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.

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.

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.

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.

Abemaciclib, a Recent Novel FDA-Approved Small Molecule Inhibiting Cyclin-Dependant Kinase 4/6 for the Treatment of Metastatic Breast Cancer: A Mini-Review

Abemaciclib (Verzerio®) is a cell cycle inhibitor of both CDK4 and CDK6. In 2017, abemaciclib was approved by the Food and Drug Administration (FDA) and, in 2018 by the European Medicines Agency (EMA) for the treatment of postmenopausal women with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2−) advanced breast cancer. In this mini-review, we provide a series of information for respectively their targets and its selectivity, results on preclinical trial, clinical phase I, II and III trials, and some perspectives. We also describe the batch and flow steps used for the synthesis of this cancer drug.

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.

P21活化激酶4在胃肠道间质瘤中的表达及意义

目的通过研究不同危险度胃肠道间质瘤(gastrointestinal stromal tumor,GIST)中P21活化激酶4(P21-activated kinase 4,PAK4)的表达量,探讨PAK4与GIST危险度的关系。方法选取2020年10月至2021年4月秦皇岛市第一医院病理诊断为GIST患者24例,根据共识将患者分为高危组、中危组、低危组,通过病理切片来确定不同危险度组的PAK4的表达量,并根据Callow计算方法对不同危险度组进行评分。结果PAK4随着GIST危险度增加表达升高,并且与正常对照组织及瘤旁组织相比,GIST中的PAK4表达量升高。结论PAK4表达量与GIST危险度相关,并且随着危险度升高,表达量也升高。

基于FAERS数据库的周期蛋白依赖性激酶4/6抑制剂血液毒性真实世界研究

目的 基于美国美国食品药品管理局(FDA)的不良事件报告系统(FAERS)分析3种周期蛋白依赖性激酶4/6(CDK4/6)抑制剂上市后的不良事件(AEs)信号,为临床用药安全提供参考。方法 提取FAERS数据库2015年第一季度至2022年第一季度共29个季度AEs,利用报告比值比法(ROR)和比例报告比值法(PRR)对CDK4/6抑制剂AEs进行数据挖掘。结果 CDK4/6抑制剂相关性血液毒性报告共有7 872份,各抑制剂血液毒性AEs占总AEs比例依次为哌柏西利(80.31%)>瑞博西利(15.36%)>阿贝西利(4.33%)。血液毒性常见中性粒细胞减少和贫血。哌柏西利(2 982/6 322,47.17%)和瑞博西利(613/1 209,50.70%)致中性粒细胞减少的报告占比较阿贝西利(117/341,34.31%)更高,血液毒性主要发生在药物开始使用后60 d内(1 630,61.86%),哌柏西利中位时间最长,且用药90 d后仍有32.9%的患者存在血液毒性,不同CDK4/6抑制剂血液毒性临床表现及发生强度存在差异。结论 哌柏西利、阿贝西利、瑞博西利均会导致明显的血液毒性,其中阿贝西利致血液毒性报告最少,但要警惕阿贝西利致贫血后导致死亡的风险。用药后的2个月内密切监测全血细胞计数,关注中性粒细胞、血红蛋白等水平,警惕CDK4/6抑制剂相关血液AEs的发生。

中晚期宫颈癌患者血清HE4、TK1、DCLK1水平变化及其与化疗效果的关系

目的 分析中晚期宫颈癌患者血清人附睾蛋白4(HE4)、胸苷激酶1(TK1)、双皮质素样激酶1(DCLK1)变化及其与化疗效果的关系。方法 收集2020年1月至2023年1月于郑州大学第一附属医院接受化疗的215例中晚期宫颈癌患者的病历资料,根据纳入患者的化疗效果将其分为良好组和不良组,其中良好组疗效评估结果为完全缓解(CR)与部分缓解(PR),共173例,不良组疗效评估结果为稳定(SD)与进展(PD),共42例。比较两组血清HE4、TK1、DCLK1水平等临床资料,分析中晚期宫颈癌患者血清HE4、TK1、DCLK1水平与化疗效果的关系。结果 良好组临床分期为Ⅱ期比例、高分化比例、无淋巴结转移比例均高于不良组,肿瘤最大直径以及血清HE4、TK1、DCLK1水平均低于不良组,差异均有统计学意义(P<0.05);经logistic多因素分析显示,肿瘤的临床分期达到Ⅳ期、分化程度为中低分化、淋巴结转移以及血清HE4、TK1、DCLK1水平升高均为影响中晚期宫颈癌患者化疗效果的独立因素(P<0.05);经Spearman相关性分析显示,中晚期宫颈癌患者临床分期、淋巴结转移以及血清HE4、TK1、DCLK1水平与其化疗效果成负相关,分化程度与其化疗效果成正相关(P<0.05)。结论 中晚期宫颈癌患者临床分期越晚、分化程度越差、临床转移以及HE4、TK1、DCLK1水平越高,越不利于患者的化疗,上述指标对其预后均具有一定预测价值。

下调PDCD4抑制MAP2K3和p38 MAPK的表达减轻LPS诱导的肾小管上皮细胞炎症和凋亡

目的研究程序性细胞死亡蛋白4(programmed cell death protein 4,PDCD4)在脓毒症诱导的急性肾损伤(acute kidney injury,AKI)中的作用机制,以及调控PDCD4表达通过丝裂原活化蛋白激酶3(mitogen-activated protein kinase 3,MAP2K3)和p38蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)对脓毒症AKI起到潜在治疗作用。方法用脂多糖(lipopolysaccharide,LPS)刺激人肾小管上皮细胞(HK-2)构建脓毒症AKI细胞模型。进一步用腺病毒介导siRNA和过表达载体抑制和上调AKI细胞模型中PDCD4的表达;CCK-8法检测细胞增殖;用DCFH-DA及激光共聚焦显微镜检测细胞中ROS水平,用总SOD活性检测试剂和MDA检测试剂盒检测细胞中SOD和MDA水平;免疫共沉淀验证PDCD4和MAP2K3之间的蛋白相互作用;TUNEL染色法检测细胞凋亡;RT-qPCR和Western blot检测PDCD4及相关基因的mRNA和蛋白表达水平;ELISA法检测患者血清中炎症相关因子水平。结果LPS诱导可以促进HK-2细胞中PDCD4表达,下调PDCD4可抑制LPS诱导的HK-2细胞的炎症、氧化应激及细胞凋亡。数据库预测及免疫共沉淀证实PDCD4可以与MAP2K3相互作用,且在LPS诱导的HK-2细胞中,MAP2K3表达水平显著增强。MAP2K3过表达和p38 MAPK激动剂可以减轻PDCD4下调对LPS诱导的细胞炎症和氧化应激的影响并抑制细胞凋亡。结论下调PDCD4可以通过抑制MAP2K3和p38 MAPK从而抑制LPS诱导的肾小管上皮细胞的炎症和凋亡。

CDK4/6抑制剂在HR^(+)晚期乳腺癌治疗中的耐药机制及进展后治疗策略

细胞周期蛋白依赖性激酶4/6(CDK4/6)抑制剂联合内分泌治疗已成为激素受体阳性(HR^(+))、人类表皮生长因子受体-2阴性(HER2^(-))乳腺癌患者的晚期一线及二线标准治疗方案。尽管CDK4/6抑制剂可实现有效的疾病控制,但对于晚期乳腺癌患者最终仍会因耐药出现疾病进展。目前CDK4/6抑制剂相关耐药机制尚不完全清楚,同时治疗失败后的最佳治疗策略仍是一个亟待解决的问题。本文就CDK4/6抑制剂的潜在耐药机制和后续治疗策略的最新研究进展做一综述。

牦牛小脑不同区域神经营养素-4及其受体的表达特征与定位研究

神经营养素-4(NT-4)及其神经营养性酪氨酸激酶受体2(NTRK2)在小脑神经元存活、生长以及功能方面发挥着重要作用。为探讨NT-4和NTRK2在牦牛高原低氧适应中的作用,本研究以高原牦牛(Bos grunniens)和平原黄牛(Bos taurus)小脑为研究对象,采用实时荧光定量PCR(qRT-PCR)、蛋白免疫印迹技术(WB)、苏木精-伊红染色(HE)和免疫组织化学(IHC)对NT-4和NTRK2在牦牛与黄牛小脑不同区域中的表达分布进行分析。qRT-PCR和WB结果表明,NT-4基因和蛋白在牦牛小脑半球皮质中表达量最高,显著高于其他组织(P<0.05);NTRK2基因和蛋白在牦牛小脑蚓部皮质中表达量最高,显著高于其他组织(P<0.05)。与黄牛相比,牦牛NT-4蛋白在小脑各区域中的表达均显著高于黄牛(P<0.05);牦牛NTRK2蛋白在蚓部髓质和小脑半球髓质中的表达量低于黄牛或无差异,其余区域均显著高于黄牛(P<0.05)。IHC结果显示,NT-4和NTRK2蛋白阳性表达特征基本一致,皮质区主要分布于分子层的篮状细胞、浦肯野细胞层以及颗粒细胞层,而髓质区则散在分布于神经胶质细胞以及神经纤维中。由上述结果可知,NT-4和NTRK2在小脑各区域的表达差异可能与其参与脑组织生理功能以及适应高原低氧环境有关。在低氧环境下,NT-4和NTRK2通过上调激活相关通路以发挥内源性神经保护作用,进而保护脑组织免受低氧损伤。本研究结果可为探究牦牛脑组织低氧适应机制提供基础。

Synthesis of 5-substituted benzyl-2,4-diamino pyrimidine derivatives as c-Fms kinase inhibitors

A serials of novel 5-substituted benzyl-2,4-diamino pyrimidine derivatives have been synthesized and evaluated as inhibitors of c-Fms kinase by the standard MTT method.The results showed that compound 15,5-[3-methoxy-4-（pyridine-3-yl）benzyl]-2,4-diamino pyrimidine,had an IC50 of 1.45μmol/L in inhibiting the proliferation of M-CSF-dependent myeloid leukemia cells in mice （NFS-60）,which was similar with GW2580,a selective inhibitor of c-Fms kinase.

Role of Toll-like receptor 4 and Janus kinase and signal transducer and activator of transcription signal transduction pathway in sepsis-induced brain damage

The Janus kinase and signal transducer and activator of transcription （JAK/STAT） signal transduction pathway is involved in sepsis-induced functional damage to the heart, liver, kidney, and other organs. However, the cellular and molecular mechanisms underlying sepsis-induced brain damage remain elusive. In the present study, we found severe loss of neurons in the hippocampal CA1 region in rats with sepsis-induced brain damage following intraperitoneal injection of endotoxin, The expression of toll-like receptor 4, tumor necrosis factor a, and interleukin-6 was significantly increased in brain tissues following lipopolysaccharide exposure. AG490 （JAK2 antagonist） and rapamycin （STAT3 antagonist） significantly reduced neuronal loss and suppressed the increased expression of toll-like receptor 4, tumor necrosis factor a, and interleukin-6 in the hippocampal CA1 region in sepsis-induced brain damaged rats. Overall, these data suggest that blockade of the JAK/STAT signal transduction pathway is neuroprotective in sepsis-induced brain damage via the inhibition of toll-like receptor 4, tumor necrosis factor a, and interleukin-6 exoression.

齐墩果酸通过miR-18a-5p/STK4轴抑制白血病K562细胞恶性进展

慢性粒细胞白血病(chronic myeloid leukemia,CML)是由于骨髓造血干细胞的恶性增生导致的疾病。虽然酪氨酸激酶抑制剂(Tyrosine kinase inhibitors,TKI)对慢性粒细胞性白血病的治疗取得长足进展,但耐药问题仍未解决。因此,寻找新的治疗药物和靶点十分关键。有研究表明,miRNAs与慢性粒细胞白血病在内的多种肿瘤有密切联系,齐墩果酸(oleanolic acid,OA)对白血病细胞有较好的抑制效果。通过对转录物组测序结果发现,齐墩果酸可以显著上调丝氨酸/苏氨酸蛋白质激酶4(serine/threonine-protein kinase 4,STK 4)的水平,而miR1a-5p是STK 4的靶miRNA。因此,本文旨在探究齐墩果酸是否可以通过miR-18a-5p/STK4影响K562细胞恶性进展。K562细胞经齐墩果酸处理后差异表达基因富集在凋亡相关通路上。EdU实验和CCK-8实验结果发现,齐墩果酸降低K562细胞增殖能力(P<0.05)。qRT-PCR,免疫荧光和Western印迹结果显示,齐墩果酸处理后,K562细胞中STK 4蛋白质水平表达显著上调(P<0.05),miR-18a-5p表达下调(P<0.05)。在细胞中转染miR-18a-5p mimics,能显著抑制STK 4的表达(P<0.05);转染miR-18a-5p inhibitor能显著增加STK 4的表达(P<0.05)。活性氧(reactive oxygen species、ROS)检测和线粒体膜电位检测结果显示,齐墩果酸可以促进K562细胞中ROS的增加,降低线粒体膜电位。过表达STK 4后,与Vector组相比,细胞的线粒体膜电位下降;敲降STK 4可以逆转齐墩果酸组导致的线粒体膜电位下降。流式细胞术检测显示,齐墩果酸能明显促进细胞凋亡的发生,而转染miR-18a-5p mimics后凋亡率显著下调(P<0.05);过表达STK 4可以促进细胞从早期凋亡向晚期凋亡转化,而同时转染miR-18a-5p mimics可以抑制这一现象。我们的研究结果证实,齐墩果酸可以通过维持miR-18a-5p的低表达和保持STK 4的高表达状态来促进K562细胞的凋亡。

Downregulation of Aquaporin 4 Expression through Extracellular Signal-regulated Kinases1/2 Activation in Cultured Astrocytes Following Scratch-injury

Objective To investigate the role of extracellular signal-regulated kinase1/2（ERK1/2） pathway in the regulation of aquaporin 4（AQP4） expression in cultured astrocytes after scratch-injury. Methods The scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase（LDH） leakages were observed to assess the degree of scratch-injury. AQP4 expression was detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2（p-ERK1/2） expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 μmol/L U0126（ERK1/2 inhibitor） was incubated in the medium at 30 min before the scratch-injury in some groups. Results Increases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expression was associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury. Conclusion These results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶4对卵巢癌细胞干性的影响及其机制

目的:探讨6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶4(PFKFB4)对卵巢癌细胞干性的影响及其机制。方法:利用慢病毒系统构建PFKFB4过表达的卵巢癌SKOV3和A2780稳定细胞系。采用Real-time PCR和Western blot检测干性相关转录因子SOX2、OCT4和NANOG的表达,成球实验检测细胞的成球能力,确定PFKFB4对卵巢癌细胞干性的影响;利用自噬激活剂雷帕霉素处理卵巢癌细胞,检测SOX2、OCT4和NANOG的表达及细胞的成球能力,评估自噬对卵巢癌细胞干性的影响;Western blot检测自噬相关蛋白LC3B-Ⅱ和p62的表达,确定PFKFB4对卵巢癌细胞自噬的影响;生物信息学分析卵巢癌中PFKFB4与BNIP3表达的相关性;Real-time PCR和Western blot检测PFKFB4对BNIP3表达的影响。结果:过表达PFKFB4促进SOX2、OCT4和NANOG的表达和卵巢癌细胞的成球能力;雷帕霉素刺激增加SOX2、OCT4和NANOG的表达和卵巢癌细胞的成球能力;过表达PFKFB4增加卵巢细胞自噬水平,包括增加LC3B-Ⅱ蛋白表达、降低p62蛋白表达;生物信息学分析显示,卵巢癌组织中PFKFB4与BNIP3表达高度正相关;过表达PFKFB4增加BNIP3的mRNA和蛋白水平。结论:PFKFB4可能通过激活BNIP3介导的细胞自噬增强卵巢癌细胞的干性。

MiR-28-5p靶向DOK4对口腔鳞状细胞癌细胞增殖的影响

目的探讨微小RNA(miR)-28-5p通过靶向酪氨酸激酶下游蛋白4(DOK4)对口腔鳞状细胞癌细胞增殖的调控作用。方法下载癌症基因组图谱(TCGA)口腔癌数据库,分析miR-28-5p、DOK4与口腔鳞状细胞癌临床表型的关系;转染miR-28-5p模拟物(mimics)、miR-28-5p抑制剂(inhibitor)及对照物(NC)、pcDNA3.1-DOK4及空载体(Vector)、DOK4干扰序列(siDOK4)。CCK-8法和克隆集落形成实验检测口腔鳞状细胞癌的细胞增殖能力;检测各组细胞的抗氧化能力和细胞活性氧(ROS)含量;双荧光素酶报告基因实验验证miR-28-5p与DOK4的靶向关系;观察DOK4过表达对细胞增殖和口腔鳞状细胞癌裸鼠移植瘤生长的影响。结果生物信息学分析显示,相较于癌旁口腔组织,miR-28-5p在口腔鳞状细胞癌组织中表达上调,DOK4 mRNA下调(P<0.05);临床分期Ⅳ期、M 1期、G 3~G 4分级患者miR-28-5p水平高于临床分期Ⅰ~Ⅲ期、M 0期、G 1~G 2分级者(P<0.05);临床分期Ⅳ期、N 1期、G_(3)~G_(4)分级患者DOK4 mRNA水平低于临床分期Ⅰ~Ⅲ期、N 0期、G_(1)~G_(2)分级者(P<0.05);DOK4高表达组无进展生存期和总生存期均高于DOK4低表达组(P<0.05)。与miR-NC组比较,miR-28-5p inhibitor组miR-28-5p水平、细胞活性和集落形成数降低(P<0.05)。与miR-NC组比较,miR-28-5p mimics组DOK4 mRNA和蛋白表达水平降低(P<0.05);与Vector组比较,DOK4过表达组细胞和移植瘤组织中DOK4 mRNA和蛋白表达升高,细胞活性、集落形成数、肿瘤体积及重量降低(P<0.05);与miR-28-5p inhibitor组比较,miR-28-5p inhibitor+siDOK4组的DOK4 mRNA和蛋白表达水平降低,细胞增殖活性、集落形成数、还原型烟酰胺腺嘌呤二核苷酸/烟酰胺腺嘌呤二核苷酸(NADPH/NADP+)、谷胱甘肽/氧化性谷胱甘肽(GSH/GSSG)升高,ROS含量降低(P<0.05)。结论miR-28-5p在口腔鳞状细胞癌中表达上调,通过靶向抑制DOK4表达,降低ROS水平,促进细胞增殖。

SPOC domain-containing protein 1 regulates the proliferation and apoptosis of human spermatogonial stem cells through adenylate kinase 4

BACKGROUND Spermatogonial stem cells(SSCs)are the origin of male spermatogenesis,which can reconstruct germ cell lineage in mice.However,the application of SSCs for male fertility restoration is hindered due to the unclear mechanisms of proliferation and self-renewal in humans.AIM To investigate the role and mechanism of SPOC domain-containing protein 1(SPOCD1)in human SSC proliferation.METHODS We analyzed publicly available human testis single-cell RNA sequencing(RNAseq)data and found that SPOCD1 is predominantly expressed in SSCs in the early developmental stages.Small interfering RNA was applied to suppress SPOCD1 expression to detect the impacts of SPOCD1 inhibition on SSC proliferation and apoptosis.Subsequently,we explored the target genes of SPOCD1 using RNA-seq and confirmed their role by restoring the expression of the target genes.In addition,we examined SPOCD1 expression in some non-obstructive azoospermia(NOA)patients to explore the correlation between SPOCD1 and NOA.RESULTS The uniform manifold approximation and projection clustering and pseudotime analysis showed that SPOCD1 was highly expressed in the early stages of SSC,and immunohistological results showed that SPOCD1 was mainly localized in glial cell line-derived neurotrophic factor family receptor alpha-1 positive SSCs.SPOCD1 knockdown significantly inhibited cell proliferation and promoted apoptosis.RNA-seq results showed that SPOCD1 knockdown significantly downregulated genes such as adenylate kinase 4(AK4).Overexpression of AK4 in SPOCD1 knockdown cells partially reversed the phenotypic changes,indicating that AK4 is a functional target gene of SPOCD1.In addition,we found a significant downregulation of SPOCD1 expression in some NOA patients,suggesting that the downregulation of SPOCD1 may be relevant for NOA.CONCLUSION Our study broadens the understanding of human SSC fate determination and may offer new theories on the etiology of male infertility.