【作者】 陈伟；

【导师】 刘付宝；

【作者基本信息】 安徽医科大学 ， 外科学（普外）， 2023， 硕士

【摘要】 背景:肝癌仍然是一个全球性的健康挑战,其中肝细胞肝癌(Hepatocellular Carcinoma,HCC)作为全球癌症相关死亡率的第四大原因,约占肝癌比例的90%,各种因素,包括黄曲霉素B1暴露、HBV和HCV感染、AALD、肥胖、NAFLD和血色病,以及遗传疾病(如肿瘤抑制因子或者癌症基因突变),均可促进HCC的发生。在我国,虽然HCC的治疗更加多样化和个体化,但5年生存率仍难以提高。大多数HCC确诊时已属晚期,因此需要阐明HCC发生发展的重要分子机制和判断HCC的预后,从而制定HCC防治的新策略。NME2(non-metastatic cells2),也被称为NM23-H2或NDPKB,位于染色体17q21上,编码核苷二磷酸激酶B亚基。NME2是一种参与基因转录调控的转移相关蛋白,在恶性肿瘤中存在差异表达,可能参与了癌症的病理过程。但NME2在HCC中的报道较少,我们前期通过生物信息学挖掘得出NME2在HCC中高表达,并且NME2的表达与自噬的调控有关。为了进一步阐明NME2对HCC恶性行为的影响和机制,本研究以NME2为出发点,探究NME2在HCC中所发挥的作用及其作用机制。研究目的:(1)明确NME2在HCC患者中的表达以及与临床预后的关系。(2)体内体外验证NME2在HCC中的作用。(3)探究NME2对HCC自噬的影响及其作用机制。研究方法:(1)免疫组化染色评估组织芯片中96例HCC患者癌和癌旁NME2表达量。完善96例HCC患者随访数据。(2)通过小干扰RNA和慢病毒转染构建NME2敲除肝癌细胞株Huh7和Hep G2。Western blot验证NME2成功敲除后,通过细胞克隆和MTT增殖验证NME2敲除对HCC细胞增殖的影响。(3)构建NME2敲除和过表达的Huh7细胞系,用于裸鼠皮下肿瘤形成。15只健康的裸鼠随机分为3组:对照组、NME2敲除组和NME2过表达组,每组5只。观察NME2在体内对肝癌增殖的影响。(4)Western blot检测敲除NME2后Huh7细胞中LC3B、Beclin-1、P62、ATG5的变化。(5)利用激光共聚焦显微镜和透射电镜观察NME2敲除对HCC细胞自噬体和自溶酶体的变化。(6)采用免疫组化方法验证裸鼠肿瘤组织中NME2与LC3B蛋白的相关性。(7)体外验证NME2对自噬相关通路蛋白的影响,并进行反向验证。研究结果:(1)NME2在HCC患者中相对高表达,与患者5年生存率呈负相关。(2)在Huh7和Hep G2中敲除NME2可以抑制HCC细胞的增殖与克隆。(3)NME2在体内裸鼠移植瘤实验中可以促进肿瘤的增殖(4)NME2敲低可以上调Huh7细胞LC3B(Ⅰ和Ⅱ)蛋白表达,氯喹(CQ)联合NME2敲低比单独NME2敲低诱导更多LC3B(Ⅰ和Ⅱ)蛋白表达。(5)在激光共聚焦显微镜和透射电镜下发现,与对照组相比,敲除NME2可使Huh7细胞自噬体和自噬溶酶体数量增加。(6)CQ可以逆转NME2敲低对Huh7细胞增殖的影响。(7)NME2过表达的细胞和裸鼠肿瘤组织中LC3B蛋白表达降低。(8)NME2可以调控4EBP1磷酸化水平,并且4EBP1 Thr37/46位点的突变抵消了NME2敲除对HCC细胞自噬激活和抑制增殖的能力。结论:(1)NME2在HCC中高表达,与患者预后呈负相关,并且具有促进肿瘤增殖的功能。(2)NME2可以通过激活自噬促进HCC细胞增殖。(3)P-4EBP1介导了NME2对HCC自噬和增殖的调控。更多还原

【Abstract】 Background: Liver cancer remains a global health challenge.Hepatocellular Carcinoma(HCC)is the fourth leading cause of cancer-related mortality worldwide,accounting for about 90% of liver cancer.These include aflatoxin B1 exposure,HBV and HCV infections,AALD,obesity,NAFLD,and hemochromatosis,as well as genetic disorders such as tumor suppressor or cancer gene mutations,which promote HCC development.In China,although the treatment of HCC is more diversified and individualized,it is still difficult to improve the 5-year survival rate.Most HCC is diagnosed at an advanced stage.Therefore,it is necessary to clarify the important molecular mechanisms of HCC occurrence and development and to judge the prognosis of HCC,to develop new strategies for HCC prevention and treatment.NME2(non-metastatic cells2),also known as NM23-H2 or NDPKB,is located on chromosome 17q21 and encodes the nucleoside diphosphate kinase B subunit.NME2 is a metastasis-associated protein involved in the regulation of gene transcription.It is differentially expressed in malignant tumors and may be involved in the pathological process of cancer.However,there are few reports of NME2 in HCC.We previously found that NME2 is highly expressed in HCC through bioinformatics mining,and the expression of NME2 is related to the regulation of autophagy.To further elucidate the effect and mechanism of NME2 on the malignant behavior of HCC,the present study used NME2 as the starting point to explore the role and mechanism of NME2 in HCC.Objective:(1)To clarify the expression of NME2 in HCC patients and its relationship with clinical prognosis(2)To verify the role of NME2 in HCC in vivo and in vitro.(3)To explore the effect and mechanism of NME2 on autophagy in HCC.Methods:(1)Immunohistochemical staining was used to evaluate the expression of NME2 in cancer and para cancer in 96 HCC patients.Follow-up data of 96 HCC patients were completed.(2)NME2 knockout hepatoma cell lines Huh7 and Hep G2 were constructed by transfection of small interfering RNA and lentivirus.After successful NME2 knockout was verified by Western blot,the effect of NME2 knockout on HCC cell proliferation was verified by cell cloning and MTT proliferation.(3)Construct NME2-knockout and overexpressed Huh7 cell lines for subcutaneous tumor formation in nude mice.Fifteen healthy nude mice were randomly divided into 3 groups: control group,NME2 knockout group,and NME2 overexpression group,with 5 mice in each group.To observe the effect of NME2 on hepatocellular carcinoma proliferation in vivo.(4)The changes of LC3 B,Beclin-1,P62 and ATG5 in Huh7 cells after NME2 knockout were detected by Western blot.(5)The effects of NME2 knockout on the autophagosome and autolysosome of HCC cells were observed by laser confocal microscopy and transmission electron microscopy.(6)Immunohistochemical method was used to verify the correlation between NME2 and LC3 B protein in tumor tissue of nude mice.(7)The effect of NME2 on autophagy-related pathway proteins was verified in vitro and reversed.Results:(1)The relatively high expression of NME2 in HCC patients was negatively correlated with the 5-year survival rate of patients.(2)Knockdown of NME2 in Huh7 and Hep G2 inhibited the proliferation and colony formation of HCC cells.(3)NME2could promote the proliferation of Huh7 cells in vivo.(4)NME2 knockdown could up-regulate the expression of LC3B(Ⅰ and Ⅱ)protein in Huh7 cells.Chloroquine(CQ)combined with NME2 knockdown induced more LC3B(Ⅰ and Ⅱ)protein expression than NME2 knockdown alone.(5)Compared with the control group,NME2 knockout increased the number of autophagosomes and autolysosomes in Huh7 cells under laser confocal microscopy and transmission electron microscopy.(6)Chloroquine reversed the effect of NME2 knockdown on the proliferation of Huh7 cells.(7)The expression of LC3 B protein was decreased in NME2 overexpression cells and tumor tissues of nude mice.(8)The phosphorylation of 4EBP1 was regulated by NME2,and4EBP1 mutation at Thr37/46 counteracted the ability of NME2 knockout to activate autophagy and inhibit proliferation in HCC cellsConclusion:(1)NME2 has high expression in HCC,and negatively correlated with the prognosis of patients,and has the function of promoting tumor proliferation.(2)NME2 promoted the proliferation of HCC cells by activating autophagy.(3)P-4EBP1 mediates the regulation of autophagy and proliferation by NME2 in HCC.更多还原