Background::The hepatitis B virus (HBV) vaccine has been efficiently used for decades. However, hepatocellular carcinoma caused by HBV is still prevalent globally. We previously reported that interferon (IFN)-induced tripartite motif-containing 25 (TRIM25) inhibited HBV replication by increasing the IFN expression, and this study aimed to further clarify the anti-HBV mechanism of TRIM25.Methods::The TRIM25-mediated degradation of hepatitis B virus X (HBx) protein was determined by detecting the expression of HBx in TRIM25-overexpressed or knocked-out HepG2 or HepG2-NTCP cells via Western blotting. Co-immunoprecipitation was performed to confirm the interaction between TRIM25 and HBx, and colocalization of TRIM25 and HBx was identified via immunofluorescence; HBV e-antigen and HBV surface antigen were qualified by using an enzyme-linked immunosorbent assay (ELISA) kit from Kehua Biotech. TRIM25 mRNA, pregenomic RNA (pgRNA), and HBV DNA were detected by quantitative real-time polymerase chain reaction. The retinoic acid-inducible gene I (RIG-I) and pgRNA interaction was verified by RNA-binding protein immunoprecipitation assay.Results::We found that TRIM25 promoted HBx degradation, and confirmed that TRIM25 could enhance the K90-site ubiquitination of HBx as well as promote HBx degradation by the proteasome pathway. Interestingly, apart from the Really Interesting New Gene (RING) domain, the SPRY domain of TRIM25 was also indispensable for HBx degradation. In addition, we found that the expression of TRIM25 increased the recognition of HBV pgRNA by interacting with RIG-I, which further increased the IFN production, and SPRY, but not the RING domain is critical in this process.Conclusions::The study found that TRIM25 interacted with HBx and promoted HBx-K90-site ubiquitination, which led to HBx degradation. On the other hand, TRIM25 may function as an adaptor, which enhanced the recognition of pgRNA by RIG-I, thereby further promoting IFN production. Our study can contribute to a better understanding of host-virus interaction.

目的:探讨TRIM25、RIG-Ⅰ在肝癌组织中的表达及二者与患者预后的关系。方法:回顾性分析2017年1月至2018年1月南京医科大学附属淮安第一医院收治的82例肝癌患者资料，收集癌组织和癌旁组织（距肿瘤边缘>5 cm）标本。采用免疫组织化学法检测癌组织和癌旁组织TRIM25、RIG-Ⅰ蛋白表达情况。分析患者癌组织TRIM25、RIG-Ⅰ表达与临床病理特征的关系，采用Kaplan-Meier法分析癌组织TRIM25、RIG-Ⅰ不同表达状态患者总生存（OS）。结果:癌组织TRIM25阳性率高于癌旁组织[68.29%（56/82）比21.95%（18/82）， P<0.001]，癌组织RIG-Ⅰ阳性率低于癌旁组织[31.71%（26/82）比74.39%（61/82）， P<0.001]。低分化患者癌组织TRIM25阳性率高于高中分化患者（ P<0.05），而RIG-Ⅰ阳性率低于高中分化患者（ P<0.05）；肝外转移患者癌组织TRIM25阳性率高于未肝外转移患者（ P<0.05），而RIG-Ⅰ阳性率低于未肝外转移患者（ P<0.05）；临床分期Ⅲ~Ⅳ期患者癌组织TRIM25阳性率高于Ⅰ~Ⅱ期患者（ P<0.05），而RIG-Ⅰ阳性率低于Ⅰ~Ⅱ期患者（ P<0.05）。中位随访27个月（4~48个月），共2例患者失访；至2022年1月随访结束，全组生存率为43.75%（35/80），癌组织TRIM25阳性和阴性患者生存率分别33.33%（18/54）和65.38%（17/26），癌组织RIG-Ⅰ阳性和阴性患者生存率分别64.00%（16/25）和34.55%（19/55）。Kaplan-Meier法分析显示，癌组织TRIM25阴性患者OS优于阳性患者，癌组织RIG-Ⅰ阳性患者OS优于阴性患者，差异均有统计学意义（均 P<0.05）。 结论:肝癌组织TRIM25表达升高，RIG-Ⅰ表达降低。肝癌组织TRIM25、RIG-Ⅰ表达与预后相关。

Background::Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The ubiquitin-specific peptidase 25 (USP25) protein has been reported to participate in the development of several cancers. However, few studies have reported its association with HCC. In this study, we aimed to investigate the function and mechanism of USP25 in the progression of HCC.Methods::We analyzed USP25 protein expression in HCC based on The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) database cohorts. Then, we constructed USP25-overexpressing and USP25-knockdown HepG2, MHCC97H, and L-O2 cells. We detected the biological function of USP25 by performing a series of assays, such as Cell Counting Kit-8 (CCK-8), colony formation, transwell, and wound healing assays. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed to detect the interaction between USP25 and the Wnt/β-catenin signaling pathway. The relationship between USP25 and tripartite motif-containing 21 (TRIM21) was assessed through mass spectrometry and co-immunoprecipitation (Co-IP) analysis. Finally, we constructed a mouse liver cancer model with the USP25 gene deletion to verify in vivo role of USP25. Results::USP25 was highly expressed in HCC tissue and HCC cell lines. Importantly, high expression of USP25 in tissues was closely related to a poor prognosis. USP25 knockdown markedly reduced the proliferation, migration, and invasion of HepG2 and MHCC97H cells, whereas USP25 overexpression led to the opposite effects. In addition, we demonstrated that USP25 interacts with TRIM21 to regulate the expression of proteins related to epithelial-mesenchymal transition (EMT; E-cadherin, N-cadherin, and Snail) and the Wnt/β-catenin pathway (β-catenin, Adenomatous polyposis coli, Axin2 and Glycogen synthase kinase 3 beta) and those of their downstream proteins (C-myc and Cyclin D1). Finally, we verified that knocking out USP25 inhibited tumor growth and distant metastasis in vivo. Conclusions::In summary, our data showed that USP25 was overexpressed in HCC. USP25 promoted the proliferation, migration, invasion, and EMT of HCC cells by interacting with TRIM21 to activate the β-catenin signaling pathway.

目的:探讨TRIM25 和EZH2 在食管鳞状细胞癌(ESCC)相关巨噬细胞浸润中的作用及其可能的作用机制.方法:利用佛波酯诱导THP-1 为M0 巨噬细胞,将其与转染处理后的KYSE510 细胞共培养,收集共培养巨噬细胞,分为Ctrl组、sh-NC 组、sh-TRIM25 组、sh-NC+oe-NC 组、sh-TRIM25+oe-NC组、sh-NC+oe-EZH2 组和sh-TRIM25 +oe-EZH2 组.收集共培养上清液,将其加入KYSE510 细胞中,分为Ctrl组、TAM组、sh-NC+TAM组、sh-TRIM25+TAM组、sh-NC+oe-NC+TAM组、sh-TRIM25+ oe-NC+TAM组、sh-NC+oe-EZH2+TAM组和sh-TRIM25+oe-EZH2 +TAM 组.qPCR 法和 WB 法检测细胞中TRIM25、EZH2 和Arg-1 表达,放线菌酮蛋白合成抑制实验检测细胞EZH2 蛋白稳定性,FCM检测F4/80+CD206+巨噬细胞比例,CCK-8 法、克隆形成实验和 Transwell 实验分别检测细胞的增殖、迁移和侵袭能力.结果:KYSE510 细胞中 TRIM25 和 EZH2 mRNA 和蛋白表达均高于人食管鳞状上皮细胞HET-1A(P<0.01).与sh-NC组和sh-NC+oe-NC组相比,sh-TRIM25 组和sh-TRIM25+oe-NC 组F4/80+CD206+巨噬细胞比例和Arg-1 蛋白表达均降低(P<0.05),sh-NC+oe-EZH2 组则升高(P<0.05).与sh-NC+TAM组和sh-NC+oe-NC+TAM 组相比,sh-TRIM25+TAM 组和 sh-TRIM25+oe-NC+TAM 组KYSE510 细胞活力、克隆形成数、迁移和侵袭细胞数均降低(P<0.05),sh-NC+oe-EZH2+TAM 组则升高(P<0.05).敲低TRIM25 可通过抑制 EZH2 蛋白半衰期,降低 EZH2 蛋白稳定性.过表达 EZH2 可部分逆转sh-TRIM25 对巨噬细胞和KYSE510 细胞的影响.结论:TRIM25 通过促进 EZH2 蛋白稳定性诱导巨噬细胞M2 极化,从而促进ESCC细胞增殖、迁移和侵袭.

目的 探究NOD样受体家族含CARD结构域5(NLRC5)在胃癌中的相互作用蛋白,并明确其对胃癌的诊断价值.方法 采用癌症基因组图谱(TCGA)数据库探究NLRC5在胃癌组织和癌旁组织中的差异,结合STRING网站分析NLRC5的相互作用蛋白,通过基因表达综合(GEO)数据库验证TCGA数据库筛选出来的蛋白对胃癌的诊断价值.引用临床模型,验证筛选出来的蛋白在胃癌组织和癌旁组织中的表达情况,确定各蛋白间的相关性,通过受试者工作特征(ROC)曲线明确其对胃癌的诊断价值.结果 TCGA结果显示,胃癌组织中NL-RC5表达水平明显高于癌旁组织(P﹤0.01).蛋白-蛋白相互作用发现,核因子κB激酶复合物抑制剂组分(CHUK)、核因子κB激酶亚单位β的抑制剂(IKBKB)、三重基序蛋白25(TRIM25)与NLRC5均呈正相关.经过GEO数据库验证得到TRIM25、CHUK、IKBKB与NLRC5可以构建联合模型诊断胃癌.临床模型中NLRC5在胃癌组织中高表达,且与TRIM25、IKBKB、CHUK表达水平均呈正相关(P﹤0.01).NLRC5联合TRIM25、IKBKB、CHUK对胃癌有较高的诊断价值.结论 TRIM25、IKBKB、CHUK参与NLRC5在胃癌发展中的相关调控机制,在胃癌诊断中具有较高的效能.

Pluripotent stem cells (PSCs) harbor constitutive DNA replication stress during their rapid proliferation and the consequent genome instability hampers their applications in regenerative medicine.It is therefore important to understand the regulatory mechanisms of replication stress response in PSCs.Here,we report that mouse embryonic stem cells (ESCs) are superior to differentiated cells in resolving replication stress.Specifically,ESCs utilize a unique Filia-Floped protein complex-dependent mechanism to efficiently promote the restart of stalled replication forks,therefore maintaining genomic stability.The ESC-specific Filia-Floped complex resides on replication forks under normal conditions.Replication stress stimulates their recruitment to stalling forks and the serine 151 residue of Filia is phosphorylated in an ATR-dependent manner.This modification enables the Filia-Floped complex to act as a functional scaffold,which then promotes the stalling fork restart through a dual mechanism:both enhancing recruitment of the replication fork restart protein,Blm,and stimulating ATR kinase activation.In the Blm pathway,the scaffolds recruit the E3 ubiquitin ligase,Trim25,to the stalled replication forks,and in turn Trim25 tethers and concentrates Blm at stalled replication forks through ubiquitination.In differentiated cells,the recruitment of the Trim25-Blm complex to replication forks and the activation of ATR signaling are much less robust due to lack of the ESC-specific Filia-Floped scaffold.Thus,our study reveals that ESCs utilize an additional and unique regulatory layer to efficiently promote the stalled fork restart and maintain genomic stability.

2018 年2 月,《N Engl J Med》杂志在线发表了关于传奇神药Larotrectinib(LOXO-101)同时进行的三项安全性和有效性的研究结果,对于年龄为4个月至76岁的患者,针对17种不同肿瘤的总体应答率为75%,再次掀起了其靶点NTRK融合基因研究的热潮,我们回顾一下55 例NTRK 融合基因融合伙伴的情况,在25例NTRK1中,融合伙伴LMNA占28.00%(7/25),TPM3 占36.00%(9/25),IRF2BP2占8.00%(2/25),CTRC 占8.00%(2/25),其余融合伙伴TPR、PDE4DIP、SQSTM1、TRIM63、PPL 各占4.00%(1/25);1例为NTRK2,其融合伙伴为STRN;29例为NTRK3,融合伙伴ETV6占96.55%(28/29),TPM4占3.45%(1/29)[1].

E3泛素连接酶三基序25(TRIM25)是E3泛素连接酶中三基序蛋白家族的成员之一,在天然免疫、防御病毒感染、调控细胞增殖和癌细胞迁移中起主要作用.研究表明TRIM25也能够结合RNA并调节Lin28a介导的let-7前体尿苷化.TRIM25作为一种新型蛋白在子宫发育、肿瘤发生发展、天然免疫和RNA代谢中发挥重要作用,本文将对其上述作用进行综述.

目的 探讨主动脉中层中α-SMA、OPN、P53、MDM2和TRIM25蛋白在主动脉夹层中的表达变化及其意义.方法将12例A型AD手术患者的升主动脉作为实验组,12例DCD供体的升主动脉作为对照组.用免疫组化和Western blot法检测α-SMA、OPN、P53、MDM2、TRIM25和p-P53在AD组及对照组中的表达情况,用RT-PCR技术检测AD组及对照组组织中P53、MDM2及TRIM-25mRNA含量.结果通过Western blot法和RT-PCR法检测结果显示,P53、MDM2和TRIM-25的蛋白、mRNA含量在AD组织中明显增加,免疫组化检测也表明P53、MDM2和TRIM25含量增加,但其p-P53水平却下降.免疫组化结果显示α-SMA阳性细胞在AD组中的含量较对照组显著减少,而OPN阳性细胞在AD组增高,迸一步Western blot法检测也证实了这一变化.结论AD主动脉中层中VSMC表型向去分化型转变,而P53、MDM2、TRIM25的表达量增高,提示TRIM25可能通过影响P53/MDM2反馈环而导致VSMC表型转变参与AD形成.

表皮生长因子受体(epidermal growth factor receptor,EGFR)基因是非小细胞肺癌(non-small cell lungcancer,NSCLC)中最常见的驱动基因之一,尤其EGFR突变和扩增都参与了肺癌的恶性进程.本文旨在探究E3泛素连接酶TRIM25 (tripartite motif25)对肺癌发生发展的作用及分子机制.应用CCK-8和Transwell实验评价TRIM25对肺癌细胞增殖和侵袭能力的影响,结果发现敲低TRIM25,显著抑制了细胞的增殖(抑制率为34％)和侵袭(抑制率为42％);采用基因集富集分析、免疫印迹和免疫组化法分析TRIM25对EGFR及其下游信号活性的影响,结果显示,TRIM25不仅上调了EGFR的表达水平,而且促进了EGFR信号活化;利用免疫共沉淀、实时定量PCR以及放线菌酮(cycloheximide,CHX)抑制蛋白质合成实验,初步探究TRIM25上调EGFR信号的分子机制.研究结果表明,TRIM25主要通过促进EGFR第63位赖氨酸位点发生泛素化修饰,进而增加EGFR蛋白稳定性、上调EGFR下游信号活性;而恢复EGFR蛋白表达后可逆转敲低TRIM25对肺癌细胞A549、H1975增殖和侵袭的抑制作用,在A549细胞中,细胞增殖率增加了1.5倍,侵袭率提高了1.6倍;同样,在H1975细胞中,细胞增殖率增加了2倍,侵袭率提高了1.7倍.以上研究结果表明,TRIM25通过维持EGFR信号持续活化,促进肺癌细胞的增殖和侵袭.本研究中所用的人肺癌组织由中国医学科学院肿瘤医院的肺癌患者提供,根据《赫尔辛基宣言》,已取得所有肺癌患者的知情同意.该研究得到了中国医学科学院肿瘤医院伦理委员会的批准.