POLO-LIKE KINASEl GENE EXPRESSION AND ITS CLINICO-PATHOLOGICAL SIGNIFICANCE IN GASTRIC CARCINOMA

Objective To clarify the polo-like kinasel （PLK1） expression in human gastric cancer tissue and its clinicopathological significance in gastric carcinoma. Methods PLK1 expression in 60 cancer tissues and their corresponding noncancerous tissues from gastric cancer patients was measured by both real-time quantitative RT-PCR and western blot assay, lmmunohistochemistry was used to detect PLK1 protein expression in eighty-nine paraffin-embedded samples. Results The PLK1 mRNA and protein expression level in the 60 fresh cancer tissues was significantly higher than that in noncancerous tissues （ P 〈 0. 0001, P = 0. 031 respectively）. In paraffin-embedded samples, apart from its increased expression level, PLK1 was found to be in both cytoplasm and nucleus, double-site location only occurred in poor-differentiated cancer, PLKI expression intensity was associated with tumor dif- ferentiation （ P = 0. 03）, invasion （ P = 0. 032 ）, TNM stage （ P = 0. 019） , ki67 expression （ P = 0. 011 ）. The patients with negative PLK1 expression had better survival rate than that with positive PLK1 expression （ P =0. 0292 ）. Conclusion PLK1 may have clinicopathological value in tumor diagnosis, and may become another new biomarker and therapeutic target for gastric cancer.

Research Progress on Targets and Selective Inhibitors of Polo-like Kinase-1(PLK-1)

In this paper,the biological function of PLK-1,the correlation between PLK-1 and tumors,and the latest research progress on PLK-1 inhibitors under study are reviewed,in order to provide references for the research and development of PLK-1 inhibitors.

Polo-like kinase 1 as a biomarker predicts the prognosis and immunotherapy of breast invasive carcinoma patients

Invasive breast carcinoma(BRCA)is associated with poor prognosis and high risk of mortality.Therefore,it is critical to identify novel biomarkers for the prognostic assessment of BRCA.Methods:The expression data of polo-like kinase 1(PLK1)in BRCA and the corresponding clinical information were extracted from TCGA and GEO databases.PLK1 expression was validated in diverse breast cancer cell lines by quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting.Single sample gene set enrichment analysis(ssGSEA)was performed to evaluate immune infiltration in the BRCA microenvironment,and the random forest(RF)and support vector machine(SVM)algorithms were used to screen for the hub infiltrating cells and calculate the immunophenoscore(IPS).The RF algorithm and COX regression model were applied to calculate survival risk scores based on the PLK1 expression and immune cell infiltration.Finally,a prognostic nomogram was constructed with the risk score and pathological stage,and its clinical potential was evaluated by plotting calibration charts and DCA curves.The application of the nomogram was further validated in an immunotherapy cohort.Results:PLK1 expression was significantly higher in the tumor samples in TCGA-BRCA cohort.Furthermore,PLK1 expression level,age and stage were identified as independent prognostic factors of BRCA.While the IPS was unaffected by PLK1 expression,the TMB and MATH scores were higher in the PLK1-high group,and the TIDE scores were higher for the PLK1-low patients.We also identified 6 immune cell types with high infiltration,along with 11 immune cell types with low infiltration in the PLK1-high tumors.A risk score was devised using PLK1 expression and hub immune cells,which predicted the prognosis of BRCA patients.In addition,a nomogram was constructed based on the risk score and pathological staging,and showed good predictive performance.Conclusions:PLK1 expression and immune cell infiltration can predict post-immunotherapy prognosis of BRCA patients.

RNA干扰技术抑制Polo-like激酶1表达对A549细胞的影响

Polo-like激酶1(Plk1)是参与细胞周期调控的重要分子,已在多种肿瘤中检测到Plk1的高表达,并发现与肿瘤细胞的增殖和预后密切关联.为明确Plk1在肺癌细胞系A549细胞增殖和周期运行中的作用,采用RNA干扰技术,构建能产生siRNA的质粒载体psiRNA-hH1-Plk1并导入A549细胞中.采用RT-PCR检测Plk1mRNA表达的变化,Western印迹检测Plk1、细胞周期蛋白B1、p53蛋白的表达变化,流式细胞术分析细胞周期变化和凋亡;免疫荧光染色检测α微管蛋白的表达.以此观察RNA干扰能否有效抑制Plk1的表达水平,以及抑制后对A549细胞生长的影响.结果表明,psiRNA-hH1-Plk1质粒能特异性地抑制Plk1基因的表达并使其活性下降,细胞周期蛋白B1及p53蛋白的表达水平升高,微管聚集障碍或形成单极的纺锤体,A549细胞增殖减慢,出现G2/M期阻滞并存在细胞凋亡.针对Plk1基因的RNA干扰有望用于肿瘤的基因治疗.

RNA干扰技术抑制Polo-like激酶1表达对A549细胞的影响(英文)

目的:观察RNA干扰技术能否有效抑制非小细胞肺癌细胞株A549细胞中Polo-like激酶1(Plk1)的表达水平,以及抑制后对A549细胞生长的影响。方法:运用脂质体法,以Plk1为靶点,构建能产生siRNA的质粒载体psiRNA-hH1-Plk1并转入A549细胞。RT-PCR检测Plk1 mRNA表达的变化、Western blotting检测Plk1、cyc-linB1、p53蛋白的表达变化、细胞计数分析细胞增殖、流式细胞术分析细胞周期变化和凋亡、免疫荧光染色检测α微管蛋白的表达。结果:psiRNA-hH1-Plk1质粒能特异地抑制Plk1基因的表达并使其活性下降,致使cyclinB1及p53蛋白的表达水平升高,微管聚集障碍或形成单极的纺锤体;A549细胞增殖减慢,出现G2/M期阻滞和凋亡。结论:上述结果提示针对Plk1基因的RNA干扰有望用于肿瘤的基因治疗。

Polo-like激酶1在肝癌细胞多药耐药中作用及其机制的初步研究

原发性肝癌是常见的恶性肿瘤之一,全球约半数的肝癌患者集中在中国,其病死率在我国恶性肿瘤中居第三位。由于早期诊断率低、术后复发率高而预后差。在肝癌的综合治疗中,化疗是重要方法之一,而肝癌的多药耐药（multi-drug reesistance,MDR）已成为制约化疗效果及影响患者生存的主要难点。Polo-like激酶（PLK-1）属于有丝分裂丝氨酸/苏氨酸激酶家族,在细胞增殖过程中起重要作用。

Apoptosis induction with polo-like kinase-1 antisense phosph-orothioate oligodeoxynucleotide of colon cancer cell line SW480

AIM： To investigate the effects of polo-like kinase-1 （PLK1） antisense phosphorothioate oligodeoxynucleotide （ASODN） on apoptosis and cell cycle of human colon cancer cell line SW480. METHODS： After SW480 colon cancer cells were transfected with PLK1 ASODN, Northern and Western blot analyses were used to examine PLK1 gene expression in cancer cells. We studied apoptosis using terminal uridine deoxynucleotidyl nick end labeling. Apoptosis and cell cycle of SW480 cells were examined by fluorescence-activated cell sorter scan. RESULTS： The levels of PLK1 mRNA and protein were greatly inhibited by PLK1 ASODN in SW480 cancer cells transfected with PLK1 ASODN. Apoptosis index （AI） induced PLK1 ASODN in a time- and dose-dependent manner. Results from FLM showed that sub-2N DNA content of transfected cancer cells was significantly increased and arrested at G2/M compared with control groups. CONCLUSION： PLK1 ASODN can induce apoptosis of human colon cancer cell line SW480.

Polo-like kinase 1 expression is a prognostic factor in human colon cancer

AIM：To clarify the expression patterns and prognostic implications of the mitotic regulator Polo-like kinase 1 （PLK1） in colon cancer. METHODS： Expression of PLK1 was investigated by immunohistochemistry （158 cases） and immunoblotting in tissue of colon adenomas and adenocarcinomas. PLK1 expression patterns were correlated with clinicopathological parameters and patient prognosis. In addition, expression of PLK1 was evaluated by immunoblot and PCR in colon carcinoma cell lines, and coexpression of PLK1 with the proliferation marker Ki-67 was investigated. RESULTS： Weak PLK1 expression was observed in normal colon mucosa and adenomas. In contrast, 66.7% of carcinomas showed strong expression of PLK1. Overexpression of PLK1 correlated positively with Dukes stage （P〈0.001）, tumor stage （P = 0.001） and nodal status （P〈0.05）. Additionally, PLK1 expression was a prognostic marker in univariate survival analysis （P〈0.01） and had independent prognostic significance （RR = 3.3, P = 0.02） in patients with Iocoregional disease. Expression of PLK1 mRNA and protein was detected in all cell lines investigated. Coexpression of PLK1 and Ki-67 was observed in the majority of colon cancer cells, but a considerable proportion of cells showed PLK1 positivity without Ki-67 expression. CONCLUSION： PLK1 is a new prognostic marker for colon carcinoma patients and may be involved in tumorigenesis and progression of colon cancer. Strategies focusing on PLK1 inhibitionin vivo might therefore represent a promising new therapeutic approach for this tumor entity. 2005 The WIG Press and Elsevier Inc. All rights reserved

Overexpression of polo-like kinase1 predicts a poor prognosis in hepatocellular carcinoma patients

AIM： To elucidate the role of overexpressed polo-like kinasel （PLK1）in hepatocellular carcinoma （HCC）. METHODS： We prospectively collected clinicopathological, immunohistochemical and semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） data from 135 HCC patients undergoing successful hepatectomy. The correlations between PLK1 mRNA expression and clinicopathologic variables were analyzed by Mann-Whitney U test. Prognostic factors were identified by univariate and multivariate analyses. RESULTS： Immunohistochemical results showed overexpression of PLK1 was mainly found in tumor tissues compared with tumor-free tissue. A similar mRNA result was obtained by semi-quantitative RT-PCR. A total of 111 samples were positive for PLK1 mRNA expression. The positive expression was correlated with venous invasion, tumor nodules and Edmondson grade. Furthermore, 1, 3, 5-year survival rates in the positive expression group were significantly lower than the negative control group. Multivariate analysis showed that positive PLK1 expression was an independent risk factor for HCC. CONCLUSION： PLK1 could be a potential biomarker for diagnosis and therapy for HCC.

Effect of Antisense RNA Targeting Polo-like Kinase 1 on Cell Growth in A549 Lung Cancer Cells

In order to investigate the effect of Polo-like kinase-1 （Plk1） depletion on cell cycle progression and cell growth in lung cancer cells, a recombinant plasmid containing antisense RNA targeting Plk1 （pcDNA3-Plk1） was transfected into A549 cells by lipofectine. RT-PCR and Western-blot were used to detect the Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine （BrdU） labeling. Cell cycle distribution and apoptosis were examined by flow cytometry, and the inhibition rate （IR） by vinorebline （NVB） was determined by MTF assay. The results showed that after transfection of pcDNA3-Plk1 into A549 cells, the expression levels of Plk1 mRNA and protein were greatly decreased. In pcDNA3-Plk1 transfected groups, abnormal morphological changes of cells and growth inhibition were observed, and the BrdU labeling index was significantly lower than in the control groups （P〈0.05）. Cells in pcDNA3-Plk1 transfected groups were arresed in G2/M phase and apoptosis was detectable 72 h post transfection. IR induced by vinorebline in pcDNA3-Plk1 transfected groups was significantly higher than in other groups. These data suggested that antisense RNA targeting Plk1 could suppress the Plk1 expression, and therefore, significantly inhibit cell proliferation and induce cell cycle arrest and apoptosis. Moreover, it sensitized lung cancer cells to chemotherapy.

Polo-like kinase 1,a new therapeutic target in hepatocellular carcinoma

AIM： To investigate the role of polo-like kinase 1 （PLK1） as a therapeutic target for hepatocellular carcinoma （HCC）. METHODS： PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA （siRNA） was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction （RT-PCR） and Western blotting, and cell proliferation using 3-（4,5-dim ethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2（4-sulf ophenyl）-2H-tetrazolium （MTS） and bromodeoxyuridine （BrdU） assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end label- ing （TUNEL） assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progres- sion was compared with controls. RESULTS： RT-PCR showed that PLK1 was overexpre- ssed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells, siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLKl-treated mice, but not in controls. CONCLUSION： Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G path- way.

Thymoquinone and Poloxin are slow-irreversible inhibitors to human Polo-like kinase 1 Polo-box domain

Objective： To provide a kinetic model（s） and reveal the mechanism of thymoquinone and Poloxin blocking an emerging anti-cancer target, human Polo-like kinase 1 （hPlkl） Polo-box domain （PBD）. Methods： The binding kinetics was determined by using a fluorescence polarization based assay. The putative mechanism was examined with a competition test. Results： Thymoquinone follows a one-step binding with an association rate constant （k1） of 6.635× 10^3 L.mol^-1 min^-1.Poloxin fit a two-step binding with a dissociation constant （Ki） of 118 μmol/L for the intermediate complex and its isomerization rate （k4） of 0.131 5 minJ to form an irreversible adduct. No significant dissociation was observed for either ligand up to 13 h. The inhibitors responded insignificantly to the presence of Michael donors as hPIkl-PBD competitors. Conclusion： Thymoquinone and Poloxin are slow-tight ligands to the hPlkl-PBD with kinetic models distinct from each other. Michael addition as the mechanism is excluded.

Dual targeting of Polo-like kinase 1 and baculoviral inhibitor of apoptosis repeat-containing 5 in TP53-mutated hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC),often diagnosed at advanced stages without curative therapies,is the fifth most common malignant cancer and the second leading cause of cancer-related mortality.Polo-like kinase 1(PLK1)is activated in the late G2 phase of the cell cycle and is required for entry to mitosis.Interestingly,PLK1 is overexpressed in many HCC patients and is highly associated with poor clinical outcome.Baculoviral inhibitor of apoptosis repeatcontaining 5(BIRC5)is also highly overexpressed in HCC and plays key roles in this malignancy.AIM To determine the expression patterns of PLK1 and BIRC5,as well as their correlation with p53 mutation status and patient clinical outcome.METHODS The expression patterns of PLK1 and BIRC5,and their correlation with p53 mutation status or patient clinical outcome were analyzed using a TCGA HCC dataset.Cell viability,cell apoptosis,and cell cycle arrest assays were conducted to investigate the efficacy of the PLK1 inhibitors volasertib and GSK461364 and the BIRC5 inhibitor YM155,alone or in combination.The in vivo efficacy of volasertib and YM155,alone or in combination,was assessed in p53-mutated Huh7-derived xenograft models in immune-deficient NSIG mice.RESULTS Our bioinformatics analysis using a TCGA HCC dataset revealed that PLK1 and BIRC5 were overexpressed in the same patient subset and their expression was highly correlated.The overexpression of both PLK1 and BIRC5 was more frequently detected in HCC with p53 mutations.High PLK1 or BIRC5 expression significantly correlated with poor clinical outcome.PLK1 inhibitors(volasertib and GSK461364)or a BIRC5 inhibitor(YM155)selectively targeted Huh7 cells with mutated p53,but not HepG2 cells with wild-type p53.The combination treatment of volasertib and YM155 synergistically inhibited the viability of Huh7 cells via apoptotic pathway.The efficacy of volasertib and YM155,alone or in combination,was validated in vivo in a Huh7-derived xenograft model.CONCLUSION PLK1 and BIRC5 are highly co-expressed in p53-mutated HCC and inhibition of both PLK1 and BIRC5 synergistically compromises the viability of p53-mutated HCC cells in vitro and in vivo.

Transcriptional regulation of human polo-like kinases and early mitotic inhibitors

Human polo-like kinases （PLK1-PLK4） have been implicated in mitotic regulation and carcinogenesis. PLK1 phosphorylates early mitotic inhibitor 1 （Emil） to ensure mitosis entry, whereas Emi2 plays a key role during the meiotic cell cycle. Transcription factor E2F is primarily considered to regulate the G1/S transition of the cell cycle but its involvement in the regulation of mitosis has also been recently suggested. A gap still exists between the molecular basis of E2F and mitotic regulation. The present study was designed to characterize the transcriptional regulation of human PLK and Emi genes. Adenoviral overexpression of E2F1 increased PLK1 and PLK3 mRNA levels in A549 cells. A reporter gene assay revealed that the putative promoter regions of PLK1, PLK3, and PLK4 genes were responsive to activators E2F, E2F1-E2F3. We further characterized the putative promoter regions of Emil and Emi2 genes, and these could be regulated by activators E2F and E2F1-E2F4, respectively. Finally, PLK1-PLK4, Emil, and Emi2 mRNA expression levels in human adult, fetal tissues, and several cell lines indicated that each gene has a unique expression pattern but is uniquely expressed in common tissues and cells such as the testes and thymus. Collectively, these results indicate that E2F can integrate G1/S and G2/M to oscillate the cell cycle by regulating mitotic genes PLK and Emi, leading to determination of the cell fate.

Polo-like Kinase1 Gene Expression and its Clinicopathological Significance in gastric Carcinoma

Objective To clarify the polo-like kinase1 (PLK1) expression in human gastric cancer tissue and its clinicopathological sig-nificance in gastric carcinoma. Methods PLK1 expression in 60 cancer tissues and their corresponding noncancerous tissues from gas-tric cancer patients was measured by both real-time quantitative RT-PCR and western blot assay. Immunohistochemistry was used to detectPLK1 protein expression in eighty-nine paraffin-embedded samples. Results The PLK1 mRNA and protein expression level in the 60fresh cancer tissues was significantly higher than that in noncancerous tissues (P<0.0001,P=0.031 respectively). In paraffin-embed-ded samples, apart from its increased expression level, PLK1 was found to be in both cytoplasm and nucleus, double-site location onlyoccurred in poor-differentiated cancer, PLK1 expression intensity was associated with tumor differentiation (P=0.03), invasion(P=0.032), TNM stage (P=0.019), ki67 expression (P=0.011). The patients with negative PLK1 expression had better survivalrate than that with positive PLK1 expression (P=0.0292). Conclusion PLK1 may have clinicopathological value in tumor diagnosis,and may become another new bio-marker and therapeutic target for gastric cancer.

原发性肝癌Plk1基因表达及其与预后的相关性

目的:分析Plk1基因在肝细胞癌(hepatocellular carcinoma,HCC)中的表达,及其与预后的关系.方法:用半定量RT-PCR及Western blot方法检测我院2003-01/2008-05原发性肝癌患者213例的Plk1基因蛋白表达,同时应用Kaplan-Meier法及多变量Cox比例险模型,分析Plk1基因的表达及临床病理因素与预后的关系.结果:Plk1基因在肝癌组织中阳性表达率为83.6%.Plk1阳性组的1、3、5年的生存率明显低于阴性组(P=0.004).原发性肝癌的预后与Plk1阳性表达,Edmondson分级,肉眼癌栓,显微癌栓及肿瘤数目相关;与HBsAg、肝硬化、AFP、假包膜、肿瘤大小无关;多因素Cox回归分析显示Edmondson分级、Plk1基因蛋白表达、肉眼癌栓及肿瘤数目4项指标反映肝癌预后情况,危险度分别为1.717、1.938、1.537及2.355.结论:Plk1基因的检测有助于有高危肝癌患者的选择,为Plk1基因靶向治疗肝癌提供了有力依据.

人Polo样激酶1活性缺失突变体和结构域在293细胞中的瞬时表达

目的:构建人Polo样激酶1(Plk1)活性缺失突变体及结构域突变体的真核表达载体,并在293细胞中表达。方法:用二次PCR方法扩增Plk1基因并点突变,将82位赖氨酸突变为精氨酸,定向克隆到pcDNA3-Flag载体中;用普通PCR方法扩增Plk1激酶区域及Polo盒区域(PBD)基因,定向克隆到pcDNA3-Flag载体中;将上述质粒转染293细胞进行瞬时表达,Western印迹检测Plk1蛋白的表达。结果:构建了Flag-Plk1(K82R)、Flag-Plk1KD、Flag-Plk1PBD真核表达质粒,在293细胞中均可有效表达,蛋白相对分子质量分别为68×103、45×103、31×103。结论:在293细胞中表达了Flag-Plk1(K82R)、Flag-Plk1KD、Flag-Plk1PBD蛋白,有助于进一步探究Plk1对底物的功能。

乳腺癌组织中FOXM1和PLK1的表达及意义

目的探讨叉头框转录因子M1(FOXM1)及Polo样激酶1(PLK1)在乳腺癌组织中的表达及临床意义。方法采用免疫组织化学法检测FOXM1及PLK1蛋白在803例乳腺浸润性导管癌和乳腺癌旁组织中的表达情况及其与乳腺癌组织临床病理特征间的关系。结果 FOXM1及PLK1在乳腺浸润性导管癌组织中的阳性表达率分别为59.78%(480/803)和27.90%(224/803),显著高于其在乳腺癌旁组织中的表达(29.89%,0),差异具有统计学意义(χ~2=145.011,χ~2=260.307,P=0.000)。FOXM1及PLK1蛋白的表达与乳腺癌的组织学分级、淋巴结转移及临床分期相关,而与肿瘤大小无关,且二者表达具有正相关关系(r=0.414,P<0.01)。结论 FOXM1及PLK-1可能协同参与了乳腺癌的发生、发展,并可能成为乳腺癌预后评估的重要指标。

丝/苏氨酸蛋白激酶Plk1研究进展

Plk1是一类从酵母到人类都高度保守的丝氨酸/苏氨酸蛋白激酶。Plk1与不同的细胞周期检查点的精密调控有关,从而确保了细胞周期事件按照严格的时间和顺序正常进行。Plk1在增殖活跃的细胞中呈高水平表达,Plk1的高度表达和肿瘤患者的低存活率之间具有显著的统计相关性。Plk1可能是非常有效的抗癌药物设计的靶点。目前已有几种Plk1的小分子抑制剂在体内外的实验中显示出了良好的应用前景。