Ubiquitin-specific protease 15 contributes to gastric cancer progression by regulating the Wnt/β-catenin signaling pathway

BACKGROUND Ubiquitin-specific protease 15(USP15)is an important member of the ubiquitinspecific protease family,the largest deubiquitinase subfamily,whose expression is dysregulated in many types of cancer.However,the biological function and the underlying mechanisms of USP15 in gastric cancer(GC)progression have not been elucidated.AIM To explore the biological role and underlying mechanisms of USP15 in GC progression.METHODS Bioinformatics databases and western blot analysis were utilized to determine the expression of USP15 in GC.Immunohistochemistry was performed to evaluate the correlation between USP15 expression and clinicopathological characteristics of patients with GC.A loss-and gain-of-function experiment was used to investigate the biological effects of USP15 on GC carcinogenesis.RNA sequencing,immunofluorescence,and western blotting were performed to explore the potential mechanism by which USP15 exerts its oncogenic functions.RESULTS USP15 was up-regulated in GC tissue and cell lines.The expression level of USP15 was positively correlated with clinical characteristics(tumor size,depth of invasion,lymph node involvement,tumor-node-metastasis stage,perineural invasion,and vascular invasion),and was related to poor prognosis.USP15 knockdown significantly inhibited cell proliferation,invasion and epithelialmesenchymal transition(EMT)of GC in vitro,while overexpression of USP15 promoted these processes.Knockdown of USP15 inhibited tumor growth in vivo.Mechanistically,RNA sequencing analysis showed that USP15 regulated the Wnt signaling pathway in GC.Western blotting confirmed that USP15 silencing led to significant down-regulation ofβ-catenin and Wnt/β-catenin downstream genes(c-myc and cyclin D1),while overexpression of USP15 yielded an opposite result and USP15 mutation had no change.Immunofluorescence indicated that USP15 promoted nuclear translocation ofβ-catenin,suggesting activation of the Wnt/β-catenin signaling pathway,which may be the critical mechanism promoting GC progression.Finally,rescue experiments showed that the effect of USP15 on gastric cancer progression was dependent on Wnt/β-catenin pathway.CONCLUSION USP15 promotes cell proliferation,invasion and EMT progression of GC via regulating the Wnt/β-catenin pathway,which suggests that USP15 is a novel potential therapeutic target for GC.

Ubiquitin-specific protease 21 promotes tumorigenicity and stemness of colorectal cancer by deubiquitinating and stabilizing ZEB1

BACKGROUND Colorectal cancer(CRC)is one very usual tumor together with higher death rate.Ubiquitin-specific protease 21(USP21)has been confirmed to take part into the regulation of CRC progression through serving as a facilitator.Interestingly,the promotive function of USP21 has also discovered in the progression of CRC.ZEB1 has illustrated to be modulated by USP7,USP22 and USP51 in cancers.However,the regulatory functions of USP21 on ZEB1 in CRC progression need more invest-igations.AIM To investigate the relationship between USP21 and ZEB1 in CRC progression.METHODS The mRNA and protein expressions were assessed through RT-qPCR,western blot and IHC assay.The interaction between USP21 and ZEB1 was evaluated through Co-IP and GST pull down assays.The cell proliferation was detected through colony formation assay.The cell migration and invasion abilities were determined through Transwell assay.The stemness was tested through sphere formation assay.The tumor growth was evaluated through in vivo mice assay.RESULTS In this work,USP21 and ZEB1 exhibited higher expression in CRC,and resulted into poor prognosis.Moreover,the interaction between USP21 and ZEB1 was further investigated.It was demonstrated that USP21 contributed to the stability of ZEB1 through modulating ubiquitination level.In addition,USP21 streng-thened cell proliferation,migration and stemness through regulating ZEB1.At last,through in vivo assays,it was illustrated that USP21/ZEB1 axis aggravated tumor growth.CONCLUSION For the first time,these above findings manifested that USP21 promoted tumorigenicity and stemness of CRC by deubiquitinating and stabilizing ZEB1.This discovery suggested that USP21/ZEB1 axis may provide novel sights for the treatment of CRC.

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.

Emerging potential of ubiquitin-specific proteases and ubiquitinspecific proteases inhibitors in breast cancer treatment

Breast cancer is the most frequently diagnosed cancer in women,accounting for 30%of new diagnosing female cancers.Emerging evidence suggests that ubiquitin and ubiquitination played a role in a number of breast cancer etiology and progression processes.As the primary deubiquitinases in the family,ubiquitin-specific peptidases(USPs)are thought to represent potential therapeutic targets.The role of ubiquitin and ubiquitination in breast cancer,as well as the classification and involvement of USPs are discussed in this review,such as USP1,USP4,USP7,USP9X,USP14,USP18,USP20,USP22,USP25,USP37,and USP39.The reported USPs inhibitors investigated in breast cancer were also summarized,along with the signaling pathways involved in the investigation and its study phase.Despite no USP inhibitor has yet been approved for clinical use,the biological efficacy indicated their potential in breast cancer treatment.With the improvements in phenotypic discovery,we will know more about USPs and USPs inhibitors,developing more potent and selective clinical candidates for breast cancer.

Inhibition of Ubiquitin-specific Protease 4 Attenuates Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells via Transforming Growth Factor Beta Receptor Type Ⅰ

Objective Ubiquitin-specific protease 4(USP4)facilitates the development of transforming growth factor-beta 1(TGF-β1)-induced epithelial-mesenchymal transition(EMT)in various cancer cells.Moreover,EMT of renal tubular epithelial cells(RTECs)is required for the progression of renal interstitial fibrosis.However,the role of USP4 in EMT of RTECs remains unknown.The present study aimed to explore the effect of USP4 on the EMT of RTECs as well as the involved mechanism.Methods In established unilateral ureteral obstruction(UUO)rats and NRK-52E cells,immunohistochemistry and Western blot assays were performed.Results USP4 expression was increased significantly with obstruction time.In NRK-52E cells stimulated by TGF-β1,USP4 expression was increased in a time-dependent manner.In addition,USP4 silencing with specific siRNA indicated that USP4 protein was suppressed effectively.Meanwhile,USP4 siRNA treatment restored E-cadherin and weakened alpha smooth muscle actin(α-SMA)expression,indicating that USP4 may promote EMT.After treatment with USP4 siRNA and TGF-β1 for 24 h,the expression of TGF-β1 receptor type I(TβRI)was decreased.Conclusion USP4 promotes the EMT of RTECs through upregulating TβRI,thereby facilitating renal interstitial fibrosis.These findings may provide a potential target of USP4 in the treatment of renal fibrosis.

Ubiquitin-specific protease 24 promotes EV71 infection by restricting K63-linked polyubiquitination of TBK1

TANK-binding kinase 1(TBK1)is an essential protein kinase for activation of interferon regulatory factor 3(IRF3)and induction of the type I interferons(IFN-I).Although the biochemical regulation of TBK1 activation has been studied,little is known about how enterovirus 71(EV71)employs the deubiquitinases(DUBs)to regulate TBK1 activation for viral immune evasion.Here,we found that EV71 infection upregulated the expression of ubiquitinspecific protease 24(USP24).Further studies revealed that USP24 physically interacted with TBK1,and can reduce K63-linked polyubiquitination of TBK1.Knockdown of USP24 upregulated TBK1 K63-linked polyubiquitination,promoted the phosphorylation and nuclear translocation of IRF3,and in turn improved IFN-I production during EV71 infection.As a consequence,USP24 knockdown dramatically inhibited EV71 infection.This study revealed USP24 as a novel regulator of TBK1 activation,which promotes the understanding of immune evasion mechanisms of EV71 and could provide a potential strategy for treatment of EV71 infection.

乳腺癌组织中泛素特异性蛋白酶18/干扰素刺激基因15表达水平及其临床意义

目的探究泛素特异性蛋白酶18(USP18)、干扰素刺激基因15(ISG15)在乳腺癌组织中的表达水平及临床意义。方法采用Ualcan数据库分析USP18、ISG15在正常乳腺组织和乳腺癌组织中的表达情况及二者与乳腺癌预后的关系;选取渭南市中心医院2012年5月至2015年7月诊治的99例乳腺癌病人癌组织、癌旁正常组织进行研究。分别检测USP18、ISG15mRNA及其蛋白表达情况;分析乳腺癌组织USP18、ISG15表达水平与病人临床病理特征、预后的关系;Cox回归分析乳腺癌病人预后的影响因素。结果Ualcan数据库中乳腺癌组织USP18为18.40±4.17、ISG15 mRNA表达水平为168.56±43.95高于正常乳腺组织(10.00±3.14、30.76±8.23)(P<0.05);乳腺癌组织USP18、ISG15 mRNA表达水平高低与乳腺癌预后无明显相关性。乳腺癌组织USP18为1.67±0.53、ISG15 mRNA为1.86±0.61及蛋白阳性表达率均高于癌旁正常组织(1.03±0.34、0.99±0.33)(P<0.05);乳腺癌组织USP18、ISG15表达水平均与淋巴结转移、乳腺癌分子亚型、肿瘤分化程度、TNM分期相关(P<0.05);乳腺癌病人癌组织中USP18表达水平与ISG15呈正相关(P<0.05);USP18阳性组、ISG15阳性组乳腺癌病人术后60个月生存率均低于USP18阴性组、ISG15阴性组(P<0.05);淋巴结转移、TNM分期、USP18、ISG15均是影响乳腺癌病人不良预后的独立危险因素(P<0.05)。结论乳腺癌病人癌组织USP18、ISG15表达水平均较高,两者均与乳腺癌病人预后关系密切,检测乳腺癌组织USP18、ISG15水平有助于评估乳腺癌病人预后。

冠心病患者血浆血管性血友病因子裂解酶、生长分化因子15水平变化及相关性分析

目的观察冠心病患者血浆血管性血友病因子裂解酶(vWF-CP)及生长分化因子(GDF15)水平的变化,探讨两者的相关性。方法选取冠心病患者96例,其中稳定性心绞痛组(SAP组)47例,急性冠脉综合征组(ACS组)49例,选取同期无血管病变的非冠心病患者49例作为对照组,入组患者均在入院后采血。用ELISA法测定血浆vWF-CP、GDF15。结果 ACS组vWF-CP为(286.22±21.75)pg/mL、SAP组为(357.47±21.98)pg/mL、对照组为(581.85±21.52)pg/mL,组间相比,P均<0.05;ACS组GDF15为(11.30±0.51)ng/mL、SAP组为(8.37±0.54)ng/mL、对照组为(1.35±0.45)ng/mL,组间相比,P均<0.01。vWF-CP水平与GDF15水平呈负相关(r=-0.558,P<0.01)。结论冠心病患者病情不稳定时血浆vWF-CP水平降低、GDF15水平升高,两者存在显著的相关性,都与冠心病的病情有关,两者的联合检测可以更好预测冠心病患者血栓事件的发生。

USP15生物学功能的研究进展

去泛素化酶(DUBs)是体内分解蛋白泛素链的一类蛋白酶体系,对蛋白泛素化降解过程起校正作用。泛素特异性蛋白酶(USP)家族是DUBs中最大的家族,通过多种途径参与免疫应答,且与一些疾病和肿瘤的发生具有明显相关性。USP15是USP家族的重要组成部分,参与调节转化生长因子β、p53、核因子κB等重要的信号转导通路,与一些肿瘤的发生发展相关。USP15也可通过调节I型干扰素产生和T细胞活化参与免疫反应,并具有多种重要生物学功能,包括维持基因稳定性、调节转录因子及抗病毒等重要的细胞活动。

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.

Ubiquitin-specific protease 47 regulates intestinal inflammation through deubiquitination of TRAF6 in epithelial cells

Deubiquitinates(DUBs) alter the stabilities, localizations or activities of substrates by removing their ubiquitin conjugates,which are closely related to the development of inflammatory response. Here, we show that ubiquitin-specific protease 47(USP47) prevents inflammation development in inflammatory bowel disease(IBD). Compared with wild-type mice, Usp47 knockout mice are more susceptible to dextran sodium sulfate(DSS)-induced acute and chronic colitis with higher inflammatory cytokines expression and severe intestinal tissue damage. Chimeric mouse experiments suggest that non-hematopoietic cells mainly contribute to the phenotype. And, DSS-induced colitis of the Usp47 knockout mice depends on commensal bacteria.Mechanistically, down-regulation of USP47 aggravates the activation of NF-κB signaling pathway by increasing the K63-linked poly-ubiquitination of tumor necrosis factor receptor-associated factor 6(TRAF6) in intestinal epithelial cells. Furthermore, the expression of USP47, negatively correlated with the degree of inflammation, is lower at colonic inflammatory lesions than that non-inflammatory sites from the intestine from ulcerative colitis(UC) and Crohn's disease(CD) patients. These data, taken together, indicate that USP47 regulates intestinal inflammation through de-ubiquitination of K63-linked poly-ubiquitination TRAF6 in intestinal epithelial cells.

泛素特异性蛋白酶15稳定着色性干皮病F蛋白并促进DNA链间交联损伤修复

着色性干皮病F蛋白(xeroderma pigmentosum group F,XPF)和切除修复交叉互补组1蛋白(excision repair cross complementing group 1,ERCC1)组成一种结构特异性的核酸内切酶(XPFERCC1)复合物,参与DNA链间交联(interstrand crosslink,ICL)损伤修复。其中,XPF蛋白的去泛素化修饰对DNA损伤修复的影响尚未见报道。本工作主要研究泛素特异性蛋白酶15 (ubiquitinspecific protease 15,USP15)对XPF的稳定性及ICL修复的影响。本研究通过蛋白质质谱和Western印迹法分析发现,XPF蛋白与USP15存在相互作用,进而使XPF蛋白去泛素化修饰;采用CRISPR-Cas9技术构建USP15基因敲除的He La细胞株(USP15 KO)并进行Western印迹分析,结果显示,敲除组XPF蛋白水平低于对照组(P<0. 001)。克隆形成试验显示,在ICL诱导剂顺铂(cisplatin,DDP)和丝裂霉素C (mitomycin,MMC)的作用下,USP15基因敲除的HeLa细胞增殖能力显著降低(P<0. 01)。本研究表明,去泛素化酶USP15是一种重要的DNA修复调节因子,该酶通过稳定XPF蛋白促进由XPF-ERCC1介导的ICL修复。本研究为改善ICL诱导剂类抗癌药物的耐药性提供了理论依据,并为肿瘤的治疗提供了潜在的新靶点。

肥胖对小鼠肝脏GDF-15、TMPRSS6、Hepcidin表达的影响

目的观察肥胖小鼠肝组织中铁代谢相关调控因子铁调素(hepcidin)mRNA,生长分化因子-15(GDF-15)和跨膜丝氨酸蛋白酶6(TMPRSS6)蛋白表达的变化,及血液和肝组织57Fe的相对含量及其意义。方法 4-6周雄性C57BL/6J小鼠20只被随机分为肥胖模型组和正常对照组,每组10只。肥胖模型组和对照组分别给予高脂饲料和普通饲料。饲喂至15周,肥胖模型建模成功。建模成功后,以0.1 ml/10 g的计量标准,对全部小鼠经口一次性灌注^(57)FeSO_4溶液。1 h后,取小鼠心脏血液及肝脏。免疫组化法检测肝脏中GDF-15、TMPRSS6的相对表达量,real-time PCR法检测肝脏hepcidin mRNA,ICP-MS法测定血液及肝脏中^(57)Fe的相对含量。结果与对照组相比,肥胖组小鼠肝脏中hepcidin表达增加(0.282±0.109 vs 0.494±0.139,P<0.01),GDF-15表达减少(0.097±0.005 vs 0.035±0.006,P<0.01),血液^(57)Fe表达减少[(22.970±4.492)μg/ml vs(15.647±1.629)μg/ml,P<0.05)],肝脏^(57)Fe表达减少[(603.103±35.511)μg/g vs(226.073±12.124)μg/mg,P<0.05],而TMPRSS6的表达尚不能认为差异有统计学意义(0.068±0.009 vs 0.085 3±0.027,P>0.05)。结论机体TMPRSS6表达量的变化不受肥胖因素影响。但肥胖可以下调机体GDF-15的表达,并由此升高肝脏hepcidin的表达,最终导致肝脏及血液铁含量减少。

The association between mutations in ubiquitin-specific protease 26(USP26)and male infertility:a systematic review and meta-analysis

During recent decades,the association between mutations in ubiquitin-specific protease 26(USP26)and male infertility remains doubtful.We conducted this meta-analysis to evaluate the association between mutations in USP26 and male infertility according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses(PRISMA)2020 guidelines.It was registered in the International Prospective Register of Systematic Reviews(PROSPERO;CRD42021225251).PubMed,Web of Science,and Scopus were systematically searched for comparative clinical studies,which were written in English and provided eligible data.Studies were included when they compared USP26 mutations in azoospermic,oligozoospermic,and asthenozoospermic patients with controls with normal sperm parameter values or whose partners had experienced spontaneous pregnancy.Pooled odds ratio(OR)with 95%confidence interval(CI)was calculated with random effect models.Overall,twelve studies with 3927 infertility patients and 4648 healthy controls were included.The association between overall USP26 mutations and infertility was not significant(OR=1.60,95%CI:0.51-5.01).For specific mutations,the pooled ORs were 1.65(95%CI:1.02-2.69)for cluster mutation(including 370-371insACA,494T>C,and 1423C>T),1.80(95%CI:0.35-9.15)for c.576G>A,1.43(95%CI:0.79-2.56)for c.1090C>T,and 3.59(95%CI:2.30-5.59)for c.1737G>A.Our results suggest that several mutations(cluster mutation,c.1737G>A)may play roles in male infertility,while others(c.576G>A and c.1090C>T)do not show notable associations with male infertility.More high-quality clinical researches are needed for validation.

Deubiquitinase ubiquitin-specific protease 3 (USP3) inhibits HIV-1 replication via promoting APOBEC3G (A3G) expression in both enzyme activity-dependent and -independent manners

Background: Ubiquitination plays an essential role in many biological processes, including viral infection, and can be reversed by deubiquitinating enzymes (DUBs). Although some studies discovered that DUBs inhibit or enhance viral infection by various mechanisms, there is lack of information on the role of DUBs in virus regulation, which needs to be further investigated.Methods: Immunoblotting, real-time polymerase chain reaction,in vivo/in vitro deubiquitination, protein immunoprecipitation, immunofluorescence, and co-localization biological techniques were employed to examine the effect of ubiquitin-specific protease 3 (USP3) on APOBEC3G (A3G) stability and human immunodeficiency virus (HIV) replication. To analyse the relationship between USP3 and HIV disease progression, we recruited 20 HIV-infected patients to detect the levels of USP3 and A3G in peripheral blood and analysed their correlation with CD4^(+) T-cell counts. Correlation was estimated by Pearson correlation coefficients (for parametric data).Results: The results demonstrated that USP3 specifically inhibits HIV-1 replication in an A3G-dependent manner. Further investigation found that USP3 stabilized 90% to 95% of A3G expression by deubiquitinating Vif-mediated polyubiquitination and blocking its degradation in an enzyme-dependent manner. It also enhances the A3G messenger RNA (mRNA) level by binding to A3G mRNA and stabilizing it in an enzyme-independent manner. Moreover, USP3 expression was positively correlated with A3G expression (r= 0.5110) and CD4^(+) T-cell counts (r= 0.5083) in HIV-1-infected patients.Conclusions: USP3 restricts HIV-1 viral infections by increasing the expression of the antiviral factor A3G. Therefore, USP3 may be an important target for drug development and serve as a novel therapeutic strategy against viral infections.

USP15及α-SMA在青光眼滤过术后瘢痕组织中表达的实验研究

目的检测家兔青光眼滤过手术(GFS)术后瘢痕组织中泛素特异性蛋白酶15(USP15)和α-平滑肌动蛋白(α-SMA)的表达,探讨USP15与α-SMA表达的关系。方法健康成年家兔20只,以右眼为实验组(20只),建立GFS的动物模型,左眼作为正常对照组(10只)。随机将实验组分为术后14 d组(10只)、术后28 d组(10只)。测量家兔正常对照组、术后14 d组、术后28 d组眼压,分析其变化。取标本行苏木精-伊红(HE)染色、Masson染色观察组织纤维化进展。对GFS术后瘢痕组织和正常结膜组织标本进行免疫组织化学染色,检测USP15和α-SMA的表达并进行相关性分析。结果术后14 d组眼压低于正常对照组[(11.40±0.69) mm Hg(1 mm Hg=0.133 k Pa)比(14.90±0.74) mm Hg](P <0.05),术后28 d组高于术后14 d组[(15.10±0.74) mm Hg比(11.40±0.69) mm Hg](P <0.05);术后14 d、28 d HE、Masson染色示滤过道纤维组织增生及胶原沉积增加,示造模成功。GFS术后14 d组、28 d组瘢痕组织中USP15和α-SMA的表达明显高于正常对照组[(5.01±1.02)、(11.80±0.76)比(1.12±0.76),(5.70±1.76)、(11.90±0.55)比(1.43±0.63)](P <0.05),USP15和α-SMA的表达随着瘢痕形成的增加而增加。GFS术后14 d、28 d瘢痕组织中USP15和α-SMA的表达呈正相关(r=0.865,P <0.001;r=0.976,P <0.001)。结论家兔GFS术后瘢痕组织中USP15和α-SMA的表达异常升高,且两者呈正相关。

USP26基因中370—371；nsACA,494T〉0和14230〉T串联突变与男性不育症相关性的荟萃分析

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.

The expression of Usp26 gene in mouse testis and brain

Deubiquitinating enzymes （DUBs） play an important role in ubiquitin-dependent processes as negative regulators of protein ubiquitination. Ubiquitin-specific protease 26 （USP26） is a member of this family. The expression of Usp26 in mammalian testis and in other tissues has yet to be fully elucidated. To study the expression of Usp26 mRNA and protein in various murine tissues, reverse transcription （RT）-PCR and immunohistochemistry analyses were carried out. The RT-PCR analysis showed that the Usp26 transcript was expressed in all of the tested tissues. USP26 protein localization was examined by immunohistochemistry, and it was shown that USP26 was not detectable at 20 days postpartum, with the expression restricted to the cytoplasm of condensing spermatids （steps 9-16）, Leydig cells and nerve fibers in the brain. In addition, the USP26 protein was detected at moderate levels in myocardial ceils, the corpus of epidydimis, epithelium of the renal tubules and the seminal gland of postnatal day 35 mice. Its spatial and temporal expression pattern suggests that Usp26 may play an important role in development or function of the testis and brain. Further research into these possibilities is in progress.

Inhibition of Ubiquitin-specific Peptidase 8 Suppresses Adrenocorticotropic Hormone Production and Tumorous Corticotroph Cell Growth in AtT20 Cells

Background： Two recent whole-exome sequencing researches identifying somatic mutations in the ubiquitin-specific protease 8 （USP8） gene in pituitary corticotroph adenomas provide exciting advances in this field. These mutations drive increased epidermal growth factor receptor （EGFR） signaling and promote adrenocorticotropic hormone （ACTH） production. This study was to investigate whether the inhibition of USP8 activity could be a strategy/br the treatment of Cushing＇s disease （CD）. Methods： The anticancer effect of USP8 inhibitor was determined by testing cell viability, colony tbrmation, apoptosis, and ACTH secretion. The immunoblotting and quantitative reverse transcription polymerase chain reaction were conducted to explore the signaling pathway by USP8 inhibition. Results： Inhibition of USP8-induced degradation of receptor tyrosine kinases including EGFR, EGFR-2 （ERBB2）, and Met leading to a suppression of ArT20 cell growth and ACTH secretion. Moreover, treatment with USP8 inhibitor markedly induced AtT20 cells apoptosis. Conclusions： Inhibition of USP8 activity could be an effective strategy for CD. It might provide a novel pharmacological approach for the treatment of CD.