Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3

BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.     

泛素-蛋白酶体系统在心肌梗死中的病理生理意义

心肌梗死后心室重塑是慢性心力衰竭的主要原因.因凋亡和坏死而导致的心肌细胞死亡可造成心肌重塑,病理性心肌肥大和左心室扩张是其主要特征.研究表明,泛素蛋白酶体系统(ubiquitin proteasome system,UPS)可能参与这一过程.     

UBA3在黑素瘤细胞的表达及其影响黑素瘤细胞生物学行为的研究

【摘要】 目的 探讨UBA3在黑素瘤细胞中的表达,初步观察干扰UBA3对黑素瘤细胞生物学行为的影响。 方法 用RT-PCR技术观察3株黑素瘤细胞中UBA3的表达,并利用siRNA技术对M14细胞中UBA3的表达进行干扰,检测转染后细胞内UBA3、连接态NEDD8以及p53的表达,并观察转染前后细胞的凋亡及迁移能力的改变。 结果 在A375、M14及MV3中UBA3的表达均较正常黑素细胞上调,其中M14细胞上调最明显,成功干扰M14中UBA3的表达后,细胞内连接态NEDD8的表达下降,p53的表达明显增加,同时M14细胞凋亡增加、迁移能力下降。 结论 构建的质粒可干扰UBA3的表达,影响细胞的Nedd化进程,从而影响细胞的生物学行为。     

恶性肿瘤中泛素化修饰对Warburg效应调控机制的研究进展

泛素化和Warburg效应在肿瘤的发生发展中均发挥重要作用。肿瘤中泛素化修饰过程与糖酵解效应密切相关,即肿瘤细胞中的泛素化修饰作用能够调控Warburg效应相关的底物蛋白的表达水平,进而调控肿瘤进展和治疗耐药。本文着重讨论肿瘤细胞中蛋白质的泛素化修饰作用对Warburg效应的调控机制。     

乙状结肠代阴道成形术后阴道黏膜中蛋白基因产物9.5、血管活性肠肽和神经肽Y的表达

目的 探讨乙状结肠代阴道成形术后阴道黏膜神经再分布的变化.方法 2002年1月至2010年12月,取在河北医科大学第二医院接受乙状结肠代阴道成形术的20例先天性无阴道患者的人工阴道黏膜组织,采用免疫组化方法检测局部组织中蛋白基因产物9.5(PGP 9.5)的表达及血管活性肠肽(VIP)能神经和神经肽Y(NPY)能神经的分布,取材时间在术后1～3年,并与原位乙状结肠黏膜进行比较.结果 (1)神经纤维密度:人工阴道黏膜组织的黏膜层、黏膜下层和肌层均可见丰富的PGP9.5阳性表达的神经纤维,VIP和NPY阳性表达的神经纤维主要分布在血管周围和肌层;人工阴道黏膜组织中PGP 9.5阳性表达的神经纤维密度为17±6,高于VIP(2.9±1.0)和NPY(2.5±0.8)阳性表达的神经纤维密度,差异有统计学意义(P＜0.05).(2)人工阴道黏膜PGP 9.5的表达:术后1年,人工阴道黏膜PGP 9.5的表达水平为14±4,低于原位乙状结肠黏膜的28±7,差异有统计学意义(P＜0.05);术后2～3年,PGP 9.5的表达水平逐步升高,术后第3年,PGP 9.5的表达水平达22±7,且这种变化以黏膜下层最为明显.(3)人工阴道黏膜VIP和NPY的表达:术后1年,阴道黏膜VIP和NPY的表达水平分别为2.3±0.7、2.5±1.1,低于原位乙状结肠黏膜的5.3±1.4、5.5±1.1,差异均有统计学意义(P＜0.05);术后2～3年,VIP表达水平逐渐回升,术后3年为3.7±0.7,明显高于术后1年的2.3±0.7,差异有统计学意义(P＜0.05),NPY表达水平在术后3年内回升不明显.结论 乙状结肠代阴道成形术后,阴道黏膜的神经分布特征与原位乙状结肠相似.在术后1～3年内,人工阴道黏膜神经纤维密度降低后又有逐步回升趋势.

Abstract：

Objective To investigate re-innervation in the neovaginal mucosa of patients underwent sigmoid colon vaginoplasty in treatment of Mayer-Rokitansky-Kistner-Hauser Syndrome (MRKHS).Methods Biopsies in the upper third of the posterior neovagina were taken in 20 patients treated by sigmoid colon vaginoplasty at 1, 2 and 3 years after surgery, respectively. Protein gene product 9. 5 ( PGP 9. 5 ),vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) were detected by immunohistochemical method and compared with those in intact sigmoid colon mucosa. Results ( 1 ) Density of nerve fiber:abundant distribution of PGP 9. 5 nerve fibers were observed in the mucosal muscle layer, submucosa, and smooth muscle layer of the neovagina. The nerve fibers of VIP and NPY immunoreactivity were mainly distributed around blood vessels and in the smooth muscles. In the neovagina, the density of nerve fibers of PGP 9. 5 of 17 ± 6 were much more than VIP of 2. 9 ± 1.0 and NPY of 2. 5 ± 0. 8 significantly ( P ＜ 0. 05 ).( 2 ) Expression of PGP 9. 5 in neovagina: at 1 year after surgery, PGP 9. 5 positive expression of 14 ± 4 was significantly lower in the neovagina than 28 ± 7 in the intact sigmoid colon( P ＜ 0. 05 ). However, after 2 to 3 years, its expression displayed an upgrade tendency in the neovagina and was significantly higher at the 3 year postoperatively than that at the 1 years postoperatively ( 22 ± 7 vs. 14 ± 4, P ＜ 0. 05 ). The changes were much more obvious in submucosa. (3) The expression of VIP and NPY in neovagina: at 1 year after surgery, VIP and NPY positive nerve fibers were also decreased in the neovagina when compared with those in the intact sigmoid colon ( 2. 3 ± 0. 7 vs. 5.3 ± 1.4, P ＜ 0. 05; 2.5 ± 1. 1 vs. 5.5 ± 1.1, P ＜ 0. 05 ) . At 2 to 3 years after surgery, the positive VIP fiber showed initially decreased and subsequently increased tendency. The density of VIP of 3.7 ± 0. 7 in the neovagina at 3 years postoperatively was higher than 2. 3 ±0. 7 at 1 years postoperatively (P ＜ 0. 05 ). No significant up-regulation was observed in NPY-positive expression in the neovagina within 3 years after operation. Conclusions Distribution of sensory PGP 9. 5,VIP and NPY immunoreactive nerve fibers was similar to the pattern observed within the intact sigmoid colon wall. The number of nerve fibers in the neovagina decreased after surgery and then increased subsequently within 3 years after surgery.     

子宫内膜异位症患者腹膜病灶中神经蛋白基因产物9.5的表达及其临床意义

目的 探讨子宫内膜异位症(内异症)患者腹膜病灶中神经蛋白基因产物9.5(PGP9.5)的表达及其与内异症疼痛的关系.方法 应用免疫组化Envision二步法检测32例内异症患者(内异症组,其中伴与不伴疼痛者各16例)的腹膜病灶组织和同期行腹腔镜手术并经病理学检查确诊为子宫肌瘤且不伴疼痛者20例(对照组)的正常腹膜组织中PGP9.5的表达.结果 内异症组伴与不伴疼痛者腹膜病灶和对照组腹膜组织中PGP9.5表达阳性率及阳性神经纤维密度分别为62%(10/16)和(3.8±1.7)条/mm~2、19%(3/16)和(1.7±0.5)条/mm~2、25%(5/20)和(1.3±0.6)条/mm~2,内异症组伴疼痛者腹膜病灶中PGP9.5阳性神经纤维密度及阳性率均显著高于内异症组不伴疼痛者和对照组,差异均有统计学意义(P均<0.05);但内异症组不伴疼痛者腹膜病灶中PGP9.5阳性神经纤维密度及阳性率与对照组比较,差异均无统计学意义(P均>0.05).内异症组伴疼痛者腹膜病灶中PGP9.5阳性神经纤维密度与患者疼痛程度呈正相关关系(r=0.855,P<0.05).内异症组伴痛经和(或)慢性盆腔痛患者腹膜病灶中,PGP9.5阳性神经纤维密度显著高于伴其他疼痛类型者,差异也有统计学意义(P<0.05),但PGP9.5阳性神经纤维密度与病灶活性、病灶部位及疾病分期均无关(P均>0.05).结论 PGP9.5阳性神经可能参与内异症疼痛的发生机制.     

TRIM25: A central factor in breast cancer

TRIM25 is emerging as a central factor in breast cancer due to its regulation and function. In particular, it has been shown that: (1) Estrogens modulate TRIM25 gene expression; (2) TRIM25 has activity as an E3-ligase enzyme for ubiquitin; and (3) TRIM25 is also an E3 ligase for interferon-stimulated gene 15 protein in the ISGylation system. Consequently, the proteome of mammary tissue is affected by TRIM25-associated pathways, involved in tumor development and metastasis. Here, we discuss the findings on the mechanisms involved in regulating TRIM25 expression and its functional relevance in breast cancer progression. These studies suggest that TRIM25 may be a biomarker and a therapeutic target for breast cancer.     

Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells

The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.     

IRS-1的表达和泛素化在KKAy及C57BL/6J小鼠糖尿病发展过程中的意义

目的:探讨胰岛素受体底物-1(IRS-1)蛋白表达和泛素化水平在糖尿病发展过程中的作用。方法:20只8周龄C57BL/6J小鼠随机分为正常对照组(正常饲料喂养,n=10)和胰岛素抵抗组(高脂饲料喂养,n=10)。8周龄KKAy小鼠8只,高脂饲料喂养,为2型糖尿病组。检测各组小鼠体重、血糖和血胰岛素水平。12周后免疫印迹检测各组动物肝组织IRS-1的表达量,同时采用剥离免疫印迹法检测肝组织IRS-1的泛素化水平。结果:高脂喂养12周后C57BL/6J小鼠出现胰岛素抵抗,KKAy小鼠出现肥胖和糖尿病。2型糖尿病组IRS-1的表达显著低于正常对照组和胰岛素抵抗组(P<0.05),而IRS-1的泛素化水平则显著高于胰岛素抵抗组(P<0.05),与对照组无显著性差异(P>0.05)。结论:2型糖尿病时IRS-1的表达下调,IRS-1泛素化水平增加是导致胰岛素信号转导障碍的重要原因。