兰州大学机构库 >生命科学学院
M2型丙酮酸激酶(PKM2)与肝细胞癌恶性增生及预后相关的功能研究
Alternative TitleStudy on Malignant Hyperplasia and Prognosis of PKM2 in Hepatocellular Carcinoma
赵睿
Thesis Advisor杨金波
2019-05-31
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Degree Discipline细胞生物学
Keyword肝细胞癌 PKM2 敲除 过表达 增殖 凋亡
Abstract背景  肝细胞癌(Hepatocellular Carcinoma, HCC)是全球常见的恶性肿瘤之一。据2008年最新全球癌症的流行病学调查中,HCC发病率占第六位,死亡率占第三位[1]。HCC具有起病隐匿、高侵袭性、转移和复发率高以及预后差的特点,同时也是肝癌中最常见的病理类型。由于上述特点,患者就诊时常处于中晚期,虽然有手术、经肝动脉栓塞化疗、局部放疗以及分子靶向药物索拉菲尼等治疗方法,但长期及远期疗效均不满意,甚至有大部分患者因癌组织多发病灶、体积较大、解剖位置离血管太近或门静脉侵犯、远处转移等给治疗上带来了很多困难。肝细胞癌的生物靶向治疗相关研究一直受到研究者们的高度重视。早期诊断也缺乏有效的肿瘤标志物,除此之外一直以来临床上除血清检测AFP外,其他的肿瘤标志物还在进一步的研究中;研究发现肿瘤细胞和正常细胞获取能量的方式有所不同,这一机制中发挥重要作用的是糖酵解限速酶—丙酮酸激酶(Pyruvate kinase Isozyme Splice Variant, PKs)。它有四种异构体,分别有不同的分泌特点,M2型是其中之一。M2型丙酮酸激酶 (Pyruvate kinase Isozyme Splice Variant 2 , PKM2) 具有在肿瘤细胞或无限繁殖的细胞中分泌的特点,在肿瘤细胞代谢过程中会取代其他同种异构体而主要发挥作用;另外,近几年的研究中还发现PKM2有类似癌基因的作用,但PKM2在个体肿瘤中的相关研究较少,在HCC中的作用仍不完善和清楚,这就是本研究的创新之处,寻找有意义的诊治靶点。目的  旨在研究M2型丙酮酸激酶(PKM2)在肝细胞癌(HCC)发生发展中的作用。通过敲低及过表达PKM2基因后研究其对肝癌细胞增殖、凋亡的影响作用、裸鼠成瘤、同时探索PKM2与HCC患者临床方面的关系,揭示出PKM2的临床意义,为PKM2成为HCC新一种生物学标记以及个体化治疗的新策略提供一定的实验依据与理论支持,为下一步PKM2与HCC相关的深入研究奠定基础。方法  通过对比七种不同的肝癌细胞株及正常肝细胞中PKM2基因的表达,筛选出进一步实验所用的肝癌细胞株,慢病毒稳转构建PKM2基因敲低及过表达的肝癌细胞株,随后进行CCK8实验、克隆形成实验、细胞凋亡,细胞周期、免疫印迹等细胞功能实验检测PKM2基因对肝细胞癌细胞增殖和凋亡的影响,初步探索PKM2在HCC细胞凋亡的作用机制;通过动物实验观察敲除PKM2基因后对肝脏肿瘤的成瘤情况;进一步通过临床实验明确PKM2基因与HCC患者的临床相关性,收集新鲜肝细胞癌组织及癌旁组织,肝细胞癌组织、肝硬化组织、正常肝组织的石蜡样本,研究PKM2基因在HCC患者中的差异性表达,通过统计学分析PKM2与HCC患者不同临床指标中的单因素及多因素的影响价值,以及揭示PKM2与HCC患者预后的关系。结果1. 细胞实验部分1.1 PKM2基因在正常肝细胞L02中为阴性表达,在七种不同肝癌细胞株中(HCCLM3、MHCC97H、MHCC97L、SMCC7721、Huh7、PLC、HepG2)均有不同程度的阳性表达,从中筛选出HCCLM3、MHCC97H两株人肝癌细胞作为下一步实验细胞株;1.2 PKM2-shRNA-LV靶向敲低MHCC97H肝癌细胞株中PKM2基因,成功构建敲低PKM2肝癌细胞株(97H-PKM2-KD);过表达HCCLM3肝癌细胞株中的PKM2基因,成功构建过表达PKM2肝癌细胞株(LM3-PKM2-OE);构建成功后分为97H-PKM2-NC, 97H-PKM2-OE, LM3-PKM2-NC, LM3-PKM2-KD四组细胞进行功能实验。1.3 PKM2在肝癌细胞株中的主要作用1.3.1 CCK8实验检测四组细胞的增殖变化,97H-PKM2-OE细胞株对比对照组NC细胞,过表达细胞株在酶标仪对波长450 nm的光的吸收率随时间变化而逐步升高,表明过表达PKM2基因使肝癌细胞活性显著升高,增强了肝癌细胞的增殖能力;相反,LM3-PKM2-KD细胞株在酶标仪对波长450 nm的光的吸收率随时间变化而逐步下降,表明敲低PKM2基因使肝癌细胞活性显著降低,降低了肝癌细胞的增殖能力;1.3.2 软琼脂克隆形成实验,相对于对照组,KD细胞株克隆形成明显减少,HCC癌细胞克隆形成能力受到抑制;OE细胞株克隆形成成倍增加,HCC癌细胞克隆形成能力明显增强,结果显示PKM2有促进肝癌细胞恶性增殖的作用。1.3.3 细胞周期实验显示与对照组比较,过表达PKM2基因导致目的细胞处于G0/G1期的细胞减少,处于S期的细胞增多,表明过表达PKM2基因后肝癌细胞停滞在细胞周期的S期;敲低的结果与之相反,PKM2敲低后将目的细胞处于G0/G1期的细胞增加,而处于S期的细胞减少,表明敲低PKM2基因将肝癌细胞停滞在细胞周期的G0/G1期;1.3.4 细胞凋亡检测结果显示,相对于对照组,过表达97H细胞中PKM2基因后,细胞凋亡率显著降低(P<0.05)。敲低LM3细胞中PKM2基因后,细胞凋亡率显著增加(P<0.05);1.3.5 免疫印迹实验结果显示,与对照组LM3-NC相比,敲低PKM2基因即PKM2低表达时,p-STAT3及c-myc的表达也是下降的,而caspase 3,8,9表达却是增加的;过表达PKM2即PKM2高表达时刚好相反,故PKM2基因在HCC中主要起到抗凋亡,从而促进细胞增殖的作用。2.动物成瘤实验采用敲低PKM2基因的肝癌细胞HCCLM3与对照组NC细胞共同注射在裸鼠皮下,观察裸鼠肿瘤体积及重量变化曲线,敲低PKM2基因可明显减缓肿瘤生长,肿瘤重量也减轻(P<0.05)。3.PKM2基因在肝癌组织中的表达及与预后分析3.1 PKM2基因在新鲜肝癌组织及癌旁组织的表达:收集兰州大学第二医院2014年1月至2017年12月40对新鲜肝细胞癌患者手术标本及对应的癌旁组织,western-blot结果显示15对(37.5%)中癌组织PKM2基因表达明显高于癌旁组织;10对(25%)中癌旁组织中PKM2高于癌组织,但大部分有血管混入存在;剩余15对(37.5%)中癌组织中与癌旁组织均增高,统计学分析无差异(P>0.05)。3.2 PKM2基因及PKM1基因在正常肝组织、肝硬化、不同分化HCC组织中的表达:PKM1在正常肝组织中阳性表达,主要表达在正常肝细胞浆中,少部分在肝细胞膜;PKM1在肝硬化组织有弱阳性表达;在不同分化的肝癌组织中基本无阳性表达。相反,PKM2在正常肝组织中为阴性表达,在肝硬化组织中呈弱表达,在不同肝癌组织中的表达均为阳性。3.3 PKM2基因在肝癌石蜡标本中的表达与临床资料的关系、预后分析:共收集兰州大学第二医院2014年1月至2017年8月86个HCC石蜡标本,实验中观察到PKM2主要在HCC细胞浆中表达,少量在HCC细胞核中表达;结合临床常用资料,PKM2基因的高表达与患者的性别、临床症状的有无、HBV-DNA定量、ALT、TBIL、AFP、肿瘤大小、血管侵犯、癌栓等多因素有关,这说明PKM2基因在肝组织的表达与临床的关系密切,但单纯使用做早期筛查的靶点是不具备特异性的,还需要联合检测;从生存分析的统计结果来看,PKM2具有较好的预测HCC预后的价值。结论  PKM2基因在肝癌组织和肝癌细胞株中均呈高表达,在肝癌组织中主要表达在HCC细胞浆;通过敲低及过表达PKM2的表达后,与对照细胞相比,PKM2有促进肝癌细胞恶性增殖,主要阻滞细胞周期在G0/G1期和S期,并有抑制肝癌细胞凋亡的作用;在抑制肝癌细胞凋亡的过程中,可能的机制是通过抑制p-STAT3的活性,由此参入并下调STAT3信号通路,抑制通路下游caspase3、8、9的表达,促进c-myc表达,进而发挥促进肿瘤细胞增殖、抑制细胞凋亡的作用。敲低PKM2表达后有抑制裸鼠成瘤的作用。PKM2在HCC组织中的表达与肝硬化组织、正常肝组织有差异性表达,不同分化程度的HCC组织中PKM2也有不同的表达,分化程度越低,PKM2的表达越高;生存分析显示PKM2有较好的评价HCC患者不良预后的作用,PKM2表达越高是预测HCC的独立危险因素之一。
Other AbstractBackgroundLiver cancer is one of the digestive malignant tumors, the global incidence of mortality accounted for fifth. Hepatocellular Carcinoma (HCC) is the most common pathological type of liver cancer, which is insidious onset, highly aggressive and poor prognosis. Because of above characteristics, the majority of patients were already in the advanced stage. Large volume, portal vein invasion and distant metastasis bring a lot of difficulty treatment. So far, the treatments of HCC have surgery, Transarterial Chemoembolization (TACE), chemotherapy, local radiotherapy and sorafenib, but the treatmental effect of long-term is not satisfied. Meanwhile, HCC lacks other effective tumor markers except AFP in clinical. Therefore, the research on biological targeted therapy of HCC has been paid much attention by researchers. In recent years, Researchers found that glucose metabolism in tumor cells and normal cells are different. Even when oxygen is enough, tumor cells are still anaerobic glycolysis. There are different theories about the mechanism, which plays an important role is a rate limiting enzyme-pyruvate kinase isozyme splice variant (PKs). It has four isomers, M2 is one of them, it has the characteristics of secretion in tumor cells or unlimited reproduction of cells. In recent years, PKM2 (pyruvate kinase isozyme splice variant 2) associated with multiple cell signaling pathways in HCC, but the role of PKM2 in HCC is still not clear.AimsThe aims of present study were to explore the PKM2 expression in human HCC tissues and cells lines, discover the relationship between PKM2 and HCC, and elucidate their molecular mechanism, in order to develop novel diagnostic and prognostic methods and potential therapeutic targets.MethodsPKM2 expression in human HCC and adjacent normal tissues were explored using immunohistochemistry, and HCC cells lines compared with normal liver cell were quantified using qRT-PCR. Next, Lentivirus-mediated RNA interference was applied to knockdown and overexpress PKM2 in HCC97H and HCCLM3. Cell growth was monitored using high content screening. Cell viability was measured by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Cell cycle progression and apoptosis were determined by flow cytometry. Using xenograft model in nude mice to test whether knockdown of PKM2 influenced the tumorigenicity of HCC cells in vivo. The expression of PKM2 in 86 cases of HCC was detected by immunohistochemical method. The relationship between PKM2 expression and clinical pathological parameters, and prognosis of patients were subsequentially analyzed.ResultsThe expression levels of PKM2 in patients with HCC and hepatic carcinoma cell lines were higher than the healthy tissues and normal liver cells. PKM2 can enhance the cell proliferation and resistance to apoptosis in the HCC. Meanwhile, PKM2 can block cell cycles into the G0/G1 and S phase. Furthermore, PKM2 can enhance the ability of HCC colony formation. The expression level of PKM2 was significantly high in the cytoplasm in HCC. Kaplan-Meier analysis revealed that PKM2 expression was strongly associated with survival in HCC patients (P<0.05). The[JY1]  high expression of PKM2 patients was significantly shorter survival time than those with low expression of PKM2 (hazard ratio for death, 1.14; 95% confidence interval, 0.92 - 1.37, P = 0.012).ConclusionsThrough knockdown and over expression, it was found that PKM2 can promote the growth and metastasis, anti-apoptosis of hepatocellular carcinoma cell lines. These functions of PKM2 may contribute to the interactions between STAT3 phosphorylation and apoptosis-related factors. We concluded that PKM2 expression in HCC tissue samples could be used as a prognosis factor for patients with HCC and the highly PKM2 expression was correlated with poor prognosis of HCC patients. [JY1]P=0.000是个什么数字?BackgroundLiver cancer is one of the digestive malignant tumors, the global incidence of mortality accounted for fifth. Hepatocellular Carcinoma (HCC) is the most common pathological type of liver cancer, which is insidious onset, highly aggressive and poor prognosis. Because of above characteristics, the majority of patients were already in the advanced stage. Large volume, portal vein invasion and distant metastasis bring a lot of difficulty treatment. So far, the treatments of HCC have surgery, Transarterial Chemoembolization (TACE), chemotherapy, local radiotherapy and sorafenib, but the treatmental effect of long-term is not satisfied. Meanwhile, HCC lacks other effective tumor markers except AFP in clinical. Therefore, the research on biological targeted therapy of HCC has been paid much attention by researchers. In recent years, Researchers found that glucose metabolism in tumor cells and normal cells are different. Even when oxygen is enough, tumor cells are still anaerobic glycolysis. There are different theories about the mechanism, which plays an important role is a rate limiting enzyme-pyruvate kinase isozyme splice variant (PKs). It has four isomers, M2 is one of them, it has the characteristics of secretion in tumor cells or unlimited reproduction of cells. In recent years, PKM2 (pyruvate kinase isozyme splice variant 2) associated with multiple cell signaling pathways in HCC, but the role of PKM2 in HCC is still not clear.AimsThe aims of present study were to explore the PKM2 expression in human HCC tissues and cells lines, discover the relationship between PKM2 and HCC, and elucidate their molecular mechanism, in order to develop novel diagnostic and prognostic methods and potential therapeutic targets.MethodsPKM2 expression in human HCC and adjacent normal tissues were explored using immunohistochemistry, and HCC cells lines compared with normal liver cell were quantified using qRT-PCR. Next, Lentivirus-mediated RNA interference was applied to knockdown and overexpress PKM2 in HCC97H and HCCLM3. Cell growth was monitored using high content screening. Cell viability was measured by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Cell cycle progression and apoptosis were determined by flow cytometry. Using xenograft model in nude mice to test whether knockdown of PKM2 influenced the tumorigenicity of HCC cells in vivo. The expression of PKM2 in 86 cases of HCC was detected by immunohistochemical method. The relationship between PKM2 expression and clinical pathological parameters, and prognosis of patients were subsequentially analyzed.ResultsThe expression levels of PKM2 in patients with HCC and hepatic carcinoma cell lines were higher than the healthy tissues and normal liver cells. PKM2 can enhance the cell proliferation and resistance to apoptosis in the HCC. Meanwhile, PKM2 can block cell cycles into the G0/G1 and S phase. Furthermore, PKM2 can enhance the ability of HCC colony formation. The expression level of PKM2 was significantly high in the cytoplasm in HCC. Kaplan-Meier analysis revealed that PKM2 expression was strongly associated with survival in HCC patients (P<0.05). The[JY1]  high expression of PKM2 patients was significantly shorter survival time than those with low expression of PKM2 (hazard ratio for death, 1.14; 95% confidence interval, 0.92 - 1.37, P = 0.012).ConclusionsThrough knockdown and over expression, it was found that PKM2 can promote the growth and metastasis, anti-apoptosis of hepatocellular carcinoma cell lines. These functions of PKM2 may contribute to the interactions between STAT3 phosphorylation and apoptosis-related factors. We concluded that PKM2 expression in HCC tissue samples could be used as a prognosis factor for patients with HCC and the highly PKM2 expression was correlated with poor prognosis of HCC patients. [JY1]P=0.000是个什么数字?BackgroundLiver cancer is one of the digestive malignant tumors, the global incidence of mortality accounted for fifth. Hepatocellular Carcinoma (HCC) is the most common pathological type of liver cancer, which is insidious onset, highly aggressive and poor prognosis. Because of above characteristics, the majority of patients were already in the advanced stage. Large volume, portal vein invasion and distant metastasis bring a lot of difficulty treatment. So far, the treatments of HCC have surgery, Transarterial Chemoembolization (TACE), chemotherapy, local radiotherapy and sorafenib, but the treatmental effect of long-term is not satisfied. Meanwhile, HCC lacks other effective tumor markers except AFP in clinical. Therefore, the research on biological targeted therapy of HCC has been paid much attention by researchers. In recent years, Researchers found that glucose metabolism in tumor cells and normal cells are different. Even when oxygen is enough, tumor cells are still anaerobic glycolysis. There are different theories about the mechanism, which plays an important role is a rate limiting enzyme-pyruvate kinase isozyme splice variant (PKs). It has four isomers, M2 is one of them, it has the characteristics of secretion in tumor cells or unlimited reproduction of cells. In recent years, PKM2 (pyruvate kinase isozyme splice variant 2) associated with multiple cell signaling pathways in HCC, but the role of PKM2 in HCC is still not clear.AimsThe aims of present study were to explore the PKM2 expression in human HCC tissues and cells lines, discover the relationship between PKM2 and HCC, and elucidate their molecular mechanism, in order to develop novel diagnostic and prognostic methods and potential therapeutic targets.MethodsPKM2 expression in human HCC and adjacent normal tissues were explored using immunohistochemistry, and HCC cells lines compared with normal liver cell were quantified using qRT-PCR. Next, Lentivirus-mediated RNA interference was applied to knockdown and overexpress PKM2 in HCC97H and HCCLM3. Cell growth was monitored using high content screening. Cell viability was measured by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Cell cycle progression and apoptosis were determined by flow cytometry. Using xenograft model in nude mice to test whether knockdown of PKM2 influenced the tumorigenicity of HCC cells in vivo. The expression of PKM2 in 86 cases of HCC was detected by immunohistochemical method. The relationship between PKM2 expression and clinical pathological parameters, and prognosis of patients were subsequentially analyzed.ResultsThe expression levels of PKM2 in patients with HCC and hepatic carcinoma cell lines were higher than the healthy tissues and normal liver cells. PKM2 can enhance the cell proliferation and resistance to apoptosis in the HCC. Meanwhile, PKM2 can block cell cycles into the G0/G1 and S phase. Furthermore, PKM2 can enhance the ability of HCC colony formation. The expression level of PKM2 was significantly high in the cytoplasm in HCC. Kaplan-Meier analysis revealed that PKM2 expression was strongly associated with survival in HCC patients (P<0.05). The[JY1]  high expression of PKM2 patients was significantly shorter survival time than those with low expression of PKM2 (hazard ratio for death, 1.14; 95% confidence interval, 0.92 - 1.37, P = 0.012).ConclusionsThrough knockdown and over expression, it was found that PKM2 can promote the growth and metastasis, anti-apoptosis of hepatocellular carcinoma cell lines. These functions of PKM2 may contribute to the interactions between STAT3 phosphorylation and apoptosis-related factors. We concluded that PKM2 expression in HCC tissue samples could be used as a prognosis factor for patients with HCC and the highly PKM2 expression was correlated with poor prognosis of HCC patients. [JY1]P=0.000是个什么数字?BackgroundLiver cancer is one of the digestive malignant tumors, the global incidence of mortality accounted for fifth. Hepatocellular Carcinoma (HCC) is the most common pathological type of liver cancer, which is insidious onset, highly aggressive and poor prognosis. Because of above characteristics, the majority of patients were already in the advanced stage. Large volume, portal vein invasion and distant metastasis bring a lot of difficulty treatment. So far, the treatments of HCC have surgery, Transarterial Chemoembolization (TACE), chemotherapy, local radiotherapy and sorafenib, but the treatmental effect of long-term is not satisfied. Meanwhile, HCC lacks other effective tumor markers except AFP in clinical. Therefore, the research on biological targeted therapy of HCC has been paid much attention by researchers. In recent years, Researchers found that glucose metabolism in tumor cells and normal cells are different. Even when oxygen is enough, tumor cells are still anaerobic glycolysis. There are different theories about the mechanism, which plays an important role is a rate limiting enzyme-pyruvate kinase isozyme splice variant (PKs). It has four isomers, M2 is one of them, it has the characteristics of secretion in tumor cells or unlimited reproduction of cells. In recent years, PKM2 (pyruvate kinase isozyme splice variant 2) associated with multiple cell signaling pathways in HCC, but the role of PKM2 in HCC is still not clear.AimsThe aims of present study were to explore the PKM2 expression in human HCC tissues and cells lines, discover the relationship between PKM2 and HCC, and elucidate their molecular mechanism, in order to develop novel diagnostic and prognostic methods and potential therapeutic targets.MethodsPKM2 expression in human HCC and adjacent normal tissues were explored using immunohistochemistry, and HCC cells lines compared with normal liver cell were quantified using qRT-PCR. Next, Lentivirus-mediated RNA interference was applied to knockdown and overexpress PKM2 in HCC97H and HCCLM3. Cell growth was monitored using high content screening. Cell viability was measured by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Cell cycle progression and apoptosis were determined by flow cytometry. Using xenograft model in nude mice to test whether knockdown of PKM2 influenced the tumorigenicity of HCC cells in vivo. The expression of PKM2 in 86 cases of HCC was detected by immunohistochemical method. The relationship between PKM2 expression and clinical pathological parameters, and prognosis of patients were subsequentially analyzed.ResultsThe expression levels of PKM2 in patients with HCC and hepatic carcinoma cell lines were higher than the healthy tissues and normal liver cells. PKM2 can enhance the cell proliferation and resistance to apoptosis in the HCC. Meanwhile, PKM2 can block cell cycles into the G0/G1 and S phase. Furthermore, PKM2 can enhance the ability of HCC colony formation. The expression level of PKM2 was significantly high in the cytoplasm in HCC. Kaplan-Meier analysis revealed that PKM2 expression was strongly associated with survival in HCC patients (P<0.05). The[JY1]  high expression of PKM2 patients was significantly shorter survival time than those with low expression of PKM2 (hazard ratio for death, 1.14; 95% confidence interval, 0.92 - 1.37, P = 0.012).ConclusionsThrough knockdown and over expression, it was found that PKM2 can promote the growth and metastasis, anti-apoptosis of hepatocellular carcinoma cell lines. These functions of PKM2 may contribute to the interactions between STAT3 phosphorylation and apoptosis-related factors. We concluded that PKM2 expression in HCC tissue samples could be used as a prognosis factor for patients with HCC and the highly PKM2 expression was correlated with poor prognosis of HCC patients. [JY1]P=0.000是个什么数字?BackgroundLiver cancer is one of the digestive malignant tumors, the global incidence of mortality accounted for fifth. Hepatocellular Carcinoma (HCC) is the most common pathological type of liver cancer, which is insidious onset, highly aggressive and poor prognosis. Because of above characteristics, the majority of patients were already in the advanced stage. Large volume, portal vein invasion and distant metastasis bring a lot of difficulty treatment. So far, the treatments of HCC have surgery, Transarterial Chemoembolization (TACE), chemotherapy, local radiotherapy and sorafenib, but the treatmental effect of long-term is not satisfied. Meanwhile, HCC lacks other effective tumor markers except AFP in clinical. Therefore, the research on biological targeted therapy of HCC has been paid much attention by researchers. In recent years, Researchers found that glucose metabolism in tumor cells and normal cells are different. Even when oxygen is enough, tumor cells are still anaerobic glycolysis. There are different theories about the mechanism, which plays an important role is a rate limiting enzyme-pyruvate kinase isozyme splice variant (PKs). It has four isomers, M2 is one of them, it has the characteristics of secretion in tumor cells or unlimited reproduction of cells. In recent years, PKM2 (pyruvate kinase isozyme splice variant 2) associated with multiple cell signaling pathways in HCC, but the role of PKM2 in HCC is still not clear.AimsThe aims of present study were to explore the PKM2 expression in human HCC tissues and cells lines, discover the relationship between PKM2 and HCC, and elucidate their molecular mechanism, in order to develop novel diagnostic and prognostic methods and potential therapeutic targets.MethodsPKM2 expression in human HCC and adjacent normal tissues were explored using immunohistochemistry, and HCC cells lines compared with normal liver cell were quantified using qRT-PCR. Next, Lentivirus-mediated RNA interference was applied to knockdown and overexpress PKM2 in HCC97H and HCCLM3. Cell growth was monitored using high content screening. Cell viability was measured by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Cell cycle progression and apoptosis were determined by flow cytometry. Using xenograft model in nude mice to test whether knockdown of PKM2 influenced the tumorigenicity of HCC cells in vivo. The expression of PKM2 in 86 cases of HCC was detected by immunohistochemical method. The relationship between PKM2 expression and clinical pathological parameters, and prognosis of patients were subsequentially analyzed.ResultsThe expression levels of PKM2 in patients with HCC and hepatic carcinoma cell lines were higher than the healthy tissues and normal liver cells. PKM2 can enhance the cell proliferation and resistance to apoptosis in the HCC. Meanwhile, PKM2 can block cell cycles into the G0/G1 and S phase. Furthermore, PKM2 can enhance the ability of HCC colony formation. The expression level of PKM2 was significantly high in the cytoplasm in HCC. Kaplan-Meier analysis revealed that PKM2 expression was strongly associated with survival in HCC patients (P<0.05). The[JY1]  high expression of PKM2 patients was significantly shorter survival time than those with low expression of PKM2 (hazard ratio for death, 1.14; 95% confidence interval, 0.92 - 1.37, P = 0.012).ConclusionsThrough knockdown and over expression, it was found that PKM2 can promote the growth and metastasis, anti-apoptosis of hepatocellular carcinoma cell lines. These functions of PKM2 may contribute to the interactions between STAT3 phosphorylation and apoptosis-related factors. We concluded that PKM2 expression in HCC tissue samples could be used as a prognosis factor for patients with HCC and the highly PKM2 expression was correlated with poor prognosis of HCC patients. [JY1]P=0.000是个什么数字?BackgroundLiver cancer is one of the digestive malignant tumors, the global incidence of mortality accounted for fifth. Hepatocellular Carcinoma (HCC) is the most common pathological type of liver cancer, which is insidious onset, highly aggressive and poor prognosis. Because of above characteristics, the majority of patients were already in the advanced stage. Large volume, portal vein invasion and distant metastasis bring a lot of difficulty treatment. So far, the treatments of HCC have surgery, Transarterial Chemoembolization (TACE), chemotherapy, local radiotherapy and sorafenib, but the treatmental effect of long-term is not satisfied. Meanwhile, HCC lacks other effective tumor markers except AFP in clinical. Therefore, the research on biological targeted therapy of HCC has been paid much attention by researchers. In recent years, Researchers found that glucose metabolism in tumor cells and normal cells are different. Even when oxygen is enough, tumor cells are still anaerobic glycolysis. There are different theories about the mechanism, which plays an important role is a rate limiting enzyme-pyruvate kinase isozyme splice variant (PKs). It has four isomers, M2 is one of them, it has the characteristics of secretion in tumor cells or unlimited reproduction of cells. In recent years, PKM2 (pyruvate kinase isozyme splice variant 2) associated with multiple cell signaling pathways in HCC, but the role of PKM2 in HCC is still not clear.AimsThe aims of present study were to explore the PKM2 expression in human HCC tissues and cells lines, discover the relationship between PKM2 and HCC, and elucidate their molecular mechanism, in order to develop novel diagnostic and prognostic methods and potential therapeutic targets.MethodsPKM2 expression in human HCC and adjacent normal tissues were explored using immunohistochemistry, and HCC cells lines compared with normal liver cell were quantified using qRT-PCR. Next, Lentivirus-mediated RNA interference was applied to knockdown and overexpress PKM2 in HCC97H and HCCLM3. Cell growth was monitored using high content screening. Cell viability was measured by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Cell cycle progression and apoptosis were determined by flow cytometry. Using xenograft model in nude mice to test whether knockdown of PKM2 influenced the tumorigenicity of HCC cells in vivo. The expression of PKM2 in 86 cases of HCC was detected by immunohistochemical method. The relationship between PKM2 expression and clinical pathological parameters, and prognosis of patients were subsequentially analyzed.ResultsThe expression levels of PKM2 in patients with HCC and hepatic carcinoma cell lines were higher than the healthy tissues and normal liver cells. PKM2 can enhance the cell proliferation and resistance to apoptosis in the HCC. Meanwhile, PKM2 can block cell cycles into the G0/G1 and S phase. Furthermore, PKM2 can enhance the ability of HCC colony formation. The expression level of PKM2 was significantly high in the cytoplasm in HCC. Kaplan-Meier analysis revealed that PKM2 expression was strongly associated with survival in HCC patients (P<0.05). The high expression of PKM2 patients was significantly shorter survival time than those with low expression of PKM2 (hazard ratio for death, 1.14; 95% confidence interval, 0.92 - 1.37, P = 0.012).ConclusionsThrough knockdown and over expression, it was found that PKM2 can promote the growth and metastasis, anti-apoptosis of hepatocellular carcinoma cell lines. These functions of PKM2 may contribute to the interactions between STAT3 phosphorylation and apoptosis-related factors. We concluded that PKM2 expression in HCC tissue samples could be used as a prognosis factor for patients with HCC and the highly PKM2 expression was correlated with poor prognosis of HCC patients.
Pages91
URL查看原文
Language中文
Document Type学位论文
Identifierhttp://ir.lzu.edu.cn/handle/262010/341864
Collection生命科学学院
Affiliation生命科学学院
First Author AffilicationSchool of Life Sciences
Recommended Citation
GB/T 7714
赵睿. M2型丙酮酸激酶(PKM2)与肝细胞癌恶性增生及预后相关的功能研究[D]. 兰州. 兰州大学,2019.
Files in This Item:
There are no files associated with this item.
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Altmetrics Score
Google Scholar
Similar articles in Google Scholar
[赵睿]'s Articles
Baidu academic
Similar articles in Baidu academic
[赵睿]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[赵睿]'s Articles
Terms of Use
No data!
Social Bookmark/Share
No comment.
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.