沉默PRR14基因抑制结肠癌HCT116细胞增殖

已有报道显示,富脯氨酸蛋白 14（proline-rich protein 14,PRR14）促进肿瘤的发生发展,但具体作用机制仍不清楚。本文以结肠癌细胞为模型,探索其对细胞增殖和细胞周期进程的影响。qPCR和Western 印迹检测发现,PRR14在4个结肠癌细胞系中呈现高水平表达。合成特异靶向PRR14基因的siRNA,转染结肠癌HCT116细胞。检测发现,PRR14基因表达下调约70%。CCK8结果显示,沉默PRR14后各时间点细胞增殖能力均显著降低,克隆形成实验细胞克隆数减少约40%;流式细胞仪结果显示,沉默PRR14后,G1期细胞比例升高约10%,S期细胞比例降低约14%;BrdU标记免疫荧光检测结果显示,BrdU阳性细胞比例减少约50%,表明细胞DNA合成速率显著降低。机制分析表明：促G1/S期转换基因周期蛋白依赖性激酶2（cyclin dependent kinase 2, CDK2）mRNA水平降低约85%,对应的蛋白质水平也明显降低,G1/S期转换抑制因子周期蛋白依赖性激酶抑制因子1A（cyclin dependent kinase inhibitor 1A,CDKN1A/P21）和周期蛋白依赖性激酶抑制因子1B（cyclin dependent kinase inhibitor 1B,CDKN1B/P27）mRNA水平分别升高约1.8倍和5倍,对应的蛋白质水平也明显升高。沉默PRR14表达,G1/S期相关基因表达紊乱,导致细胞G1期阻滞并抑制细胞增殖。结肠癌细胞中PRR14高表达可促进癌细胞恶性增殖。     

沉默PRR14基因抑制结肠癌HCT116细胞增殖

已有报道显示,富脯氨酸蛋白 14（proline-rich protein 14,PRR14）促进肿瘤的发生发展,但具体作用机制仍不清楚。本文以结肠癌细胞为模型,探索其对细胞增殖和细胞周期进程的影响。qPCR和Western 印迹检测发现,PRR14在4个结肠癌细胞系中呈现高水平表达。合成特异靶向PRR14基因的siRNA,转染结肠癌HCT116细胞。检测发现,PRR14基因表达下调约70%。CCK8结果显示,沉默PRR14后各时间点细胞增殖能力均显著降低,克隆形成实验细胞克隆数减少约40%;流式细胞仪结果显示,沉默PRR14后,G1期细胞比例升高约10%,S期细胞比例降低约14%;BrdU标记免疫荧光检测结果显示,BrdU阳性细胞比例减少约50%,表明细胞DNA合成速率显著降低。机制分析表明：促G1/S期转换基因周期蛋白依赖性激酶2（cyclin dependent kinase 2, CDK2）mRNA水平降低约85%,对应的蛋白质水平也明显降低,G1/S期转换抑制因子周期蛋白依赖性激酶抑制因子1A（cyclin dependent kinase inhibitor 1A,CDKN1A/P21）和周期蛋白依赖性激酶抑制因子1B（cyclin dependent kinase inhibitor 1B,CDKN1B/P27）mRNA水平分别升高约1.8倍和5倍,对应的蛋白质水平也明显升高。沉默PRR14表达,G1/S期相关基因表达紊乱,导致细胞G1期阻滞并抑制细胞增殖。结肠癌细胞中PRR14高表达可促进癌细胞恶性增殖。     

植物细胞周期调控因子及其功能的研究进展

细胞周期调控因子能通过影响细胞周期对植物细胞的生长、分裂和分化产生作用,进而调节植物的生长发育。本文综述了近几年来植物细胞周期调控因子中细胞周期蛋白（cyclin,CYC）、周期蛋白依赖激酶（cyclin-dependent kinase,CDK）等的作用机理及研究进展,阐述了各调控因子在植物生长发育过程中的作用。     

CDK 抑制剂在抗肿瘤领域的研发进展

细胞周期蛋白依赖性激酶（cyclin dependent kinase，CDK）为细胞周期调节的关键激酶，参与细胞增殖、转录、存活等生理过程。 CDK 在多种肿瘤中异常活化，是抗肿瘤药物研发的重要靶点之一。目前已有 1 个 CDK 抑制剂（palbociclib, CDK4/CDK6 抑制剂）被美国 食品药品监督管理局批准于 2015 年上市，数十个 CDK 抑制剂处于针对实体瘤和血液系统肿瘤的临床或临床前研究阶段。综述目前抗肿瘤 领域 CDK 抑制剂的研发现状、遇到的问题和可能的解决方案，并讨论其临床应用的可能。     

酵母RNA聚合酶II羧基端结构域激酶CTDK-I及其亚基的结构与功能

RNA聚合酶Ⅱ最大亚基Rpb1的羧基端结构域（carboxyl-terminal repeat domain,CTD）是RNA聚合酶Ⅱ发挥转录延伸功能所必需的,对其执行精确的转录调节功能至关重要。酵母细胞周期蛋白依赖性激酶CTDK-I（carboxyl-terminal repeat domain kinase,CTDK-I）由CTK1、CTK2和CTK3组成,作用于RNA聚合酶Ⅱ羧基端结构域,动态磷酸化CTD的七肽重复序列（YSPTSPS）来调控转录和翻译。酵母中的特异性蛋白CTK3与特殊的细胞周期蛋白CTK2结合形成异二聚体,再与CTDK-I的催化亚基CTK1结合以调节其活性。CTK1作为细胞周期蛋白CDK（cyclin dependent kinase,CDK）的同源蛋白,其结构与功能的研究可拓展人们对CDK蛋白家族的认识;CTK2-CTK3复合物对CTK1调控机制的研究也可为细胞周期蛋白抑制剂的研发提供新的思路。本文简述了酵母CTDK-I的功能特点及其亚基的结构与功能以及亚基间的相互作用,并展望了CTDK-I复合物的研究前景。     

猪miR-331-3p过表达载体的构建及其对细胞增殖的影响

本研究旨在阐明猪miR-331-3p对细胞增殖的影响,探讨其对细胞增殖的作用机制首先构建了miR-331-3p的过表达载体pcDNA 3.1 (+)-miR-331-3p,并将将PK15细胞分为4组,分别为实验组、实验组对照组、抑制剂组和抑制剂对照组。实验组和对照组分别转染pcDNA 3.1(+)-miR-331-3p和pcDNA 3.1(+)。抑制剂组和抑制剂对照组分别转染miR-331-3p Inhibitor和miR-331-3p阴性对照(miR-331-3p NC)。通过在各组添加CCK-8试剂绘制细胞增殖曲线,并使用PI染色检测细胞所处周期比例。同时,利用实时荧光定量PCR(Quantitative real-time PCR,qPCR)检测生长抑制蛋白家族成员5 (Inhibitor of growth family member 5,ING5)、细胞周期蛋白依赖性激酶2 (Cyclin dependent kinase 2,CDK2)、细胞周期蛋白依赖性激酶3 (Cyclin dependent kinase 3,CDK3)、细胞周期蛋白依赖性激酶4 (Cyclin dependent kinase 4,CDK4)、细胞周期蛋白B (Cyclin B)和细胞周期蛋白依赖性激酶抑制剂1A(Cyclindependentkinaseinhibitor1A,CDKN1A)的表达变化。结果表明,实验组miR-331-3p表达量显著升高,细胞增殖曲线表明48 h和72 h时细胞数目均呈现出实验组实验对照组和抑制剂对照组抑制剂组的趋势(P0.05)。与实验对照组相比,实验组处于G0/G1期的细胞比例下调,S期和G2/M细胞的比例上调,抑制剂对照组趋势与之相反;同时,实验组中与促进增殖的基因CDK2、CDK3、CDK4和CyclinB的mRNA表达水平均显著升高,而抑制增殖的基因ING5和CDKN1A均表现出显著下降的趋势。本研究成功构建了miR-331-3p过表达载体,且发现miR-331-3p具有促进猪肾上皮细胞增殖的能力,研究结果为深入研究miR-331-3p在猪生长发育中的作用机制奠定了基础。     

CDK1 siRNA干扰在Taxol诱导细胞凋亡中的作用

目的研究转染细胞周期依赖性蛋白激酶1（cyclin．dependent kinase1,CDK1）siRNA、以及转染后进行凋亡刺激对细胞周期和凋亡的影响,探讨CDK1在细胞凋亡中的确切作用,揭示细胞周期与细胞凋亡协调的分子机制。方法以人宫颈癌细胞株HeLa细胞为研究对象,脂质体转染CDK1siRNA,转染后48h加紫杉醇（Tax01）（20μg／m1）刺激凋亡,Western印迹检测CDK1和抗凋亡蛋白BCL2表达,AnnexinV／PI法检测细胞的凋亡,流式细胞仪分析DNA含量检测细胞周期。结果转染CDK1 siRNA后,CDK1蛋白的表达下降,细胞周期G2／M期比例增加,细胞凋亡率与对照相比没有明显升高。只加Taxol刺激12h后细胞凋亡率增加并伴有S期和G2／M期比例增加。转染CDKlsiRNA后再用Taxol刺激,其细胞凋亡率没有明显改变,G2／M期阻滞效应也没有叠加。BCL2蛋白只在加Taxol刺激组表达下降,与CDK1表达减少没有相关性。结论siRNA沉默导致的CDK1表达降低只导致细胞周期G2／M期阻滞,没有引起细胞凋亡;CDK1的表达降低对紫杉醇所诱导的细胞周期阻滞和细胞凋亡效应没有明显影响。     

TRAF4调控人结直肠癌细胞增殖的机制研究

目的:研究TRAF4影响结直肠癌细胞增殖的分子机制。方法:MTS和软琼脂集落形成实验检测基因沉默TRAF4后对结直肠癌细胞生长及相关信号通路的影响,检测TRAF4表达下调后结直肠癌细胞的糖酵解变化及己糖激酶II的表达和结直肠细胞对5-Fu的敏感性。结果:基因沉默TRAF4抑制结直肠癌细胞的停泊依赖和停泊非依赖增殖,抑制EGF诱导的Akt活化,下调结直肠癌细胞糖酵解,抑制己糖激酶II的表达,增加结直肠癌细胞对5-Fu的敏感性。结论:TRAF4通过调控糖代谢影响结直肠癌细胞增殖。     

Cdk6在维持ES细胞自我更新中的作用

细胞周期蛋白依赖性激酶6（cyclin dependent kinase 6,Cdk6）对胚胎早期发育有着重要的作用.然而,它在胚胎干（embryonic stem,ES）细胞中的生物学功能仍不清楚.在该研究中,我们运用RNA干扰技术和基因表达分析方法探索了Cdk6在小鼠胚胎干细胞中的功能及分子机制.结果表明：Cdk6的表达水平与小鼠ES细胞的自我更新密切相关.首先,维甲酸（RA）处理和白血病抑制因子（LIF）去除实验显示 ,随着ES细胞的分化,Cdk6的表达水平明显降低.更为重要的是,RNA干扰介导的Cdk6表达抑制导致ES细胞自我更新相关基因的显著下调,同时伴随细胞分化基因的表达激活,提示Cdk6对维持ES细胞自我更新至关重要.     

p-MAPK、CyclinD1和CDK4蛋白在大肠癌中的表达及相关性

目的 检测p-MAPK、CyclinD1和CDK4蛋白在大肠癌中的表达,并探讨其相关性.方法 选取78例大肠癌组织,距癌灶3 cm以外的癌旁组织,进行组织切片,HE染色进行病理分型,应用S-P免疫组化法对组织中的p-MAPK、Cyclin D1和CDK4进行检测.结果 病理分型后,78例大肠癌中p-MAPK、CyclinD1和CDK4蛋白的阳性表达率分别为67.9 %（53/78）、57.7 %（45/78）和39.7 %（31/78）;78例相应癌旁组织中阳性表达率分别为6.4 %（5/78）、10.3 %（8/78）和19.2 %（15/78）.三者阳性表达率和阳性强度均以大肠癌中为高（P＜0.01）;大肠癌中p-MAPK与CyclinD1表达呈明显正相关（P＜0.01）,而与CDK4蛋白表达无明显相关性（P＞0.05）,CyclinD1和CDK4表达呈正相关（P＜0.05）,RT-PCR检测结果与免疫组化相似,p-MAPK、CyclinD1和CDK4蛋白的阳性表达率分别为8.2 %（61/78）、67.9 %（53/78）和53.8 %（42/78）;相应癌旁组织中阳性表达率分别为3.8 %（3/78）、2.6 %（2/78）和6.4 %（5/78）.结论 MAPK活化可能是cylinD1激活的原因之一,并在大肠癌发生过程中可能起重要作用;而Cyclin D1 在细胞周期中可与CDK4 结合,从而促进细胞周期进程.     

Activation of Cdk2/Cyclin E complexes is dependent on the origin of replication licensing factor Cdc6 in mammalian cells

Cyclin E-associated CDK2 activity is required for the initiation of DNA synthesis in human cells. CDK2 activity is tightly regulated; CDK2 must be in the nucleus, bound to a cyclin, phosphorylated on T160, and dephosphorylated on T14/Y15 for complete kinase activation. Nuclear localization exposes CDK2 to activating enzymes (CAK, Cdc25A) in stimulated cells. Previous studies from our lab indicate CDK2 nuclear localization and cyclin E co-expression are insufficient to cause CDK2 activation or T160 phosphorylation in stimulated IIC9 cells; these activities still require serum stimulation and ERK kinase activity. Recent studies have implicated a role for origin of replication (ORC) licensing proteins in the activation of G1/S Cdks. In this study, we show that CDK2 associates with chromatin and Cdc6 in an ERK-dependent manner following stimulation of IIC9 CHEF cells. We show that nuclear-localized CDK2 (CDK2-NLS) ectopically expressed with cyclin E requires mitogenic stimulation and ERK activation for chromatin association, in addition to previously shown kinase activation and T160 phosphorylation in IIC9 cells. Additionally, we show that expression of Cdc6 in stimulated IIC9 cells treated with ERK inhibitor rescues CDK2-NLS chromatin association, kinase activation, and T160 phosphorylation. From the above data, we deduce ERK-dependent CDK2 activation is due in part to ERK-dependent Cdc6 expression. To examine the role of Cdc6 directly in stimulated primary human fibroblasts, we used RNA interference to attenuate the expression of Cdc6. We show that Cdc6 expression is required for CDK2 chromatin association and kinase activation in stimulated primary human fibroblasts. Additionally, we show that Cdc6 expression is required for the initiation of DNA synthesis and S phase entry in stimulated primary human fibroblasts. Ultimately, this data implicates Cdc6 expression as an important mitogen-induced mechanism in the activation of CDK2/cyclin E, the initiation of DNA synthesis, and the regulation of G1-S phase progression.     

活性依赖的突触抑制和突触稳态的分子机制

常规RNA干涉或基因敲除的功能缺失手段仅仅只是简单地移除某个基因或蛋白,而这个过程常常会掩盖磷酸化对某个特定蛋白的调节。在树突发育和突触功能活性依赖的调节过程中,突触后致密蛋白磷酸化的机制仍然是未知的领域。突触后Rap GTP酶激活蛋白SPAR与PSD95结合,可以促进树突棘的生长并加强突触。Plk2（polo-like kinase2,也称为Snk）是一种受突触活性诱导表达的蛋白激酶,它可以磷酸化SPAR,磷酸化的SPAR通过泛素化．蛋白酶体途径降解,从而导致树突棘和突触的减少。Plk2的诱导表达和随后SPAR的降解是长时间神经活性增强过程中突触强度的稳态抑制（突触剥落）所必需的。有趣的是,SPAR需要被另外一种激酶cDK5磷酸化后才能被Plk2所降解。这种机制通过CDK5对一部分突触进行标记,为由Plk2-SPAR通路抑制或去除这些突触提供了可能的途径,但其分子机制在神经退行性疾病突触丢失中的作用仍需进一步探讨。     

A functional role for Smad7 in sustaining colon cancer cell growth and survival

Initially identified as an inhibitor of transforming growth factor (TGF)-β mainly owing to its ability to bind TGF-β receptor type I and abrogate TGF-β-driven signaling, Smad7 can interact with additional intracellular proteins and regulate TGF-β-independent pathways, thus having a key role in the control of neoplastic processes in various organs. Genome-wide association studies have shown that common alleles of Smad7 influence the risk of colorectal cancer (CRC), even though the contribution of Smad7 in colon carcinogenesis is not fully understood. In this study, we assessed the expression and role of Smad7 in human and mouse models of sporadic CRC. We document a significant increase of Smad7 in human CRC relative to the surrounding nontumor tissues and show that silencing of Smad7 inhibits the growth of CRC cell lines both in vitro and in vivo after transplantation into immunodeficient mice. Knockdown of Smad7 results in enhanced phosphorylation of the cyclin-dependent kinase (CDK)2, accumulation of CRC cells in S phase and enhanced cell death. Smad7-deficient CRC cells have lower levels of CDC25A, a phosphatase that dephosphorylates CDK2, and hyperphosphorylated eukaryotic initiation factor 2 (eIF2)α, a negative regulator of CDC25 protein translation. Consistently, knockdown of Smad7 associates with inactivation of eIF2α, lower CDC25A expression and diminished fraction of proliferating cells in human CRC explants, and reduces the number of intestinal tumors in Apc^min/+ mice. Altogether, these data support a role for Smad7 in sustaining colon tumorigenesis.     

DPP3/CDK1 contributes to the progression of colorectal cancer through regulating cell proliferation,cell apoptosis,and cell migration

At present, colorectal cancer (CRC) has become a serious threat to human health in the world. Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase that may be involved in several physiological processes. However, whether DPP3 affects the development and progression of CRC remains a mystery. This study is the first to demonstrate the role of DPP3 in CRC. Firstly, the results of immunohistochemistry analysis showed the upregulation of DPP3 in CRC tissues compared with normal tissues, which is statistically analyzed to be positively correlated with lymphatic metastasis, pathological stage, positive number of lymph nodes. Moreover, the high expression of DPP3 predicts poor prognosis in CRC patients. In addition, the results of cell dysfunction experiments clarified that the downregulation of DPP3 significantly inhibited cell proliferation, colony formation, cell migration, and promoted apoptosis in vitro. DPP3 depletion could induce cell apoptosis by upregulating the expression of BID, BIM, Caspase3, Caspase8, HSP60, p21, p27, p53, and SMAC. In addition, downregulation of DPP3 can reduce tumorigenicity of CRC cells in vivo. Furthermore, CDK1 is determined to be a downstream target of DPP3-mediated regulation of CRC by RNA-seq, qPCR, and WB. The interaction between DPP3 and CDK1 shows mutual regulation. Specifically, downregulation of DPP3 can accentuate the effects of CDK1 knockdown on the function of CRC cells. Overexpression of CDK1 alleviates the inhibitory effects of DPP3 knockdown in CRC cells. In summary, DPP3 has oncogene-like functions in the development and progression of CRC by targeting CDK1, which may be an effective molecular target for the prognosis and treatment of CRC.Subject terms: Cancer, Diseases     

CyclinD1、CDK4、P16和结核菌L型感染在前列腺癌中的表达及临床意义

目的探讨CyclinD1、CDK4和P16在前列腺癌中的表达以及结核菌L型感染率及临床意义。方法应用免疫组化和抗酸染色等方法检测了65例前列腺癌（carcinoma of prostate,PCa）和30例良性前列腺增生（benignprostatic hyperplasia,BPH）中的CyclinD1、CDK4和P16的表达,以及结核菌L型的检出率。并对前列腺肿瘤主要临床资料和病理分级参数进行比较,用χ^2检验进行统计学处理。结果 CyclinD1、CDK4阳性表达前列腺癌明显高于前列腺增生（P〈0.01）;并与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.01-0.05）呈正相关。P16阳性表达前列腺增生明显高于前列腺癌（P〈0.01）;与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.01-0.05）呈负正相关。结核菌L型检出率前列腺癌明显高于前列腺增生;与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.05）。结论 CyclinD1、CDK4和P16在前列腺肿瘤中不同程度异常表达以及结核菌L型检出率与肿瘤的临床分期、病理分级和转移相关,因此研究CyclinD1、CDK4和P16的阳性表达和结核菌L型感染与前列腺癌发生发展中可能有协同作用,具有重要的临床应用价值。