Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility

Aim： To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 （USP26） gene and its involvement in idiopathic male infertility in China. Methods： Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. Results： Of 41 infertile men, 9 （22.0%, P = 0.01） had changes in USP26 gene on the X chromosome. A compound mutation （364insACA; 460G→A） was detected in 8 patients （19.5%, P = 0.01） and a 1044T→A substitution was found in 1 patient （2.4%, P 〉 0.05）. All three variations led to changes in the coding amino acids. Two substitutions predict some changes： 460G→ A changes a valine into an isoleucine, and 1044T → A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. Conclusion： The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.

Ubiquitin-specific protease 22 enhances intestinal cell proliferation and tissue regeneration after intestinal ischemia reperfusion injury

BACKGROUND Intestinal ischemia reperfusion(I/R) injury is a serious but common pathophysiological process of many diseases, resulting in a high mortality rate in clinical practice. Ubiquitin-specific protease 22(USP22) acts as regulator of cell cycle progression, proliferation, and tumor invasion. Depleted USP22 expression has been reported to contribute to arrested cell cycle and disrupted generation of differentiated cell types in crypts and villi. However, the role of USP22 in intestinal damage recovery has not been investigated. Therefore, elucidation of the underlying mechanism of USP22 in intestinal I/R injury may help to improve the tissue repair and patient prognosis in clinical practice.AIM To investigate the role of USP22 in intestinal cell proliferation and regeneration after intestinal I/R injury.METHODS An animal model of intestinal I/R injury was generated in male Sprague-Dawley rats by occlusion of the superior mesenteric artery followed by reperfusion.Chiu's scoring system was used to grade the damage to the intestinal mucosa. An in vitro model was developed by incubating rat intestinal epithelial IEC-6 cells in hypoxia/reoxygenation conditions in order to simulate I/R in vivo. siRNA and overexpression plasmid were used to regulate the expression of USP22. USP22,Cyclin D1, and proliferating cell nuclear antigen(PCNA) expression levels were measured by Western blot analysis and immunohistochemistry staining. Cell survival(viability) and cell cycle were evaluated using the Cell Counting Kit-8and flow cytometry, respectively.RESULTS USP22 expression was positively correlated with the expression levels of PCNA and Cyclin D1 both in vivo and in vitro, which confirmed that USP22 was involved in cell proliferation and intestinal regeneration after intestinal I/R injury. Decreased levels of Cyclin D1 and cell cycle arrest were observed in the USP22 knockdown group(P < 0.05), while opposite results were observed in the USP22 overexpression group(P < 0.05). In addition, increased expression of USP22 was related to improved intestinal pathology or IEC-6 cell viability after I/R or hypoxia/reoxygenation. These results suggested that USP22 may exert a protective effect on intestinal I/R injury by regulating cell proliferation and facilitating tissue regeneration.CONCLUSION USP22 is correlated with promoting intestinal cell proliferation and accelerating intestinal tissue regeneration after intestinal I/R injury and may serve as a potential target for therapeutic development for tissue repair during intestinal I/R injury.

Association of 370-371insACA, 494T〉C, and 1423C〉T haplotype in ubiquitin-specific protease 26 gene and male infertility： a meta-analysis

Whether the 370-371insACA, 494T〉C, and 1423C〉T haplotype in ubiquitin-specific protease 26 （USP26） gene is associated with male infertility is controversial. To clarify this issue, we conducted a meta-analysis based on the most recent studies. Eligible studies were screened by using PubMed and Embase. Pooled odd ratio （OR） with 95% confidence interval （CI） was calculated with fixed effect models. Ten studies with 1603 patients and 2505 controls were included, Overall, the results indicated that there was an association between the haplotype and male infertile risk （OR = 1.74, 95% CI： 1.09-2.77）. The OR calculated based on the five studies in Asia and three in Europe was 1.96 （95% CI： 1,05-3.67） and 1.54 （95% Ch 0.75-3.16） respectively, however, the OR was 0.86 （95% Ch 0.05-15,29） based on the two investigations in America. In addition, the data from the patients with azoospermia （AZO） showed an increased pooled OR of 2.35 （95% Cl： 1.22-4.50）. In contrast, the studies with oligoasthenoteratozoospermia （OAT） exhibited that the pooled OR was 0,97 （95% Ch 0.43-2.16）. Our analyses indicate that there is an association of alteration in USP26 with male infertility, especially in AZO and Asian population.

Small ubiquitin-like modifier protein-specific protease 1 and prostate cancer

Small ubiquitin-like modifier protein （SUMO） modification is a highly dynamic process, catalyzed by SUMO- specific activating （El）, conjugating （E2） and ligating （E3） enzymes, and reversed by a family of SUMO-specific proteases （SENPs）. There are six members of the human SENP family, and each SENP has different cellular locations and substrate specificities. However, the precise roles of SENPs in cellular processes have not been elucidated to date. This brief review will focus on recent advances pertaining to the identified targets of SENP 1 and its potential role in prostate cancer.

Molecular mechanism of modulating the liquefaction epididymal protease inhibitor of human semen

Aim： To study the molecular mechanism of epididymal protease inhibitor （Eppin） modulating the process of prostate specific antigen （PSA） digesting semenogelin （Sg）. Methods： Human Sg cDNA （nucleotides 82-849） and Eppin cDNA （nucleotides 70-423） were generated by polymerase chain reaction （PCR） and cloned into pET-100D/TOPO. Recombinant Eppin and Sg （rEppin and rSg） were produced by BL21 （DE3）. The association of Eppin with Sg was studied by far-western immunoblot and radioautography. In vitro the digestion of rSg by PSA in the presence or absence of rEppin was studied. The effect of anti-Q20E （N-terminal） and C-terminal of Eppin on Eppin-Sg binding was monitored. Results： Eppin binds Sg on the surface of human spermatozoa with the C-terminal of Eppin （amino acids 75-133）. rSg was digested with PSA and many low molecular weight fragments were produced. When rEppin is bound to rSg, then digested by PSA, incomplete digestion and a 15-kDa fragment results. Antibody binding to the N-terminal of rEppin did not affect rSg digestion. Addition of antibodies to the C-terminal of rEppin inhibited the modulating effect of rEppin. Conclusion： Eppin protects a 15-kDa fragment of rSg from hydrolysis by PSA.

Molecular mechanism of epididymal protease inhibitor modulating the liquafication of human semen

To study the molecular mechanism of epididymal protease inhibitor （Eppin） modulating the liquafication of semen. Methods： Human semenogelin cDNA （nucleotides 82-849） and Eppin cDNA （nucleotides 70-423） were generated by PCR and cloned into pET-100D/TOPO.Recombinant Eppin and Sg were produced by BL21 （DE3）. The association of Eppin with Sg was studied by far-western and radioautography.In vitro the digestion of Sg by PSA in the presence or absence of recombinant Eppin was studied. The effect of anti-Q20E （N-terminal） and C-terminal of Eppin on Eppin-Sg binding was monitored. Results： Eppin binds Sg on the surface of human spermatozoa with C-terminal Eppin （aa75-133）.Recombinant Sg was digested with PSA ,many low molecular weight fragments were produced, when Eppin is bound to Sg ,then digested by PSA ,producing incomplete digestion and a 14.5-14.8 kDa fragmen. Antibody binding to the N-terminal of Eppin did not affect Sg digestion. Addition of antibodies to the C-terminal of Eppin inhibited the modulating effects of Eppin. Conclusion： Eppin modulates the digestion activity of PSA through binding Sg.The active site locates at C-terminal.

Development and Characterization of Monoclonal Antibody Specifically Against TSP50

Testis-specific protease 50（TSP50） has been identified as a testis-specific protein that is expressed abnormally in most human breast cancer samples,which makes it an attractive molecular marker and a potential target for diagnosis and therapy.In the present study,we prepared a panel of monoclonal antibodies（mAbs） with high specificity and sensitivity against TSP50 by hybridoma method and characterized them by ELISA,Western blot,immunofluroescence and immunohistochemical analyses.The results show that all of the 9 different clones can specifically bind to TSP50.The mAbs against TSP50 we generated could be good tools for both basic and clinical studies.

Hepatitis C virus resistance to new specifically-targeted antiviral therapy: A public health perspective

Until very recently, treatment for chronic hepatitis C virus(HCV) infection has been based on the combination of two non-viral specific drugs: pegylated interferon-α and ribavirin, which is effective in, overall, about 40%-50% of cases. To improve the response to treatment, novel drugs have been designed to specifically block viral proteins. Multiple compounds are under development, and the approval for clinical use of the first of such direct-acting antivirals in 2011(Telaprevir and Boceprevir), represents a milestone in HCV treatment. HCV therapeutics is entering a new expanding era, and a highly-effective cure is envisioned for the first timesince the discovery of the virus in 1989. However, any antiviral treatment may be limited by the capacity of the virus to overcome the selective pressure of new drugs, generating antiviral resistance. Here, we try to provide a basic overview of new treatments, HCV resistance to new antivirals and some considerations derived from a Public Health perspective, using HCV resistance to protease and polymerase inhibitors as examples.

A protease-activated receptor 1 antagonist protects against global cerebral ischemia/reperfusion injury after asphyxial cardiac arrest in rabbits

Cerebral ischemia/reperfusion injury is partially mediated by thrombin, which causes brain damage through protease-activated receptor 1（PAR1）. However, the role and mechanisms underlying the effects of PAR1 activation require further elucidation. Therefore, the present study investigated the effects of the PAR1 antagonist SCH79797 in a rabbit model of global cerebral ischemia induced by cardiac arrest. SCH79797 was intravenously administered 10 minutes after the model was established. Forty-eight hours later, compared with those administered saline, rabbits receiving SCH79797 showed markedly decreased neuronal damage as assessed by serum neuron specific enolase levels and less neurological dysfunction as determined using cerebral performance category scores. Additionally, in the hippocampus, cell apoptosis, polymorphonuclear cell infiltration, and c-Jun levels were decreased, whereas extracellular signal-regulated kinase phosphorylation levels were increased. All of these changes were inhibited by the intravenous administration of the phosphoinositide 3-kinase/Akt pathway inhibitor LY29004（3 mg/kg） 10 minutes before the SCH79797 intervention. These findings suggest that SCH79797 mitigates brain injury via anti-inflammatory and anti-apoptotic effects, possibly by modulating the extracellular signal-regulated kinase, c-Jun N-terminal kinase/c-Jun and phosphoinositide 3-kinase/Akt pathways.

Ubiquitin-Specific Protease 14 （UBP14） Is Involved in Root Responses to Phosphate Deficiency in Arabidopsis

A mutant isolated from a screen of EMS-mutagenized Arabidopsis lines, per1, showed normal root hair development under control conditions but displayed an inhibited root hair elongation phenotype upon Pi deficiency. Additionally, the per1 mutant exhibited a pleiotropic phenotype under control conditions, resembling Pi-deficient plants in several aspects. Inhibition of root hair elongation upon growth on low Pi media was reverted by treatment with the Pi analog phosphite, suggesting that the mutant phenotype is not caused by a lack of Pi. Reciprocal grafting experiments revealed that the mutant rootstock is sufficient to cause the phenotype. Complementation analyses showed that the PER1 gene encodes an ubiquitin-specific protease, UBP14. The mutation caused a synonymous substitution in the 12th exon of this gene, resulting in a lower abundance of the UBP14 protein, probably as a consequence of reduced translation efficiency. Transcriptional profiling of per1 and wild-type plants subjected to short-term Pi starvation revealed genes that may be important for the signaling of Pi deficiency. We conclude that UBP14 function is crucial for adapting root development to the prevailing local availability of phosphate.

长链非编码RNA核富集转录本1对瘢痕成纤维细胞增殖、凋亡和迁移的影响

背景:已有研究阐明核富集转录本1(nuclear enriched abundant transcript 1,NEAT1)下调抑制了瘢痕成纤维细胞的进展,但具体机制尚不完全清楚。目的:探讨长链非编码RNA NEAT1调节miR-136-5p/泛素特异性蛋白酶4(ubiquitin specific protease 4,USP4)轴对瘢痕成纤维细胞生物学行为的影响。方法:将瘢痕成纤维细胞分为5组:si-NC组、空白对照组、si-NEAT1组、si-NEAT1+miR-136-5p inhibitor组、si-NEAT1+inhibitor-NC组,qRT-PCR检测NEAT1、miR-136-5p表达;CCK-8法及EDU染色检测细胞增殖能力;流式细胞术检测细胞凋亡情况;划痕愈合实验检测细胞迁移情况;Western blot检测USP4、p27、Bax、基质金属蛋白酶9、α-平滑肌肌动蛋白、Ⅰ型胶原蛋白α1链蛋白表达;双荧光素酶实验检测NEAT1与miR-136-5p、miR-136-5p与USP4的关系。结果与结论:①与si-NC组比较,si-NEAT1组NEAT1表达、A450值、EDU阳性细胞百分比、划痕愈合率以及USP4、基质金属蛋白酶9、α-平滑肌肌动蛋白、Ⅰ型胶原蛋白α1链蛋白表达降低(P<0.05),miR-136-5p表达、细胞凋亡率及p27、Bax蛋白表达升高(P<0.05);②miR-136-5p inhibitor逆转了沉默NEAT1对瘢痕成纤维细胞生物学行为的影响;③miR-136-5p与NEAT1、miR-136-5p与USP4存在靶向调控关系。结果表明,沉默NEAT1可能通过调控miR-136-5p/USP4轴抑制瘢痕成纤维细胞的增殖和迁移,诱导其凋亡。

The desumoylating enzyme sentrin-specific protease 3 contributes to myocardial ischemia reperfusion injury

Sentrin-specific protease 3（SENP3）, a member of the desumoylating enzyme family, is known as a redox sensor and could regulate multiple cellular signaling pathways. However, its implication in myocardial ischemia reperfusion（MIR） injury is unclear. Here, we observed that SENP3 was expressed and upregulated in the mouse heart depending on reactive oxygen species（ROS） production in response to MIR injury. By utilizing si RNA-mediated cardiac specific gene silencing, SENP3 knockdown was demonstrated to significantly reduce MIR-induced infarct size and improve cardiac function. Mechanistic studies indicated that SENP3 silencing ameliorated myocardial apoptosis mainly via suppression of endoplasmic reticulum（ER） stress and mitochondrial-mediated apoptosis pathways. By contrast, adenovirusmediated cardiac SENP3 overexpression significantly exaggerated MIR injury. Further molecular analysis revealed that SENP3 promoted mitochondrial translocation of dynamin-related protein 1（Drp1） in reperfused myocardium. In addition, mitochondrial division inhibitor-1（Mdivi-1）, a pharmacological inhibitor of Drp1, significantly attenuated the exaggerated mitochondrial abnormality and cardiac injury by SENP3 overexpression after MIR injury. Taken together, we provide the first direct evidence that SENP3 upregulation pivotally contributes to MIR injury in a Drp1-dependent manner, and suggest that SENP3 suppression may hold therapeutic promise for constraining MIR injury.

脓毒症合并急性肾损伤患者外周血USF2、USP10表达水平及临床意义

目的 探讨脓毒症合并急性肾损伤(AKI)患者外周血上游转录因子2(USF2)、泛素特异性蛋白酶10(USP10)的表达水平及临床意义。方法 选择2018年1月至2022年12月该院收治的259例脓毒症患者,根据是否合并AKI将患者分为AKI组(107例)和非AKI(NAKI)组(152例)。收集临床一般资料,检测外周血中USF2、USP10的表达水平。Pearson分析USF2、USP10与肾功能的相关性。二元Logistic回归分析影响脓毒症患者合并AKI的因素。绘制受试者工作特征(ROC)曲线分析USF2、USP10诊断脓毒症患者合并AKI的价值。结果 AKI组血清USF2表达水平高于NAKI组,差异有统计学意义(P<0.05),USP10表达水平低于NAKI组,差异有统计学意义(P<0.05)。AKI组USF2表达与尿素氮(BUN)、血清肌酐(Scr)、胱抑素C(CysC)呈正相关(P<0.05),USP10表达与BUN、Scr、CysC呈负相关(P<0.05)。高序贯器官衰竭(SOFA)评分、脓毒症休克、高表达USF2是脓毒症患者发生AKI的危险因素(P<0.05),高表达USP10是保护因素(P<0.05)。USF2、USP10诊断脓毒症患者发生AKI的曲线下面积(AUC)分别为0.742(95%CI:0.676~0.808)、0.781(95%CI:0.724~0.839),联合USF2和USP10诊断脓毒症患者发生AKI的AUC为0.907(95%CI:0.865~0.948),高于单独诊断(P<0.05)。结论 脓毒症患者外周血中USF2表达增加,USP10表达下降与合并AKI风险增加以及肾功能下降有关。

MicroRNA-137-5p靶向USP30改善阿尔茨海默病

目的 探索miR-137-5p对阿尔茨海默病(AD)的保护机制。方法 首先用qRT-PCR评估AD患者和健康对照组人血清中miR-137和USP30的表达。用D-半乳糖和氯化铝建立AD小鼠模型,用水迷宫试验检测小鼠的行为,确认AD小鼠模型的成功。用Aβ1-42寡聚体诱导的SH-SY5Y细胞建立AD细胞模型,通过实时定量聚合酶链反应检测AD模型中miR-137-5p和USP30的表达。双重荧光素酶试验用于验证miR-137-5p和USP30之间的靶向结合关系。结果 miR-137-5p的表达在AD患者中与健康对照组相比有所下降(P<0.05),而USP30则明显增加(P<0.05)。miR-137-5p能改善AD细胞模型中的细胞凋亡,USP30的过表达部分废除了miR-137-5p对Aβ1-42-处理的SH-SY5Y细胞的影响,miR-137-5p通过靶向USP30改善AD小鼠的认知能力和Aβ的沉积。结论 miR-137-5p可以通过下调USP30来改善AD症状,miR-137-5p可能能成为治疗AD的一个靶点。

黑色素瘤SENP1蛋白质参与达卡巴嗪耐药性的探究

目的探究与黑色素瘤耐药性相关的基因及信号通路,揭示其与黑色素瘤耐药性的相关性。方法以A375及M14黑色素瘤细胞为研究对象,通过逐渐提高达卡巴嗪(dacarbazine,DTIC)的浓度获得耐药性黑色素瘤细胞株,采用转录物组学研究耐药性黑色素瘤细胞系中显著性变化的基因及通路,利用实时荧光定量聚合酶链反应(real time quantitative polymerase chain reaction,RT-qPCR)、蛋白质杂交(Western blotting,WB)等对变化的基因进行验证。结果(1)成功构建了黑色素瘤耐药细胞株:通过DTIC小剂量逐步增加的方法成功建立了耐药型的黑色素瘤细胞系A375与M14,通过计算其对DTIC的半抑制浓度值(the half maximal inhibitory concentration,IC50),确定了细胞对DTIC的敏感性发生了显著的变化;通过流式细胞技术检测,发现耐药的黑色素瘤细胞具有显著抗DTIC引发的凋亡的能力。(2)发现了黑色素瘤耐药性相关的基因及信号通路:利用建立的耐DTIC的黑色素瘤细胞系,进行了全基因组转录测序和分析,发现了类泛素特异性蛋白酶1(SUMO-specific protease 1,SENP1)的高表达和蛋白激酶Hippo信号通路的异常活化相关。(3)SENP1异常表达可能参与DTIC耐药:WB检测野生型和耐药型黑色素瘤细胞系发现在耐药的细胞中,SENP1与YAP表达都上调。(4)通过基因敲除证实SENP1参与DTIC耐药,蛋白质相互作用实验初步证实SENP1对YAP存在去泛素化调控作用。结论SENP1与DTIC耐药性存在正相关关系,其异常上调可能导致Hippo信号通路发生变化使得黑色素瘤对DTIC耐受性的提升。

UBR5对肝细胞癌临床病理特征和预后的影响及靶向治疗的潜在价值分析

目的:探讨肝细胞癌组织中泛素蛋白连接酶E3成分N-识别蛋白5(UBR5)的表达对患者临床病理特征和预后的影响及临床价值。方法:收集肝细胞癌患者的肿瘤组织和癌旁组织,免疫组织化学检测组织中UBR5的表达并将患者分为UBR5阴性组和阳性组。比较UBR5阳性表达对患者临床病理特征及对术后3年生存率的影响。Cox回归分析影响患者预后的危险因素。利用转染技术敲低HepG2细胞中UBR5的表达,分析敲低UBR5后对细胞周期和凋亡率的影响。结果:UBR5阴性表达组62例,阳性组105例,UBR5阳性表达对肿瘤分化程度、肿瘤长径和肿瘤分期有影响(均P<0.05)。UBR5阳性表达组术后3年存活率为70.5%,低于UBR5阴性表达组的85.5%(χ^(2)=4.441,P=0.035)。Cox多因素回归分析显示,肿瘤低分化程度、肿瘤长径>5 cm、肿瘤分期Ⅲ~Ⅳ期、UBR5阳性表达为影响患者预后的危险因素(均P<0.05)。敲低HepG2细胞中UBR5的表达后G1期细胞增加、G2期细胞减少,细胞凋亡率显著增加(均P<0.05)。结论:UBR5阳性表达影响患者的预后,敲低肝细胞癌细胞中UBR5的表达可能是有效的靶向治疗方法。

子宫动脉血流多普勒超声联合血清PIGF、Vaspin、ESM-1诊断HDP价值

目的:探讨妊娠高血压疾病(HDP)患者子宫动脉血流多普勒超声、血清促血管生成因子(PIGF)、丝氨酸蛋白酶抑制剂(Vaspin)、内皮细胞特异分子-1(ESM-1)水平及其诊断价值。方法:回顾性收集2020年6月-2021年12月本院接诊的HDPL患者86例为病例组,产前检查健康孕妇75例为对照组,检测两组血清PIGF、Vaspin、ESM-1水平及子宫动脉血流参数,分析在诊断HDP价值。结果:病例组血清PIGF(271.56±45.56 pg/ml)低于对照组(330.15±50.35 pg/ml),Vaspin(0.46±0.04 ng/ml)、ESM-1(1.38±0.51μg/L)高于对照组(0.39±0.08 ng/ml、1.01±0.07μg/L),多普勒超声子宫动脉血流阻力指数(RI)(0.79±0.14)、搏动指数(PI)(0.83±0.21)、收缩期峰值流速与舒张末期血流速度比值(S/D)(2.26±0.74)均高于对照组(0.51±0.20、0.44±0.19、1.41±0.35),且病例组妊娠期高血压、轻度子痫前期、重度子痫前期患者上述各指标均有差异(均P<0.05)。受试者工作特征曲线分析,血清PIGF、Vaspin、ESM-1、RI、PI、S/D及各项联合,诊断HDP的曲线下面积分别为0.811、0.780、0.691、0.944、0.860、0.813、0.999,截断值分别为291.56 pg/ml、0.43 ng/ml、1.12μg/L、0.58、0.53、1.87,联合检测的特异度(96.5%)、准确度(93.4%)最高(均P<0.05)。结论:HDP患者血清PIGF、Vaspin、ESM-1、子宫动脉血流参数异常改变,且对HDP有较好诊断价值,本次研究也为靶向药物治疗HDP提供了新思路。

USP7-MDM2-p53信号轴对子宫内膜癌细胞增殖、凋亡和细胞周期的影响

目的:探讨泛素特异性蛋白酶7(USP7)调节Mdm2 p53结合蛋白同源物(MDM2)-p53轴对子宫内膜癌细胞增殖、凋亡和细胞周期的影响。方法:Western blot检测人子宫内膜癌组织、癌旁组织、人子宫内膜上皮细胞hEEC及人子宫内膜癌细胞系Ishikawa、HEC-1-A、KLE中USP7蛋白表达。将Ishikawa细胞分为NC组、P22077(USP7抑制剂)组、pcDNA组、pcDNA-MDM2组、P22077+pcDNA组、P22077+pcDNA-MDM2组,CCK-8法和克隆形成实验检测Ishikawa细胞增殖;流式细胞术检测Ishikawa细胞凋亡与细胞周期变化;Western blot检测Ishikawa细胞中USP7、细胞周期蛋白D1(CyclinD1)、周期素依赖性激酶2(CDK2)、Bcl-2相关X蛋白(Bax)、MDM2、p53蛋白表达。以RG7388(MDM2抑制剂)或PFT-α(p53抑制剂)与20μmol/L P22077共处理Ishikawa细胞48 h以验证USP7-MDM2-p53信号轴上下游关系。结果:USP7蛋白在子宫内膜癌组织和细胞中高表达,且Ishikawa细胞中USP7蛋白表达量最高,因此,选择Ishikawa细胞为研究对象。与NC组比较,P22077组Ishikawa细胞OD 450值、克隆形成率、S期和G 2/M期细胞数、USP7、CyclinD1、CDK2、MDM2蛋白表达降低,细胞凋亡率、G_(0)/G_(1)期细胞数、p53、Bax蛋白表达升高(P<0.05);与NC组、pcDNA组比较,pcDNA-MDM2组Ishikawa细胞OD 450值、克隆形成率、S期和G 2/M期细胞数、USP7、CyclinD1、CDK2、MDM2蛋白表达升高,细胞凋亡率、G_(0)/G_(1)期细胞数、p53、Bax蛋白表达降低(P<0.05);与P22077组、P22077+pcDNA组比较,P22077+pcDNA-MDM2组Ishikawa细胞OD 450值、克隆形成率、S期和G 2/M期细胞数、USP7、CyclinD1、CDK2、MDM2蛋白表达升高,细胞凋亡率、G_(0)/G_(1)期细胞数、p53、Bax蛋白表达降低(P<0.05)。p53为USP7-MDM2通路下游分子。结论:抑制USP7表达可能通过下调MDM2来激活p53进而抑制Ishikawa细胞增殖、促进细胞凋亡及周期停滞。

血管内皮生长因子A与小泛素相关修饰物特异性蛋白酶5在前列腺癌中的表达及临床意义

目的:探讨血管内皮生长因子A(VEGFA)与小泛素相关修饰物特异性蛋白酶5(SENP5)在前列腺癌中的表达情况及临床意义。方法:使用GEPIA数据库,对VEGFA、SENP5基因的相关性及与前列腺癌病人的生存期进行分析;采用qRT-PCR、Western blotting从转录、蛋白质翻译水平检测肿瘤细胞中VEGFA、SENP5的表达情况;收集前列腺癌病人组织标本和临床资料,采用免疫组织化学法检测前列腺癌组织中VEGFA、SENP5的蛋白表达,分析其表达情况与肿瘤病人临床病理参数间的关系;使用STRING网站进行VEGFA、SENP5蛋白质互作网络图构建。结果:VEGFA与前列腺癌病人总生存时间有关(P<0.05),SENP5与病人总生存时间、无病进展生存时间均相关(P<0.05);与正常细胞相比,VEGFA与SENP5在前列腺癌细胞中mRNA、蛋白质均高表达(P<0.05);VEGFA与SENP5蛋白在肿瘤病理组织中可见淡黄色或棕黄色染色,VEGFA主要于细胞质中表达,SENP5主要于细胞核中表达;VEGFA强阳性表达与临床分期、有无远处转移相关(P<0.05),SENP5强阳性表达与Gleason评分、有无远处转移相关(P<0.05);VEGFA与SENP5间显著相关(P<0.01);SENP5可能通过SUMO1-HSP90AA1轴与VEGFA相互作用。结论:VEGFA、SENP5在前列腺癌中高表达,两者显著相关,且与肿瘤临床病理参数有关,可能是前列腺癌病人治疗的潜在靶点。