目的:检测卵巢肿瘤(ovarian tumor,OTU)结构域线性链接特异性去泛素化酶(OTU domain deubiquitinase with linear linkage specificity,OTULIN)在胃癌组织中的表达情况,并探讨敲除OTULIN基因表达对胃癌MKN45和AGS细胞增殖的影响,及可能...
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目的:检测卵巢肿瘤(ovarian tumor,OTU)结构域线性链接特异性去泛素化酶(OTU domain deubiquitinase with linear linkage specificity,OTULIN)在胃癌组织中的表达情况,并探讨敲除OTULIN基因表达对胃癌MKN45和AGS细胞增殖的影响,及可能的作用机制。方法:采用免疫组织化学法检测73例胃癌组织与24例正常胃黏膜组织中OTULIN的表达水平,分析OTULIN表达与胃癌临床病理特征和患者预后的相关性。收集并分析癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库和基因表达综合数据库(Gene Expression Omnibus database,GEO)中的胃癌数据验证上述结论。采用CRISPR/Cas9基因编辑技术构建敲除OTULIN表达的胃癌MKN45和AGS细胞并用蛋白质印迹法检测基因敲除效率;采用CCK-8法和软琼脂克隆检测敲除OTULIN表达对MKN45和AGS细胞增殖的影响。蛋白免疫沉淀-质谱技术筛选OTULIN和线性泛素分子(INT-Ub.7KR)的共同互作蛋白,发现线性泛素化修饰底物蛋白。利用丙酮酸激酶(pyruvate kinase,PK)活性试剂盒检测敲除OTULIN表达后糖酵解限速酶的活性变化,乳酸定量试剂盒检测乳酸生成情况。最后采用免疫共沉淀及蛋白质印迹法验证OTULIN对底物蛋白线性泛素化作用。结果:免疫组织化学法检测结果显示,OTULIN在胃癌组织中的表达水平明显高于正常胃黏膜组织(P=0.0041),肿瘤组织中OTULIN高表达者生存期较短(P=0.0077)。TCGA和GEO数据库数据分析也显示胃癌组织OTULIN表达水平升高(P<0.05),且OTULIN高表达与患者不良预后相关(P=0.011)。统计结果显示,OTULIN高表达与TNM分期晚密切相关(P=0.0273),预测患者较短生存期(P=0.04)。敲除OTULIN基因可显著抑制胃癌细胞的增殖能力(P均<0.001),降低胃癌细胞中PK活性和乳酸生成水平(P均<0.01)。OTULIN可结合糖酵解途径的多个限速酶并下调它们的线性泛素化水平,包括丙酮酸激酶M1(pyruvate kinase M1,PKM1)和PKM2以及乳酸脱氢酶A(lactate dehydrogenase A,LDHA)和LDHB。结论:胃癌中OTULIN高表达可能是评估患者预后的一个新的生物标志物,OTULIN可能通过下调糖酵解限速酶的线性泛素化修饰,激活胃癌糖酵解途径,促进胃癌发展。
Background & Aims:The circadian clock presents daily rhythmicity to numerous crucial biological processes and its disruption is related to carcinogenesis in several kinds of *** a core circadian rhythm component,b...
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Background & Aims:The circadian clock presents daily rhythmicity to numerous crucial biological processes and its disruption is related to carcinogenesis in several kinds of *** a core circadian rhythm component,brain and muscle arnt-like protein 1(BMAL1)dysregulation has been shown to contribute to aberrant metabolism in human ***,the biological functions of BMAL1,especially its involvement in aberrant lipid metabolism in hepatocellular carcinoma(HCC),remain ***:The expression of BMAL1 and its clinical significance was determined in tissues and cell lines of *** addition,biological functions of BMAL1 in HCC cells and the underlying molecular mechanisms were explored both in vitro and in ***,therapeutic application of BMAL1 in the treatment of HCC was also ***:We established that BMAL1was often down-regulated in HCC cells primarily due to the up-regulation of ***-regulation of BMAL1 was remarkably linked to dismal survival in individuals with ***1 down-regulation enhanced HCC cell growth,as well as ***,through cooperating with EZH2,BMAL1 transcriptionally suppressed glycerol-3-phosphate acyltransferase mitochondrial(GPAM) expression,a pivotal enzyme involved in the regulation of lipid biosynthesis,leading to reduced levels lysophosphatidic acid(LPA),which has long been known as mediator of ***,treatment with SR8278,an activator of BMAL1,exhibited a therapeutic influence on ***:BMAL1 has as critical anti-oncogenic role in HCC,providing a strong research evidence for BMAL1 as a prospective HCC therapeutic target.
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