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.

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.

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 labeling (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 progression was compared with controls.RESULTS:RT-PCR showed that PLK1 was overexpressed 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 siPLK1-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 pathway.

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.

Diabetes and high-glucose could upregulate the expression of receptor for activated C kinase 1 in retina

BACKGROUND Diabetic retinopathy(DR)is a major ocular complication of diabetes mellitus,leading to visual impairment.Retinal pigment epithelium(RPE)injury is a key component of the outer blood retinal barrier,and its damage is an important indicator of DR.Receptor for activated C kinase 1(RACK1)activates protein kinase C-ε(PKC-ε)to promote the generation of reactive oxygen species(ROS)in RPE cells,leading to apoptosis.Therefore,we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS,thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR.AIM To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR.METHODS In this study,Sprague-Dawley rats and adult RPE cell line-19(ARPE-19)cells were used as in vivo and in vitro models,respectively,to explore the role of RACK1 in mediating PKC-εin early DR.Furthermore,the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model.RESULTS Streptozotocin-induced diabetic rats showed increased apoptosis and upregulated expression of RACK1 and PKC-εproteins in RPE cells following a prolonged modeling.Similarly,ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε,accompanied by an increases in ROS production,apoptosis rate,and monolayer permeability.However,silencing RACK1 significantly downregulated the expression of PKC-εand ROS,reduced cell apoptosis and permeability,and protected barrier function.CONCLUSION RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.

MicroRNA-298 determines the radio-resistance of colorectal cancer cells by directly targeting human dual-specificity tyrosine(Y)-regulated kinase 1A

BACKGROUND Radiotherapy stands as a promising therapeutic modality for colorectal cancer(CRC);yet,the formidable challenge posed by radio-resistance significantly undermines its efficacy in achieving CRC remission.AIM To elucidate the role played by microRNA-298(miR-298)in CRC radio-resistance.METHODS To establish a radio-resistant CRC cell line,HT-29 cells underwent exposure to 5 gray ionizing radiation that was followed by a 7-d recovery period.The quantification of miR-298 levels within CRC cells was conducted through quantitative RT-PCR,and protein expression determination was realized through Western blotting.Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and proliferation by clonogenic assay.Radio-induced apoptosis was discerned through flow cytometry analysis.RESULTS We observed a marked upregulation of miR-298 in radio-resistant CRC cells.MiR-298 emerged as a key determinant of cell survival following radiation exposure,as its overexpression led to a notable reduction in radiation-induced apoptosis.Intriguingly,miR-298 expression exhibited a strong correlation with CRC cell viability.Further investigation unveiled human dual-specificity tyrosine(Y)-regulated kinase 1A(DYRK1A)as miR-298’s direct target.CONCLUSION Taken together,our findings underline the role played by miR-298 in bolstering radio-resistance in CRC cells by means of DYRK1A downregulation,thereby positioning miR-298 as a promising candidate for mitigating radioresistance in CRC.

Polo-like kinase 1,on the rise from cell cycle regulation to prostate cancer development

Polo-like kinase 1(Plk1),a well-characterized member of serine/threonine kinases Plk family,has been shown to play pivotal roles in mitosis and cytokinesis in eu-karyotic cells.Recent studies suggest that Plk1 not only controls the process of mitosis and cytokinesis,but also,going beyond those previously described functions,plays critical roles in DNA replication and Pten null prostate cancer initiation.In this review,we briefly summarize the functions of Plk1 in mitosis and cytokinesis,and then mainly focus on newly discov-ered functions of Plk1 in DNA replication and in Pten-null prostate cancer initiation.Furthermore,we briefly introduce the architectures of human and mouse pros-tate glands and the possible roles of Plk1 in human prostate cancer development.And finally,the newly chemotherapeutic development of small-molecule Plk1 inhibitors to target Plk1 in cancer treatment and their translational studies are also briefly reviewed.

Effect of antisense RNA targeting polo-like kinase 1 on cell cycle and proliferation in A549 cells

Background Expression of polo-like kinase 1 (Plk1) is elevated in lung cancer and has been proposed as having prognostic value and related to resistance to chemotherapy and radiation. In addition, Plk1 has several functions in mitotic progression. In this study, the authors investigated t he effect of Plk1 depletion on cell cycle progression and proliferation in A549 cells, a lung cancer cell line.Methods A recombinant plasmid containing antisense RNA targeting Plk1 (pcDNA3-Plk1) was transfected into A549 cells. Reverse transcription-polymerase chain reaction and Western blot were used to examine Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine (BrdU) labelling. Cell cycle and apoptosis were examined by flow cytometry. Expression of α-tubulin was detected by immunofluorescence, and the inhibition rate (IR) by chemotherapeutic agents was determined by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide assay.Results After transfection into A549 cells, pcDNA3-Plk1 reduced Plk1 mRNA by 46.75% for 24 hours and by 61.84% for 48 hours. Plk1 protein was significantly decreased simultaneously (P<0.05). Abnormal morphological changes of cells and growth inhibition were observed in pcDNA3-Plk1 transfected groups. The BrdU labelling index was 25.59% 48 hours after transfection, which was significantly lower than that of the control groups (P<0.05). Forty-eight hours after transfection, there showed absence of microtubule polymerization and spindle abnormalities in staining for α-tubulin. A549 cells showed a strong G 2/M arrest and apoptosis 72 hours post transfection. IR of vinorelbine in pcDNA3-Plk1 transfected groups was significantly higher than that of the other groups (P<0.05, respectively).Conclusions Plk1 depletion interferes with spindle formation, induces cell cycle arrest and apoptosis, and consequently inhibits cell proliferation in A549 cells. Moreover, it sensitizes lung cancer cells to chemotherapy.

Polo-like kinase 1短发卡RNA重组腺病毒载体构建及其生物学应用

目的构建针对人类Polo-like kinase1（Plk1）基因的短发卡RNA（shRNA）真核表达载体，并进行腺病毒包被。方法设计并合成含有小发夹结构的Plk1 shRNA对应模板序列，退火并与pYr-1．1载体重组质粒；通过LR体外同源重组将pYr-1．1-Plk1—shRNA表达框构建至pAd—DEST腺病毒表达载体上；包装重组腺病毒；感染人类胰腺癌细胞BxPC-3验证干扰效率。实验分3组：对照组（Control）、空载组[rAd-增强绿色荧光蛋白（tAd—EGFP）]、实验组（rAd—shPlk1），实时定量反转录聚合酶链反应（RT-qPCR）和Western blot检测Plk1基因的表达，流式细胞术检测细胞周期的变化及对细胞凋亡的影响。结果腺病毒感染BxPC-3细胞后，RT-qPCR检测Plk1 mRNA表达量：对照组1．047±0．315、空载组1．121±0．199、实验组0．119±0．050；Western blot检测Plk1蛋白表达量：对照组0．760±0．088、空载组0．743±0．101、实验组0．050±0．043，实验组较对照组及空载组Plk1 mRNA和蛋白水平均明显降低（P〈0．01）。流式细胞术检测G2期细胞比例：对照组6．077±0．056、空载组6．533±1．644、实验组33．427±7．774，实验组G：期细胞数比例显著高于空载组和对照组（P〈0．01）。感染24h后实验组的细胞凋亡率明显高于空载组和对照组（P〈0．01）。结论成功构建包被有Plk1 shRNA的腺病毒表达载体，且重组腺病毒载体可以抑制Plk1基因的表达，引起细胞周期G2／M期停滞和促进细胞凋亡。

三氧化二砷对结肠癌SW-480细胞凋亡及polo-like kinase-1基因表达的影响

目的观察三氧化二砷对人结肠癌细胞SW-480并探讨其作用机理。方法采用不同浓度的三氧化二砷作用结肠癌SW-480细胞后,采用MTT法检测细胞的恶性增殖,采用琼脂糖凝胶电泳和流式细胞仪检测凋亡及细胞周期情况,采用定量PCR检测polo-likekinase-1(PLK1)mRNA水平。结果三氧化二砷能抑制结肠癌细胞的恶性增殖,且与浓度相关。三氧化二砷能诱导结肠癌细胞凋亡,且呈浓度依赖性。流式细胞仪检测发现,三氧化二砷将细胞阻滞在G2/M期。三氧化二砷能下调结肠癌PLK1mRNA水平,且呈药物浓度和作用时间依赖性。结论三氧化二砷有效地抑制结肠癌SW-480细胞增殖,其机制可能与其下调PLK1表达,从而诱导凋亡有关。

RNAi沉默Polo-like kinase-1基因表达对胰腺癌细胞增殖的影响

目的:探讨polo-like kinase-1(PLK1)基因在胰腺癌细胞中的作用.方法:采用PLK1小干扰核糖核酸分子(small interfering RNA,siRNA)转染人胰腺癌Mi- aPaCa-2细胞后,分别采用实时定量PCR和Western blot检测PLK1基因mRNA和蛋白表达水平,观察PLK1 siRNA转染对胰腺癌细胞体内外增殖的影响.于转染不同时间后收集细胞,分别采用琼脂糖凝胶电泳和TUNEL方法检测胰腺癌细胞凋亡情况.结果:胰腺癌MiaPaCa-2细胞经siRNA转染处理后,PLK1 mRNA和蛋白表达水平明显下降(P<0.05).PLK1基因siRNA可明显抑制癌细胞体外生长(P<0.05)和体内裸鼠模型增殖(P<0.05).细胞凋亡检测发现,DNA电泳出现明显的梯度图谱,且与浓度相关(r=0.836,P<0.05).TUNEL结果显示,转染组癌细胞凋亡指数明显增加,且呈时间和浓度依赖性(r= 0.875,P<0.05).结论:PLK1 siRNA转染可明显抑制胰腺癌细胞增殖,其机制可能与诱导细胞凋亡有关.

丝/苏氨酸蛋白激酶Polo-like Kinase 1研究进展

Plk1是真核生物细胞分裂的重要调控因子。RNA干扰和小分子抑制剂等新技术的应用增加了对Plk1在有丝分裂中功能、底物和调控机制的认识。Plk1不仅在有丝分裂早期参与调节中心体成熟和双极纺锤体的形成,而且在胞质分裂、DNA损伤检验点甚至DNA复制中也有重要作用。该综述比较全面地讨论了Plk1在细胞分裂周期中的作用,重点介绍了Plk1在有丝分裂后期和间期作用的新发现,并就Plk1作为抗肿瘤药物作用靶点的研究进展做了总结。

MCC1019,a selective inhibitor of the Polo-box domain of Polo-like kinase 1 as novel,potent anticancer candidate

Polo-like kinase(PLK1) has been identified as a potential target for cancer treatment.Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain(PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethylbenzofuran-2-carboxylic acid ethyl ester(designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis.This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest—a phenomenon known as mitotic catastrophe, which is followed by immediate cell death via apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in vivo in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.

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 5W480.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.

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

AIM: To elucidate the role of overexpressed polo-like kinase1 (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 over-expression 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 expres-sion was an independent risk factor for HCC. CONCLUSION: PLK1 could be a potential biomarker for diagnosis and therapy for HCC.

Diversity evolution and jump of Polo-like kinase 1 inhibitors

Polo-like kinase 1(Plk1),a member of a family of serine/threonine kinases,is an attractive target for the development of anticancer drugs because it is involved in the regulation of cell-cycle progression and cytokinesis.This kinase provides two pockets for developing Plk1 inhibitors:the N-terminal catalytic domain(NCD)and the polo-box domain(PBD).For both of the two pockets,some natural products were identified as Plk1 inhibitors and some synthetic Plk1 inhibitors were developed by mimicking ATP and phosphopeptides,natural products binding to NCD and PBD respectively.This article not only reviews the progression of Plk1 inhibitors binding to these two pockets,but also discusses diversity evolution and jump in the process of drug development using Plk1 inhibitors as examples and how they impact on drug design and pharmacophore modeling.

Effect of polo-like kinase 1 gene silence on cell cycle and drug resistance in K562/A02 cell

Polo-like kinase 1（PLK1） plays an important role in many cell-cycle-related events. At G2/Mtransition, PLK1 contributes to the activation of cyclinB/Cdc by phosphorylation of Cdc25C, centrosome functional maturation, bipolar spindle formation. In later stage of mitosis, PLK1 is involved in regulating components of the anaphase-promoting complex （APC） for mitotic exit and in the execution of cytokinesis. Moreover, recent reports have shown that PLK1 is involved in both G2 and mitotic DNA damage checkpoints. When G2/M DNA damage occurs, PLK1 activity is suppressed and cell cycle arrests to repair the damaged DNA. So far, the deregulated expression of PLK1 has been detected in many types of human tumors and PLK1 is considered as a novel prognostic marker for several tumors.

Overexpression of mitogen-activated protein kinase phosphatase-1 in endothelial cells reduces blood-brain barrier injury in a mouse model of ischemic stroke

Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.

Tousled-like kinase 1 promotes gastric cancer progression by regulating the tumor growth factor-beta signaling pathway

BACKGROUND The role of Tousled-like kinase 1(TLK1)in in gastric cancer(GC)remains unclear.AIM To investigate the expression,biological function,and underlying mechanisms of TLK1 in GC.METHODS We measured TLK1 protein expression levels and localized TLK1 in GC cells and tissues by western blot and immunofluorescence,respectively.We transfected various GC cells with lentiviruses to create TLK1 overexpression and knockdown lines and established the functional roles of TLK1 through in vitro colony formation,5-ethynyl-2`-deoxyuridine,and Transwell assays as well as flow cytometry.We applied bioinformatics to elucidate the signaling pathways associated with TLK1.We performed in vivo validation of TLK1 functions by inducing subcutaneous xenograft tumors in nude mice.RESULTS TLK1 was significantly upregulated in GC cells and tissues compared to their normal counterparts and was localized mainly to the nucleus.TLK1 knockdown significantly decreased colony formation,proliferation,invasion,and migration but increased apoptosis in GC cells.TLK1 overexpression had the opposite effects.Bioinformatics revealed,and subsequent experiments verified,that the tumor growth factor-beta signaling pathway was implicated in TLK1-mediated GC progression.The in vivo assays confirmed that TLK1 promotes tumorigenesis in GC.CONCLUSION The findings of the present study indicated that TLK1 plays a crucial role in GC progression and is,therefore,promising as a therapeutic target against this disease.