW480 cells.Cell proliferation,cell cycle progression and cell invasiveness were quantified by MTT assays,flow cytometry and transwell assays,respectively.RNAi-mediated inhibition of MSH2 expression in SW480 cells resulted in decreased cell proliferation,cell cycle arrest at the G0/G1 phase and decreased cell invasiveness.Taken together,these results provide evidence that MSH2 stimulates cell proliferation,promotes cell cycle progression and positively regulates cell invasiveness.     

BAP31 is frequently overexpressed in patients with primary colorectal cancer and correlates with better prognosis

We previously showed that B cell receptor associated protein 31(BAP31) was significantly upregulated in colorectal cancer compared with normal mucosa epithelia. However, its expression pattern and pathological role in colorectal cancer are not clearly understood. In this study, we investigated whether the expression of BAP31 was associated with the clinicopathological parameters of colorectal cancer. The expression pattern of BAP31 was detected by immunohistochemistry on a tissue microarray in both primary ...     

长非编码RNA LINC00941在结直肠癌中的表达及对细胞增殖的影响研究

为了研究与细胞分化相关的长非编码RNA(long non-coding RNA, lncRNA) LINC00941在肿瘤发生发展中的作用,通过实时荧光定量PCR技术检测LINC00941在6种不同类型的人类癌细胞和正常胚胎肾细胞HEK-293细胞中的表达水平,结果表明,LINC00941在结直肠癌细胞HCT116和HCT116 p53-/-、肺癌细胞A549和NCI-H1299、黑素瘤细胞Stilling中均有较高的表达水平,在结直肠癌细胞中表达水平最高. 以结直肠癌患者肿瘤组织和癌旁正常组织为材料,实时荧光定量PCR检测LINC00941的表达水平发现,肿瘤组织中LINC00941 RNA的表达水平显著高于癌旁组织. 通过shRNA(short hairpin RNA)干扰技术降低HCT116细胞中的LINC00941 RNA水平,导致细胞增殖速度下降,说明LINC00941与结直肠癌的发生发展相关.     

Napabucasin对结直肠癌细胞增殖及迁移的影响

为了探究新型小分子抑制剂Napabucasin对结直肠癌细胞增殖以及迁移的影响. 首先通过分子模拟对接分析了Napabucasin与STAT3蛋白的互作机制. 然后利用克隆形成实验、细胞划痕实验等方法在多种结直肠癌细胞系中证明了Napabucasin能够显著抑制结直肠癌细胞的集落形成能力以及迁移能力. 进而使用Napabucasin与Wnt信号通路激活剂Wnt agonist 1共处理结直肠癌细胞HCT116,结合蛋白质印迹实验发现,Wnt信号通路介导了Napabucasin对结直肠癌细胞的迁移以及增殖的抑制过程. 研究结果显示,Napabucasin能够在体外抑制结直肠癌细胞的增殖能力以及迁移能力,并且Wnt信号通路参与介导了这一抑制过程.     

H13对HT-29细胞周期和caspase-3蛋白表达的影响

探讨新型Topo I抑制剂H13对体外培养的人结肠癌细胞HT-29细胞周期和caspase-3表达的影响.以人结肠癌细胞HT-29为研究对象,PI染色,流式细胞仪检测H13处理48 h后细胞周期的分布;Western Blot法检测H13处理48 h后caspase-3表达的变化.2.5和5 g/mL H13处理的HT-29处理48 h后,G1期细胞百分率下降,S期细胞百分率上升,并且有剂量依赖性;2.5,5,10 g/mL H13处理的HT-29处理48 h后,caspase-3蛋白表达升高.H13对诱导HT-29细胞周期阻滞于S期,其机制可能与其上调caspase-3表达有关.     

RACK1依赖的miRNA途径参与调节植物叶绿素的生物合成

为深入探索活化C激酶受体1(receptor of activated C kinase 1,RACK 1)在调节植物microRNA(miRNA)的生物发生,以及其靶基因中的关键作用,对在美国国家生物技术信息中心(National Center for Biotechnology Information,NCBI)网站上共享的基因表达综合数据库(Gene Expression Omnibus,GEO)中有关rack1突变体的小RNA序列数据重新进行了系统分析和挖掘,找到了19个新miRNA.通过解析差异miRNA表达,鉴定到了特异调控叶绿素合成相关基因HEMA和HEMC表达的miRNA,为今后深入系统地研究RACK1在调节叶绿体发育和光合作用机理提供了关键的理论依据,也为利用公共数据库挖掘潜在的数据信息提供了基本的研究设想和思路.     

Killin介导p53信号通路的机理与其在细胞周期不同时期特异性分布的相关性研究

本研究中,通过检测killin在其它p53下游相关基因缺失的情况下能否激活细胞凋亡,证明了p53通过killin介导的S期抑制以及细胞凋亡与p21、puma和bax通路没有直接关系.另外通过将EGFP-PCNA和RFP-killin表达质粒共同转染到cosE5细胞中,并观察细胞处于不同时期时Killin蛋白的分布情况,发现在细胞S期中Killin与PCNA的核定位呈现相互排斥的点状分布,与先前BrdU标记结果吻合.这一结果再次印证了Killin在S期可能抑制DNA复制.同时Killin被观察到在非S期时聚集于核仁内,从而可能影响核糖体RNA的合成.这些发现意味着killin作为主要的p53靶基因之一,可能在细胞周期不同检查点进行调控.     

蜂胶黄酮Pinobanksin-3-acetate对结肠癌HCT-116细胞增殖及凋亡的影响

 为探讨蜂胶黄酮(Pinobanksin-3-acetate, PB3A)对结肠癌HCT-116 细胞增殖及细胞凋亡的影响, 采用四甲基偶氮唑盐(MTT)比色法, 检测不同浓度、不同时间PB3A、5-氟尿嘧啶(5-FU)及两种药物联合作用对HCT-116 细胞生长所产生的影响, 倒置显微镜观察细胞的形态学变化特点, Annexin V-FITC/PI 双染色、流式细胞仪检测药物作用24 h 后的细胞凋亡率。结果显示, PB3A 对HCT-116 细胞增殖有明显的抑制作用, 并呈浓度和时间依赖性。其抑制活性与5-FU 几乎没有显著性差异, 联合用药具有协同效应; 在一定剂量范围内, 可见凋亡细胞明显增多。流式细胞仪分析结果表明, 细胞凋亡率明显上升, 呈剂量依赖性。PB3A 对HCT-116 细胞具有明显的增殖抑制和诱导细胞凋亡作用。     

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function

Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.     

ATR-Chk1-Cdc25信号通路参与盘基网柄菌G2/M转变期的调控

显微镜观察盘基网柄菌野生型KAx--3细胞和突变型RNAi-allC细胞,计数结果表明后者的单细胞繁殖速度约为前者的8倍.为探究该突变型盘基网柄菌细胞周期缩短的原因,用荧光定量PCR和western blot研究了ATR-Chk1Cdc25信号通路在其中的可能作用.实验结果表明:RNAi-allC细胞中cdc25基因相对表达量约为KAx-3细胞的8倍,而其Chk1与ATR基因的相对表达量却明显低于KAx-3细胞.突变细胞中Cdc25蛋白含量高于KAx-3细胞,但其Chk1蛋白含量却显著低于KAx-3细胞.这些数据表明,两种类型细胞之间的ATR、Chk1、Cdc25在mRNA水平和蛋白表达上均存在差异,特别是ATR基因表达量的不同明显影响ChK1和Cdc25的表达量,提示ATR-Chk1-Cdc25信号通路应该在一定程度上参与了盘基网柄菌细胞周期G2/M期的调控.     

Androgen regulation of cell proliferation and expression of viral sequences in mouse mammary tumour cells

The role of steroids in promoting cell proliferation is well established but the molecular mechanisms are not clear. The S115 mouse mammary tumour cell line provides a model system for molecular studies in vitro in that it exhibits in tissue culture both a positive proliferative response to androgens and a change from a transformed phenotype in the presence of androgen to a normal phenotype when androgen is removed. We have considered here the possible involvement of mouse mammary tumour virus (MMTV) in these processes. We have demonstrated the presence in S115 cells of MMTV-related sequences which are transcribed into RNA only in the long-term presence of androgen. Prolonged culture in the absence of androgen, which results in loss of proliferative response to androgen, is accompanied by loss of MMTV-related RNA and increased methylation of MMTV-related sequences.     

RNA干扰Nucleostemin基因对人结肠癌细胞株HT-29细胞增殖及细胞周期的影响

将Nucleostemin(NS)siRNA利用脂质体2000转染人结肠癌细胞株HT 29,分别利用CCK-8试剂盒和流式细胞术检测NS siRNA对HT 29细胞增殖和细胞周期的影响,进一步利用Real-time PCR和Western blotting技术检测NS基因和细胞周期相关基因p21 mRNA和蛋白的表达.结果表明,NS siRNA的转入能有效下调NS mRNA和蛋白的表达(P0.05),并能明显抑制人结肠癌细胞株HT 29的细胞增殖(P0.05),并诱导细胞周期静止在G_0/G_1期,转染NS siRNA后,p21 mRNA和蛋白的表达均明显升高,提示细胞增殖抑制和细胞周期静止与p21基因表达的升高密切相关.     

Overexpression of N-cadherin is correlated with metastasis and worse survival in colorectal cancer patients

N-cadherin is related to the progression and metastases of several solid carcinomas. However, it was still unclear whether N-cadherin is overexpressed in colorectal malignant tumors that have stronger malignant tendency. In this study, we used immunohistochemistry to detect the expression patterns of N-cadherin in both the primary tumors and their normal mucosa tissues of 120 patients with colorectal cancer. We revealed that N-cadherin was expressed in 78.3% (94/120) of colorectal tumor tissues and in only 9.2% (11/120) of paired distant normal mucosa tissues with a significant difference (P=0.000). The low, moderate, and high expression of N-cadherin protein was 42.5%, 30.8%, and 26.7%, respectively. N-cadherin overexpression was associated with advanced TNM stage, lymph nodes metastasis and distant metastasis (P<0.05). Patients with N-cadherin overexpressed showed the obvious lower overall survival rate than those with moderate and low expression, and patients with low expression had a better survival rate than those with moderate and high expression (P<0.05). In conclusion, high N-cadherin expression may lead to tumor aggressiveness and metastatic potential in colorectal cancer, and may prove to be a possible prognostic factor.     

ATR-Chkl-Cdc25信号通路参与盘基网柄菌G2/M转变期的调控简

显微镜观察盘基网柄菌野生型KAx-3细胞和突变型RNAi-allC细胞,计数结果表明后者的单细胞繁殖速度约为前者的8倍. 为探究该突变型盘基网柄菌细胞周期缩短的原因,用荧光定量PCR和western blot研究了ATR-Chk1-Cdc25信号通路在其中的可能作用. 实验结果表明：RNAi-allC细胞中cdc25基因相对表达量约为KAx-3细胞的8倍,而其Chk1与ATR基因的相对表达量却明显低于KAx-3细胞. 突变细胞中Cdc25蛋白含量高于KAx-3细胞,但其Chk1蛋白含量却显著低于KAx-3细胞. 这些数据表明,两种类型细胞之间的ATR、Chk1、Cdc25在mRNA水平和蛋白表达上均存在差异,特别是ATR基因表达量的不同明显影响ChK1和Cdc25的表达量,提示ATR-Chk1-Cdc25信号通路应该在一定程度上参与了盘基网柄菌细胞周期G2/M期的调控.     

Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation

The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110alpha, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110alpha kinase activity. Homozygosity for this kinase-dead p110alpha led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110alpha derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110beta, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110alpha was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110alpha in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110alpha in a variety of cancers.     

SGK and 14-3-3 protein are involved in the serine/ threonine phosphorylation mechanism for TPO/MPL signal transduction

Thrombopioetin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Yeast two-hybrid screening was performed to isolate the proteins interacting with the cytoplasmic domain of c-Mpl. 48 positive clones were isolated from 5 × 10⁶ independent transformants. The results of sequence analysis demonstrate that they represent 13 different protein encoding sequences. Among them there are a partial coding sequence of serine/threonine protein kinase SGK (serum and glucocorticoid-inducible kinase) and 14-3-3 theta protein partial coding sequence. GST-pull-down assay and co-immunoprecipitation in mammal cells have confirmed the interaction between these two proteins and c-Mpl. By constructing a series of deleted c-Mpl cytoplasmic domain, the interaction region in c-Mpl cytoplasmic tail was localized in amino acids 523–554. At the same time, the directed interaction between SGK and 14-3-3 proteins also has been verified by yeast two-hybrid assay. The present note is the first time to report that two proteins act with c-Mpl at the same time and put forward that SGK and 14-3-3 protein may be involved in the serine/threonine phosphorylation mechanism for signal transduction.     

Frequent 3p21 allelic loss and methylation-associated RASSF1A inactivation in non-small cell lung cancer and its clinical implication

A total of 110 primary NSCLCs (non-small cell lung cancers) were recruited in this study to characterize the pattern of 3p21 LOH together with the RASSF1A methylation status and their clinical implication. 3p21 LOH by 8 microsatellite markers, RASSF1A methylation status by methylation-specific PCR (MSPCR) as well as bisulfite genomic sequencing (BGS), and RASSF1A expression level by real-time quantitative PCR was performed. 3p21 LOH is frequent in NSCLC with a mean frequency of (41.2±3.7)%. Significant associations between 3p21 LOH and gender, smoking history, histological type, and tumor size were observed. Cases with LOH have a slightly lower RASSF1A expression than cases without LOH but not statistically significant. Comparison of RASSF1A methylation that resulted from the three analyses shows significant correlations from one another. Higher frequency of methylation was observed in larger tumors and in smokers compared with smaller tumors and non-smokers, respectively. A significant correlation was also observed in extent between methylation and RASSF1A expression, illustrating that epigenetic mechanism could affect gene expression. The significant clinicopathological relations of 3p21 LOH may be of great use for both early detection and therapeutic interventions.     

大肠癌中MTA1和nm23 - H1蛋白的表达及其意义

目的：探讨大肠癌中MTA1、nm23-H1蛋白的表达及其意义。方法：用免疫组化留法检测84例大肠癌MTA1、nm23-H1蛋白的表达。结果：84例大肠癌中MTA1、nm23-H1阳性表达率分别为61．9％和51．2％;MTA1高表达与大肠癌组织分化程度、Duke’s分期和淋巴结转移关系密切（P〈0．05）;nm23-H1低表达与大肠癌组织分化程度、Duke’s分期和淋巴结转移关系密切（P〈0．05）;大肠癌中MTA1和nm23-H1蛋白表达呈负相关（P〈0．05）。结论：MTA1和nm23-H1蛋白表达与大肠癌分化程度、淋巴结转移和预后密切相关,可作为判断大肠癌患者转移复发的参考指标。     

Protein tyrosine phosphatase is possibly involved in cellular signal transduction and the regulation of ABA accumulation in response to water deficit in Maize L.coleoptile

Water deficit-induced ABA accumulation is an ideal model or “stimulus-response”system to investigate cellular stress signaling in plant cels,using such a model the cellular stress signaling triggered by water deficit was investigated in Maize L.coleoptile.Water deficit-induced ABA accumulation was sensitively blocked by NaVO3,a potent inhibitor both to plasma membrane H^ -ATPase(PM-H^ -ATPase)and protein tyrosine phosphatase(PTPase).However,while PM-H^ -ATPase activity was unaffected under water deficit and PM-H^ -ATPase activator did not induce an ABA accumulation instead of water deficit,water deficit induced an increase in the protein phosphatase activity,and furthermore,ABA accumulation was inhibited by PAO,a specific inhibitor of PTPase.These results indicate that protein phosphtases may be involved in the cellular signaling in response to water deficit.Further studies identifiled at least four species of protein phosphtase as assayed by using pNPP as substrate,among which one component was especially sensitive to NaVO3.The NaVO3-sensitive enzyme was purified and finally showed a protein band about 66kD on SDS/PAGE.The purified enzyme showed a great activity to some specific PTPase substrates at pH 6.0.In addition to NaVO3,the enzyme was also sensitive to some other PTPase inhibitors such as Zn^2 and MO3^3 ,but not to Ca^2 and Mg^2 ,indicating that it might be a protein tyrosine phosphatase.Interestingly,the purified enzyme could be deactivated by some reducing agent DTT.which was previously proved to be an inhibitor of water deficit-induced ABA accumulation.This result further proved that PTPase might be involved in the cellular signaling of ABA accumulation in response to water deficit.