Cyclin-dependent kinase 7 inhibitor THZ1 in cancer therapy

Current cancer therapies have encountered adverse response due to poor therapeutic efficiency,severe side effects and acquired resistance to multiple drugs.Thus,there are urgent needs for finding new cancer-targeted pharmacological strategies.In this review,we summarized the current understanding with THZ1,a covalent inhibitor of cyclin-dependent kinase 7(CDK7),which demonstrated promising anti-tumor activity against different cancer types.By introducing the anti-tumor behaviors and the potential targets for different cancers,this review aims to provide more effective approaches to CDK7 inhibitor-based therapeutic agents and deeper insight into the diverse tumor proliferation mechanisms.

Inhibition of Cyclin F Promotes Cellular Senescence through Cyclin-dependent Kinase 1-mediated Cell Cycle Regulation

Objective Kidney renal clear cell carcinoma(KIRC)is a common renal malignancy that has a poor prognosis.As a member of the F box family,cyclin F(CCNF)plays an important regulatory role in normal tissues and tumors.However,the underlying mechanism by which CCNF promotes KIRC proliferation still remains unclear.Methods Bioinformatics methods were used to analyze The Cancer Genome Atlas(TCGA)database to obtain gene expression and clinical prognosis data.The CCK8 assay,EdU assay,and xenograft assay were used to detect cell proliferation.The cell senescence and potential mechanism were assessed by SA-β-gal staining,Western blotting,as well as ELISA.Results Our data showed that CCNF was highly expressed in KIRC patients.Meanwhile,downregulation of CCNF inhibited cell proliferation in vivo and in vitro.Further studies showed that the reduction of CCNF promoted cell senescence by decreasing cyclin-dependent kinase 1(CDK1),increasing the proinflammatory factors interleukin(IL)-6 and IL-8,and then enhancing the expression of p21 and p53.Conclusion We propose that the high expression of CCNF in KIRC may play a key role in tumorigenesis by regulating cell senescence.Therefore,CCNF shows promise as a new biomarker to predict the clinical prognosis of KIRC patients and as an effective therapeutic target.

Expression of cyclin-dependent kinase inhibitor 2A 16,tumour protein 53 and epidermal growth factor receptor in salivary gland carcinomas is not associated with oncogenic virus infection

It is known that human papillomavirus （HPV） infection can cause squamous cell neoplasms at several sites, such as cervix uteri carcinoma and oral squamous carcinoma. There is little information on the expression of HPV and its predictive markers in tumours of the major and minor salivary glands of the head and neck. We therefore assessed oral salivary gland neoplasms to identify associations between HPV and infection-related epidermal growth factor receptor （EGFR）, cyclin-dependent kinase inhibitor 2A （CDKN2A/p16） and tumour protein p53 （TP53）. Formalin-fixed, paraffin-embedded tissue samples from oral salivary gland carcinomas （n=51） and benign tumours （n=26） were analysed by polymerase chain reaction （PCR） analysis for several HPV species, including high-risk types 16 and 18. Evaluation of EGFR, CDKN2A, TP53 and cytomegalovirus （CMV） was performed by immunohistochemistry. Epstein-Barr virus （EBV） was evaluated by EBV-encoded RNA in situ hybridisation. We demonstrated that salivary gland tumours are not associated with HPV infection. The expression of EGFR, CDKN2A and TP53 may be associated with tumour pathology but is not induced by HPV. CMV and EBV were not detectable. In contrast to oral squamous cell carcinomas, HPV, CMV and EBV infections are not associated with malignant or benign neoplastic lesions of the salivary glands.

Expression of cyclin-dependent protein kinase 5 in the hippocampus of vascular dementia mice after cerebral ischemia and reperfusion

BACKGROUND： The p25-activated cyclin-dependent protein kinase 5 （Cdk5） may induce neuronal cell death and cause the development of dementia following cerebral ischemia and reperfusion. OBJECTIVE： To observe changes in the expression of Cdk5 and p25 in hippocampal tissue of vascular dementia mice at different time points following cerebral ischemia and reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed in the clinical trial center of Hebei Provincial People＇s Hospital between September 2007 and October 2008. MATERIALS： Cdk5 rabbit anti-mouse polyclonal antibody, p35 rabbit anti-mouse polyclonal antibody, and β-actin mouse monoclonal antibody were purchased from Santa Cruz Biotechnology, Inc., USA; horseradish peroxidase-labeled goat anti-rabbit IgG and horseradish peroxidase-labeled goat anti-mice IgG were offered by Beijing Zhongshan Geldenbridye Biotechnology Co.,Ltd., China; the protein quantitative kit was produced by Applygen Gene Technology Corp., Beijing, China; cDNA reverse transcription and PCR amplification reagents were products of TianGen＆ Biotech （Beijing） Co.,Ltd., China. METHODS： One hundred and sixty male Kunming mice were randomly divided into two groups： a sham-operated group （n = 65） and a model group （n = 95）. Vascular dementia was induced with three periods of transient ischemia and reperfusion of the bilateral common carotid arteries. In the sham-operated group, the bilateral common carotid arteries were not blocked. MAIN OUTCOME MEASURES： Behavioral tests were done at four and six weeks post surgery. Pathological changes in the hippocampal CA1 region were observed with hematoxylin-eosin staining Cdk5 mRNA expression was examined by RT-PCR, and Western blots were used to evaluate Cdk5 and p25 expression. Learning and memory performance were assayed using the Morris water maze. RESULTS： Vascular dementia reduced learning and memory performance at 4 and 6 weeks post surgery. Vascular dementia also caused severe, time-dependent neuronal damage and death in the hippocampal CA1 region. Dementia induction also increased mRNA and protein expression of Cdk5 and p25 at both 4 and 6 weeks after surgery. CONCLUSION： Cdk5/p25 is involved in the development of vascular dementia in mice following cerebral ischemia and reperfusion.

Cyclin-dependent kinase 5 is required for suppressing D1-dependent signaling mediated through muscarinic 4 in isolated medium spiny neurons

OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.

Can cyclin-dependent kinase 4/6 inhibitors convert inoperable breast cancer relapse to operability? A case report

BACKGROUND Pathological complete response(pCR) is rare in hormone receptor-positive(HR+)HER2-negative breast cancer(BC) treated with either endocrine therapy(ET) or chemotherapy. Radical resection of locoregional relapse, although potentially curative in some cases, is challenging when the tumor invades critical structures.The oral cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with ET has obtained a significant increase in objective response rates and progression-free survival in patients with advanced BC and is now being evaluated in the neoadjuvant setting. We present a clinical case of a patient with an inoperable locoregional relapse of HR+ HER2-negative BC who experienced p CR after treatment with palbociclib.CASE SUMMARY We report the clinical case of a 60-year-old patient who presented with an inoperable locoregional relapse of HR+, HER2-negative BC 10 years after the diagnosis of the primary tumor. During a routine follow-up visit, breast magnetic resonance imaging and positron emission tomography/computed tomography revealed a 4-cm lesion in the right subclavicular region, infiltrating the chest wall and extending to the subclavian vessels, but without bone or visceral involvement. Treatment was begun with palbociclib plus letrozole, converting the disease to operability over a period of 6 mo. Surgery was performed and a p CR achieved. Of note, during treatment the patient experienced a very uncommon toxicity characterized by burning tongue and glossodynia associated with dysgeusia, paresthesia, dysesthesia, and xerostomia. A reduction in the dose of palbociclib did not provide relief and treatment with the inhibitor was thus discontinued, resolving the tongue symptoms. Laboratory exams were unremarkable. Given that this was a late relapse, the tumor was classified asendocrine-sensitive, a condition associated with high sensitivity to palbociclib.CONCLUSION This case highlights the potential of the cyclin-dependent kinase 4/6 inhibitor plus ET combination to achieve pCR in locoregional relapse of BC, enabling surgical resection of a lesion initially considered inoperable.

Expression of cyclin-dependent kinases in HL-60 cells during differentiation induced by retinoic acid

This study was designed to investigate the relationship of the expression of cyclin-dependent kinases (CDKs) with theeffects of all-trans retinoic acid (ATRA) on the proliferation of HL-cells. HL-60 cells were treated with ATRA for 1-4 d. Then thecapacity of DNA Synthesis was evaluated with 3H-TdR incorporation and the expression of cyclin E, cyclin D, CDK2 and CDK4protein determined with immunocytochemical staining. In addition, the expression Of CDC2, CDK2 and CDK4 mRNA was deter-mined with in situ hybridization. It was found that ATRA suppressed the proliferation of HL-60 cells and decreased their capacityof DNA synthesis to result in a down-regulation of the expression of cyclin E, cyclin D and CDC2 without comcomittant suppressionon the expression of CDK2 and CDK4. It is concluded that the effects of ATRA on the proliferation of HL-60 cells may be relatedto the down-regulation of the expression of cyclin E, cyclin D and CDC2.

SOX7靶向ERK1/2/PD-L1通路抑制结直肠癌血管生成

目的探讨性别决定区Y框蛋白7(SOX7)对结直肠癌血管生成的影响及潜在作用机制。方法应用免疫荧光检测结直肠癌患者组织样本中SOX7表达水平,之后通过裸鼠、转染SOX7 mimic的人结直肠癌细胞系SW480细胞和人脐静脉内皮细胞(HUVEC)共培养进一步研究。用Western-blot验证SOX7与ERK1/2/PD-L1对结直肠癌细胞的相关蛋白表达的影响。用CCK8检测SOX7与ERK1/2/PD-L1对HUVEC增殖的影响。通过体外内皮细胞成管实验测定SOX7与ERK1/2/PD-L1对肿瘤血管生成的影响。结果SOX7在人结直肠癌组织中表达被抑制(P<0.01),同时SOX7的过表达抑制了小鼠体内肿瘤生长(P<0.01)。SW480细胞中SOX7的过表达抑制了ERK1/2、c-Jun的表达,并在ERK1/2的激动剂Senkyunolide I的作用下上调了SW480细胞的ERK1/2、c-Jun蛋白表达(P<0.01),逆转了SOX7对SW480细胞中ERK1/2、c-Jun蛋白表达的影响(P<0.01)。HUVEC中SOX7抑制了PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,Senkyunolide I上调了HUVEC的PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,并逆转了SOX7对HUVEC中上述相关蛋白表达的影响(P<0.01)。PD-1/PD-L1 Inhibitor 3抑制了PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,SOX7过表达在PD-1/PD-L1 Inhibitor 3的影响下并没有表现出抑制作用。CCK8实验结果显示SOX7过表达显著抑制了HUVEC的增殖能力,Senkyunolide I作用下的两组HUVEC增殖能力较SOX7 NC组与SOX7 mimic组明显上升,PD-1/PD-L1 Inhibitor 3作用下的两组HUVEC增殖能力较SOX7 NC组与SOX7 mimic组明显下降,以上均有明显统计学差异(P<0.01)。成管实验结果显示SOX7过表达抑制了HUVEC的血管生成,Senkyunolide I强烈加速了血管生成,而PD-1/PD-L1 Inhibitor 3血管生成则被显著抑制,以上均有明显统计学差异(P<0.01)。结论SOX7通过ERK1/2/PD-L1通路抑制结直肠肿瘤的增殖和血管生成,SOX7可能是晚期CRC患者临床治疗中潜在的抗血管生成靶点。

Chromodomain-helicase-DNA binding protein 5, 7 and pronecrotic mixed lineage kinase domain-like protein serve as potential prognostic biomarkers in patients with resected pancreatic adenocarcinomas

Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomainhelicase-DNA binding protein 5, chromodomain-helicaseDNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation,and should be validated in future studies.

TLR7/8 signalling affects X-sperm motility via the GSK3α/β-hexokinase pathway for the efficient production of sexed dairy goat embryos

Background:Goat milk is very similar to human milk in terms of its abundant nutrients and ease of digestion.To derive greater economic benefit,farmers require more female offspring(does);however,the buck-to-doe offspring sex ratio is approximately 50%.At present,artificial insemination after the separation of X/Y sperm using flow cytometry is the primary means of controlling the sex of livestock offspring.However,flow cytometry has not been successfully utilised for the separation of X/Y sperm aimed at sexing control in dairy goats.Results:In this study,a novel,simple goat sperm sexing technology that activates the toll-like receptor 7/8(TLR7/8),thereby inhibiting X-sperm motility,was investigated.Our results showed that the TLR7/8 coding goat Xchromosome was expressed in approximately 50%of round spermatids in the testis and sperm,as measured from cross-sections of the epididymis and ejaculate,respectively.Importantly,TLR7/8 was located at the tail of the Xsperm.Upon TLR7/8 activation,phosphorylated forms of glycogen synthase kinaseα/β(GSK3α/β)and nuclear factor kappa-B(NF-κB)were detected in the X-sperm,causing reduced mitochondrial activity,ATP levels,and sperm motility.High-motility Y-sperm segregated to the upper layer and the low-motility X-sperm,to the lower layer.Following in vitro fertilisation using the TLR7/8-activated sperm from the lower layer,80.52±6.75%of the embryos were XX females.The TLR7/8-activated sperm were subsequently used for in vivo embryo production via the superovulatory response;nine embryos were collected from the uterus of two does that conceived.Eight of these were XX embryos,and one was an XY embryo.Conclusions:Our study reveals a novel TLR7/8 signalling mechanism that affects X-sperm motility via the GSK3α/β-hexokinase pathway;this technique could be used to facilitate the efficient production of sexed dairy goat embryos.

血清CXCR7、SGK1水平与急性心肌梗死经皮冠状动脉介入治疗术后预后不良的关系

目的探讨血清C-X-C基序趋化因子受体7(CXCR7)、血清/糖皮质激素调节激酶1(SGK1)水平与急性心肌梗死(AMI)经皮冠状动脉介入治疗(PCI)术后预后不良的关系。方法选取2020年9月—2022年12月于南京医科大学第二附属医院急诊科接受PCI术的AMI患者100例为AMI组,同期医院健康体检者50例为健康对照组,根据PCI术后1年预后情况将AMI患者分为预后不良亚组30例和预后良好亚组70例。采用酶联免疫吸附法检测血清CXCR7、SGK1水平;多因素Logistic回归分析AMI患者PCI术后预后不良的影响因素;建立受试者工作特征(ROC)曲线评价血清CXCR7、SGK1水平对AMI患者PCI术后预后不良的预测价值。结果与健康对照组比较,AMI组血清CXCR7水平降低,SGK1水平升高(t/P=9.613/<0.001、9.955/<0.001);100例AMI患者PCI术后1年不良预后发生率为30.00%(30/100);与预后良好亚组比较,预后不良亚组血清CXCR7水平降低,SGK1水平升高(t/P=6.254/<0.001、5.329/<0.001)。多因素Logistic回归显示,Gensini评分高、KILLIP分级≥Ⅲ级、SGK1升高为AMI患者PCI术后预后不良的独立危险因素[OR(95%CI)=1.071(1.025~1.119)、4.501(1.172~17.282)、1.132(1.046~1.224)],CXCR7升高为独立保护因素[OR(95%CI)=0.956(0.926~0.987)]。血清CXCR7、SGK1及二者联合预测AMI患者PCI术后预后不良的AUC分别为0.794、0.779、0.902,二者联合的AUC大于血清CXCR7、SGK1单独预测(Z/P=3.062/0.002、2.930/0.003)。结论AMI患者血清CXCR7水平降低、SGK1水平升高,是PCI术后不良预后的影响因素,二者联合对其预测价值较高。

脑出血患者脑组织中NEK7、NLRP3的表达水平及其与疾病严重程度的关系

目的 探究脑出血患者脑组织中Nod样受体蛋白-3 (NLRP3)、NIMA相关蛋白激酶7 (NEK7)表达水平与疾病严重程度的相关性。方法 前瞻性选取2017年1月至2020年12月郑州颐和医院诊治的80例脑出血患者进行研究,取皮层造瘘通道靠近血肿0.5 cm处的脑组织为靠近组,另取远离血肿位置的脑组织为远离组。依据脑出血患者出血量将其分为少量组(出血量<15 mL) 29例、中量组(出血量15~30 m L) 27例和大量组(出血量>30 mL)24例;按美国国立卫生研究院卒中量表(NIHSS)评分将患者分为轻型组(1~4分) 30例、中型组(5~15分) 27例和重型组(>15分) 23例。采用实时荧光定量PCR (qRT-PCR)法测定各组脑组织中NEK7 m RNA、NLRP3 m RNA表达水平;采用Pearson法分析脑出血患者血肿0.5 cm处脑组织中NEK7 m RNA表达水平与NLRP3 m RNA表达水平的相关性;比较不同出血量、不同严重程度的脑出血患者距离血肿0.5 cm处脑组织中NEK7 m RNA、NLRP3 m RNA表达水平。结果 靠近组患者脑组织中NEK7 m RNA、NLRP3 m RNA表达水平分别为1.72±0.58、1.69±0.57,明显高于远离组的1.03±0.34、1.01±0.33,差异均有统计学意义(P<0.05);脑出血患者血肿0.5 cm处脑组织中NEK7 m RNA表达水平与NLRP3 mRNA表达水平呈正相关(r=0.563,P<0.05);脑出血患者距离血肿0.5 cm处脑组织中NEK7m RNA、NLRP3 m RNA表达水平随着出血量的增加而升高,差异均有统计学意义(P<0.05);脑出血患者距离血肿0.5 cm处脑组织中NEK7 mRNA、NLRP3 m RNA表达水平随着NIHSS评分的增加而升高,差异均有统计学意义(P<0.05)。结论 脑出血患者距离血肿0.5 cm处脑组织中NEK7、NLRP3表达水平明显升高,两者均与出血量和疾病严重程度显著相关,检测距离血肿0.5 cm处脑组织NEK7、NLRP3有利于判断脑出血严重程度及出血情况。

PTK7在口腔鳞癌中的表达分析及其生物学功能研究

目的探讨PTK7在口腔鳞癌(oral squamous cell carcinoma,OSCC)中的表达水平、潜在的生物学功能及其临床意义。方法采用实时荧光PCR(quantitive real-time polymerase chain reaction,qPCR)和Western blot检测PTK7在OSCC细胞系和口腔鳞癌组织标本中的表达情况,利用siRNA干扰技术下调PTK7在HN6和Cal27细胞系中的表达,通过CCK-8实验、平板克隆实验、细胞划痕实验及Transwell实验检测PTK7下调后其对OSCC细胞系增殖、迁移、侵袭的影响;同时采用免疫组织化学染色法检测75例口腔鳞癌组织中PTK7蛋白的表达水平,分析其相关的临床意义。结果qPCR及Western blot结果显示PTK7基因及其编码蛋白在口腔鳞癌细胞系HN6和Cal27中高表达,并在6例新鲜口腔鳞癌标本中的表达高于其配对的癌旁正常组织;下调PTK7的表达可抑制OSCC细胞增殖、迁移和侵袭功能;在75例口腔鳞癌组织中,PTK7的表达水平与OSCC患者的发病年龄、吸烟情况、病理分化程度等相关,其差异有统计学意义(P<0.05)。Kaplan-Meier生存分析发现,高表达PTK7的OSCC患者的预后较差。结论PTK7是口腔鳞癌发生发展过程的潜在癌基因,其表达水平影响OSCC细胞的生物学功能,临床上可根据PTK7表达水平了解口腔鳞癌的特性,PTK7可作为口腔鳞癌预后判定的独立指标。

前列腺癌组织细胞周期蛋白依赖性激酶2、7、9表达与临床病理特征及预后的关系研究

目的:研究前列腺癌(PC)组织细胞周期蛋白依赖性激酶CDK2、CDK7、CDK9表达与患者临床病理特征及预后的关系。方法:选取2016年3月至2018年3月哈尔滨二四二医院收治的167例PC患者,术中收集癌组织及癌旁正常前列腺组织。应用免疫组织化学染色法检测CDK2、CDK7、CDK9蛋白表达情况,分析CDK2、CDK7、CDK9与PC临床病理特征的关系。随访5年,收集患者的生存情况资料,采用Kaplan-Meier分析CDK2、CDK7、CDK9阴性和阳性表达患者生存率情况,采用单因素和多因素Cox回归分析PC患者预后的影响因素。结果:与癌旁正常前列腺组织比较,PC组织CDK2、CDK7、CDK9阳性表达率升高(P<0.05)。有淋巴结转移、术前游离PSA含量越高、Gleaso评分越高、TNM分期越高,CDK2、CDK7、CDK9阳性表达率越高(P<0.05)。Kplan-Meier分析结果显示,CDK2、CDK7、CDK9阳性表达组生存率低于阴性表达组(P<0.05)。单因素Cox回归分析显示,术前游离PSA、Gleaso评分、淋巴结转移、TNM分期及CDK2、CDK7、CDK9表达情况与PC患者预后有关(P<0.05)。多因素Cox分析结果显示,术前游离PSA≥1.9 ng/mL、Gleaso评分≥7分、有淋巴结转移、TNM分期Ⅲ期、CDK2阳性表达、CDK7阳性表达、CDK9阳性表达均为PC预后不良的危险因素(P<0.05)。结论:CDK2、CDK7、CDK9在PC组织中呈高表达,其表达水平与PC患者术前游离PSA、Gleaso评分、淋巴结转移、TNM分期及预后相关。

视黄醇结合蛋白7调控Akt/mTOR信号通路对乳腺癌细胞增殖和迁移侵袭的影响

目的探讨视黄醇结合蛋白7(RBP7)调控蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对乳腺癌细胞增殖和迁移侵袭的影响。方法GEPIA网站用于分析乳腺癌组织的RBP7表达,实时定量PCR(qPCR)用于检测乳腺癌细胞(MCF7、SKBR-3、BT474、T47D、BT549和MDA-MB-231)的RBP7表达。将MDA-MB-231细胞分为pcDNA3.1组(阴性对照)、pcDNA3.1-RBP7组(上调RBP7表达)和pcDNA3.1-RBP7+3BDO组(上调RBP7表达且增强mTOR活性)。qPCR和Western blot用于检测磷酸化Akt(p-Akt)、磷酸化mTOR(p-mTOR)和c-Myc的表达。MTT法、划痕愈合实验和Transwell小室实验检测MDA-MB-231细胞的增殖、迁移和侵袭情况。结果RBP7在乳腺癌组织和细胞中低表达(P<0.05)。生物信息学分析显示RBP7低表达乳腺癌患者的预后较差。pcDNA3.1-RBP7组p-Akt、p-mTOR和c-Myc的表达低于pcDNA3.1组(P<0.05);而pcDNA3.1-RBP7+3BDO组p-Akt、p-mTOR和c-Myc的表达高于pcDNA3.1-RBP7组(P<0.05)。此外,pcDNA3.1-RBP7组MDA-MB-231细胞的增殖、迁移和侵袭能力均弱于pcDNA3.1组(P<0.05),而pcDNA3.1-RBP7+3BDO组MDA-MB-231细胞的增殖、迁移和侵袭能力均强于pcDNA3.1-RBP7组(P<0.05)。结论RBP7通过调控Akt/mTOR通路抑制乳腺癌细胞的增殖、迁移和侵袭,可能为乳腺癌患者提供一种新的治疗干预手段。

基于嘌呤配体P2X门控离子通道型受体7的资生肾气丸镇痛机制研究

目的:探索资生肾气丸对醋酸致小鼠扭体模型的镇痛作用。方法:将42只小鼠随机分为7组,分别给予相应药液灌胃,末次灌胃1 h后造模,建立醋酸致小鼠扭体模型。观察小鼠扭体次数,计算其扭体抑制率,应用酶联免疫吸附试验(ELISA)检测各组小鼠血清白细胞介素-1β(IL-1β)、前列腺素E 2(PGE 2)、神经生长因子(NGF)含量,采用实时PCR(RT-PCR)检测嘌呤配体P2X门控离子通道型受体7(P2X7R)mRNA、Nod样受体蛋白3(NLRP3)mRNA、NIMA相关激酶7(NEK7)mRNA的表达情况。结果:与模型组比较,随着时间的增加,资生肾气丸低剂量、中剂量、高剂量均可抑制小鼠扭体反应,降低扭体次数,增加小鼠扭体抑制率,降低小鼠血清中IL-1β、PGE_(2)、NGF的含量,降低P2X7RmRNA、NLRP3mRNA、NEK7mRNA的表达以抑制疼痛反应,以高剂量效果最优(P<0.05),与吲哚美辛、痛风舒片疗效相近。结论:资生肾气丸高剂量对小鼠血清疼痛因子的降低效果显著,其机制可能与嘌呤配体P2X门控离子通道型受体7信号通路有关。

Long non-coding RNA H19 promotes proliferation inhepatocellular carcinoma cells via H19/miR-107/CDK6 axis

Hepatocellular carcinoma (HCC) is the leading cause of cancer death worldwide;nevertheless, currenttherapeutic options are limited or ineffective for many patients. Therefore, elucidation of molecular mechanisms inHCC biology could yield important insights for the intervention of novel therapies. Recently, various studies havereported dysregulation of long non-coding RNAs (lncRNAs) in the initiation and progression of HCC, including H19;however, the biological function of H19 in HCC remains unclear. Here, we show that knockdown of H19 disruptedHCC cell growth, impaired the G1-to-S phase transition, and promoted apoptosis, while overexpression of H19yielded the opposite results. Screening for expression of cell cycle-related genes revealed a significant downregulationof CDK6 at both RNA and protein levels upon H19 suppression. Bioinformatic analysis of the H19 sequence and the3′ untranslated region (3′ UTR) of CDK6 transcripts showed several binding sites for microRNA-107 (miR-107), andthe dual luciferase reporter assay confirmed their direct interaction with miR-107. Consistently, blockage of miR-107activity alleviated the growth suppression phenotypes induced by H19 downregulation, suggesting that H19 serves asa molecular sponge for miR-107 to promote CDK6 expression and cell cycle progression. Together, this studydemonstrates a mechanistic function of H19 in driving the proliferation of HCC cells and suggests H19 suppressionas a novel approach for HCC treatment.

PTK7在口腔鳞状细胞癌中的表达及临床意义

目的:通过在口腔鳞状细胞癌(oral squamous cell carcinoma, OSCC)组织样本中研究酪氨酸蛋白激酶7(protein tyrosine kinase 7, PTK7)的表达,分析PTK7蛋白的表达在OSCC中的作用及对患者预后的影响。方法:选取OSCC组织样本总共113例,采用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶连结(streptavidin perosidase, SP)法检测PTK7蛋白的表达,结合OSCC发展、预后等临床相关因素进行卡方检验及Kaplan-Meier生存分析等统计学分析。结果:113例OSCC组织标本中有70例PTK7蛋白表达阳性,在癌旁正常鳞状上皮组织中,PTK7蛋白表达为阴性。进一步的分析结果表明,PTK7的表达与OSCC患者的相关影响因素(年龄、性别、分化程度、临床分期、肿瘤大小及局部浸润范围、淋巴结转移等)相比较,差异无统计学意义(P≥0.05);Kaplan-Meier生存分析结果显示,PTK7蛋白表达阴性组的总生存率显著高于阳性组(P<0.05)。结论:PTK7的检测有助于综合判断OSCC的临床预后,提示PTK7作为OSCC的预后指标值得深入研究。

沉默CDK7通过下调YAP表达诱导子宫内膜癌细胞凋亡

目的 研究沉默细胞周期蛋白依赖性激酶7(CDK7)诱导Yes相关蛋白(YAP)表达下调对子宫内膜癌HEC-1-A细胞凋亡的影响。方法 利用siRNA干扰技术沉默HEC-1-A细胞中CDK7的表达,采用qPCR和Western blotting检测siRNA干扰后CDK7的表达水平,CCK-8实验检测沉默CDK7对HEC-1-A细胞活力的影响,流式细胞术检测沉默CDK7对HEC-1-A细胞凋亡的影响,Western blotting检测沉默CDK7对YAP和磷酸化YAP蛋白及其下游蛋白Cyr16、CTGF表达的影响,凋亡实验检测共转染si-CDK7和pcDNA3.1-YAP对细胞凋亡的影响。结果 转染si-CDK7后,HEC-1-A细胞中CDK7 mRNA和蛋白表达水平均显著下降(P<0.01),细胞活性显著降低(P<0.01),细胞凋亡率显著升高(P<0.01),YAP和磷酸化YAP蛋白及其下游蛋白Cyr16、CTGF表达水平明显下降(P<0.01);共转染pcDNA3.1-YAP可逆转沉默CDK7导致的细胞凋亡(P<0.01)。结论 沉默CDK7可诱导YAP蛋白表达下调,促进子宫内膜癌HEC-1-A细胞凋亡。

MicroRNA-7-5p通过mTORC2/SGK-1信号通路负向调控人肺泡上皮细胞钠离子通道

目的:探讨microRNA-7-5p通过哺乳动物雷帕霉素靶蛋白复合物2(mTORC2)/血清糖皮质激素诱导激酶-1(SGK-1)信号通路负向调控人肺泡上皮细胞钠离子通道(ENaC)的机制。方法:对人类非小细胞肺癌肺泡上皮细胞系A549细胞转染microRNA-7-5p模拟剂,设为microRNA-7-5p组,阴性对照组A549细胞转染与目的基因序列无同源性的不表达microRNA-7-5p的阴性对照剂。雷帕霉素组在A549细胞培养基中加入雷帕霉素。PP242组在A549细胞培养基中加入PP242。空白对照组仅加入lipo fectamineTM 2000试剂。采用RT-qPCR检测各组SGK-1 mRNA;免疫印迹法检测SGK-1蛋白、ENaC蛋白的表达量。比较各组的microRNA-7-5p表达水平、SGK-1 mRNA及蛋白表达量、ENaC蛋白表达量。结果:microRNA-7-5p组的microRNA-7-5p表达水平高于空白对照组、阴性对照组、雷帕霉素组和PP242组,A549细胞转染成功上调microRNA-7-5p表达水平(P<0.05)。microRNA-7-5p组中,SGK-1 mRNA水平、SGK-1及ENaC-α、ENaC-β、ENaC-γ蛋白表达量均低于空白对照组、阴性对照组、雷帕霉素组(P<0.05)。结论:microRNA-7-5p可能通过mTORC2/SGK-1信号通路负向调控ENaC。