糖酵解相关通路对索拉非尼治疗肝细胞肝癌疗效的影响

发布时间：2018-04-16 17:48

本文选题：有氧糖酵解 + PKM2　；参考：《北京协和医学院》2017年博士论文

【摘要】：背景:肝细胞肝癌(Hepatocellular carcinoma,HCC)是肝脏最常见的恶性肿瘤之一。多数肝癌患者初诊时已经处于病程的中晚期,多靶点酪氨酸激酶抑制剂索拉非尼(Sorafenib,NexavarR)是治疗不可手术的进展期HCC的一线靶向用药。但是由于肝癌复杂的生物学特性,索拉非尼临床疗效异质性较大。因此探讨索拉非尼耐药的机制对于改善进展期肝癌患者的预后显得极为重要。有氧糖酵解是肿瘤细胞糖代谢的主要方式,糖酵解中的关键酶如丙酮酸激酶(Pyruvate kinase M2,PKM2)和乳酸脱氢酶(Lactate dehydrogenase,LDH)对于肿瘤的发生发展有重要作用,糖酵解的关键酶PKM2和LDHA等与索拉非尼耐药的相关性有待进一步深入研究。Extracellular signal-regulated kinase(ERK)5 通路也称为 BMK1 通路,是Mitogen-activated protein kinase(MAPK)家族中新近发现并加以研究的分子通路,近年来研究发现ERK5的表达水平和多种恶性肿瘤的发生发展具有一定关系。既往研究提示ERK1/2可调节PKM2基因入核而影响细胞恶性生物行为。作为索拉非尼主要作用靶点之一,ERK1/2基因表达变化在索拉非尼耐药中具有重要作用。而同作为MAPK家族的ERK5对PKM2是否有调节作用,及其与索拉非尼耐药的关系还亟待进一步深入研究。材料和方法:1.回顾性分析119例行索拉非尼治疗的晚期乙肝相关肝癌患者临床资料。根据治疗前血清乳酸脱氢酶水平将患者分为2组。分析血清乳酸脱氢酶水平的预后预测价值及与其他临床病理因素的关系;2.应用免疫组织化学方法检测肝癌肿瘤组织、癌旁组织中LDHA的蛋白表达水平。3.构建PKM2基因过表达及敲减的慢病毒,并经筛选构建稳定敲减及过表达PKM2基因的肝癌细胞株。4.分别观察下调及上调PKM2基因表达对索拉非尼引起的细胞增殖、凋亡、迁移、葡萄糖消耗率、乳酸产率等的影响,并使用RT-PCR及Western Blot检测LDHA的mRNA及蛋白质表达变化。5.观察ERK5特异性抑制剂XMD8-92对索拉非尼引起的肝癌细胞增殖、凋亡、迁移、葡萄糖消耗率、乳酸产率等的影响。并使用RT-PCR及Western Blot检测PKM2及LDHA的mRNA及蛋白质表达变化。6.观察上调PKM2基因对XMD8-92及XMD8-92联合索拉非尼处理的肝癌细胞的增殖、凋亡、迁移、葡萄糖消耗率、乳酸产率等的影响,并使用RT-PCR及Western Blot检测LDHA的mRNA及蛋白质表达变化。结果:1.患者分组的界值定为221U/L。中位随访时长15(范围:3-73)个月,91个患者达到随访终点。多因素分析发现治疗前乳酸脱氢酶水平是影响总生存时间和无进展生存时间的独立危险因素。对于治疗前乳酸脱氢酶水平高于221U/L的患者,治疗后3个月乳酸脱氢酶升高者预后更差。治疗前LDH偏高与低白蛋白、大血管癌栓、高Child-Pugh分级、高T分级、高AFP水平和高总胆红素有关。2.接受索拉非尼治疗的肝癌组织中LDHA表达显著高于癌旁组织。3.成功构建了 PKM2的敲减及过表达慢病毒,并成功转染Huh7细胞得到稳转株。4.下调及上调PKM2基因表达可分别增强和减弱索拉非尼引起的肝癌细胞抑制增殖、促进凋亡、抑制迁移、减少葡萄糖消耗率、减少乳酸产率等的影响。下调及上调PKM2基因表达可分别下调或上调LDHA的mRNA及蛋白质表达变化。5.ERK5特异性抑制剂XMD8-92可增强索拉非尼引起的对肝癌细胞抑制增殖、促进凋亡、抑制迁移、减少葡萄糖消耗率、减少乳酸产率等的影响,且呈剂量依赖效应。XMD8-92可降低肝癌细胞核中PKM2表达,以及细胞中LDHA的表达,且XMD8-92与索拉非尼联合用药较索拉非尼单药处理对上述基因的表达抑制效果更为显著,且呈剂量依赖效应。6.上调PKM2基因可减弱XMD8-92及XMD8-92联合索拉非尼处理的引起的肝癌细胞增殖、促进凋亡、抑制迁移、减少葡萄糖消耗率、减少乳酸产率等的影响,且PKM2过表达后XMD8-92组及XMD8-92联合索拉非尼组细胞中LDHA升高。结论:PKM2介导的糖酵解通路改变可能是引起索拉非尼耐药机制之一。血清乳酸脱氢酶水平是接受索拉非尼治疗的晚期肝细胞肝癌患者的预后预测因素。PKM2介导的糖酵解通路可被ERK5特异性抑制剂XMD8-92调节。
[Abstract]:Background: hepatocellular carcinoma (Hepatocellular, carcinoma, HCC) is one of the most common malignant tumor of the liver. The majority of patients with hepatocellular carcinoma diagnosed at an advanced stage of disease, multitargeted tyrosine kinase inhibitor Sola Fini (Sorafenib, NexavarR) is a target for the treatment of unresectable advanced HCC to medication. But due to the biological characteristics of hepatocellular carcinoma the Sola Fini complex, clinical heterogeneity. Therefore to explore the mechanism of sorafenib resistant to improve the prognosis of patients with advanced HCC is extremely important. Aerobic glycolysis is the main way of tumor cells in sugar metabolism, key enzyme in glycolysis such as pyruvate kinase (Pyruvate kinase, M2, PKM2) and lactic acid dehydrogenase (Lactate dehydrogenase, LDH) plays an important role in the occurrence and development of tumor related key glycolytic enzymes PKM2 and LDHA and Sola Fini further resistance Study of.Extracellular signal-regulated kinase (ERK) 5 pathway known as BMK1 pathway, Mitogen-activated protein kinase (MAPK) is a newly discovered family and molecular pathways to study, recent studies have found that the expression of ERK5 and tumor development has certain relationship. Previous studies suggested that ERK1/2 can regulate the PKM2 gene into the nucleus effect of biological behavior of malignant cells. As one of the main targets of Sola Fini, ERK1/2 gene expression plays an important role in the sorafenib resistance. But the same as the MAPK family of ERK5 whether the regulation of PKM2, and its relationship with Sola Fini resistance also need further research. Materials and methods: a retrospective analysis of 119 cases of Sora 1. sorafenib in the treatment of advanced HBV related HCC patients. According to the clinical data before treatment, serum lactate dehydrogenase levels in patients Divided into 2 groups. Analysis of the relationship between the prognostic value of serum lactate dehydrogenase level and other clinical pathological factors; detection of HCC tumor tissue 2. by immunohistochemical method LDHA in cancer tissue protein expression level of.3. gene over expression construct PKM2 and lentiviral knockdown, and screened stable knockdown and construction expression of PKM2 gene in hepatocellular carcinoma cell line.4. were observed by Sola Fini and upregulation of PKM2 gene expression on induced cell proliferation, apoptosis, migration, glucose consumption rate, lactic acid yield, and the use of RT-PCR and Western Blot to detect the expression of LDHA mRNA and.5. protein changes observed in cancer cells proliferation, ERK5 specific inhibitor of XMD8-92 Sola Fini induced apoptosis, migration, glucose consumption rate, the yield of lactic acid. The effect of the expression of mRNA and protein and using RT-PCR and Blot detection of Western PKM2 and LDHA .6. observed upregulation of PKM2 gene on proliferation of hepatocellular carcinoma cells XMD8-92, and combined treatment of Sola Fini XMD8-92 apoptosis, migration, glucose consumption rate, lactic acid yield, and protein expression of mRNA RT-PCR and Western Blot changes and the detection of LDHA. Results: 1. patients were grouped as 221U/L. value median follow-up duration 15 (range: 3-73 months), 91 patients at follow-up. Multivariate analysis showed that the end point before the treatment of lactate dehydrogenase levels are independent risk factors for overall survival and progression free survival time. Before treatment for lactate dehydrogenase level is higher than that of 221U/L patients, 3 months after the treatment of lactate dehydrogenase increased poorer prognosis LDH. Before the treatment with the high low albumin, vascular tumor thrombus, high grade Child-Pugh, high grade T, LDHA high expression level of AFP and high total bilirubin.2. received the Sola Fini treatment in hepatocellular carcinoma Compared with paracancerous tissues.3. was successfully constructed PKM2 knockdown and overexpression of lentivirus and transfected into Huh7 cells successfully expressed stable strain downregulation of.4. and upregulation of PKM2 gene can increase and decrease caused by Sola Fini hepatocellular carcinoma cells inhibit proliferation and promote apoptosis, inhibit migration, reduced glucose consumption rate, decrease the yield of lactic acid wait. Downregulation and upregulation of PKM2 gene were down regulated or up-regulated expression of LDHA mRNA and protein changes of.5.ERK5 specific inhibitor XMD8-92 can enhance sorafenib caused on hepatocellular carcinoma cell proliferation inhibition, apoptosis, inhibition of migration, reduced glucose consumption rate, reduce the effect of lactic acid yield, dose-dependent.XMD8-92 reduce the expression of PKM2 in hepatocellular carcinoma cells, and the expression of LDHA in the cells, and the XMD8-92 and sorafenib in combination with sorafenib monotherapy treatment on the gene expression. As the inhibitory effect is more significant, dose-dependent upregulation of.6. PKM2 gene can decrease XMD8-92 and induced the proliferation of hepatoma cells, combined with Sola Fini XMD8-92 promote apoptosis, inhibit migration, reduced glucose consumption rate, reduce the effect of lactic acid yield, and PKM2 LDHA increased in XMD8-92 group and XMD8-92 combined with sorafenib group cells expression. Conclusion: PKM2 mediated glycolytic pathway changes may be caused by one of the resistance mechanisms of Sola Fini. Serum lactate dehydrogenase level is sorafenib therapy in patients with advanced hepatocellular carcinoma and the prognostic factors of.PKM2 mediated glycolytic pathway by ERK5 specific inhibitor XMD8-92 regulation.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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