【作者】 王菲；

【导师】 尹震宇；

【作者基本信息】 厦门大学 ， 微生物学， 2017， 硕士

【摘要】 背景原发性肝癌在世界范围内都有很高的发病率与死亡率,是常见的恶性肿瘤之一,肝癌的发生具有早期症状不明显,后期发展迅速的特点,这为肝癌的早期治疗提高了难度,随着医学的进步,尽管肝癌的诊断与治疗都有了很大进步,但肝癌患者的总体生存率还是不理想,所以寻找新的治疗靶点越来越迫切。近年来关于自噬在肿瘤发生发展中的研究越来越多,自噬在肿瘤的形成、增殖、转移以及能量代谢等诸多方面具有调节作用。自噬活性在肝癌的发生发展中起着重要作用。LAPTM4B(Lysosomal Transmembrane Protein4β)在肿瘤中发挥多种生物学功能,如促进肿瘤细胞的增殖、迁移、侵袭、抑制细胞的凋亡等,LAPTM4B还能诱导蒽环类药物的胞浆滞留,减少药物对DNA的损伤。LAPTM4B降解会影响溶酶体膜的稳定性,参与细胞自噬过程,在自噬体成熟过程中发挥重要作用,通过促进自噬能增加细胞对饥饿等应激状态的耐受性。但关于LAPTM4B如何在肝癌细胞中调控自噬知之甚少,因此在肝癌细胞中进行了LAPTM4B与自噬的相关研究。方法与结果1、利用qPCR检测了60对临床肝癌样本中LAPTM4B的表达,结果表明LAPTM4B在癌组织中明显高于癌旁组织。2、构建LAPTM4B的沉默与过表达稳转肝癌细胞系,检测LAPTM4B对肝癌细胞增殖能力的影响。实验表明LAPTM4B的表达量与肝癌细胞的增殖能力呈正相关。3、通过western blot,免疫荧光等检测LAPTM4B对肝癌细胞自噬的影响。结果表明在细胞处于饥饿状态下时LAPTM4B能够增加自噬水平。4、通过qPCR与western blot实验检测干扰LAPTM4B表达后自噬相关蛋白表达量的改变。结果发现LAPTM4B会影响自噬相关蛋白ATG3的表达,二者表达量呈正相关。并且发现在60对临床肝癌样本中LAPTM4B与ATG3具有显著相关性。5、在肝癌细胞中LAPTM4B通过调控ATG3对自噬产生影响。在过表达LAPTM4B细胞中沉默ATG3细胞自噬水平下降,在沉默LAPTM4B的细胞中过表达ATG3自噬水平会有所恢复。结论LAPTM4B在肝癌细胞不仅能增加细胞的增殖能力而且在饥饿条件下还会促进肝癌细胞自噬的发生,试验中通过验证LAPTM4B表达量与多种自噬相关蛋白表达的关系,发现LAPTM4B通过调控ATG3实现在肝癌细胞中对自噬水平的影响。更多还原

【Abstract】 BackgroundHepatocellular carcinoma(HCC)is one of the most common malignances with high morbidity and mortality worldwide.There are not obvious symptoms in the early stage of HCC.Although advancement of the diagnosis and treatment of HCC have been made,the overall survival rate of patients with HCC remains poor.Therefore,it is urgently needed to identify novel therapeutic targets.Autophagy play crucial role in the turmorgenesis and development of cancers.It has been reported that autophagy can regulate the growth,metastasis and energy metabolism of HCC.LAPTM4B(Lysosomal Transmembrane Protein 4 beta)exerts oncogenic activity in tumors,such as promoting cell proliferation,migration,invasion and inhibition of cell apoptosis.LAPTM4B also induce anthracycline intracellular retention,reduce injury drugs on DNA,and affect the stability of the lysosomal membrane.Recent studies demonstrated that LAPTM4B is involved in cell autophagy.However,the underlying molecular mechanisms by which LAPTM4B regulates autophagy remain elucidated.Methods and results1.We performed qPCR to detect the expression of LAPTM4B in 60 pairs of liver cancer samples and found that the expression level of LAPTM4B was significantly higher in tumor tissues than that in paired nontumor tissues.2.We performed gain-and loss-of-function assays to investigate the effect of LAPTM4B on the proliferation of HCC cells.The results showed that LAPTM4B significantly promoted cell proliferation both in vivo and in vitro.3.The effects of LAPTM4B on autophagy in HCC cells were detected by western blot and immunofluorescence assay.The results showed that LAPTM4B could increase starvation-induced autophagy.4.qPCR and western blot assays were used to detect the ATGs expression following LAPTM4B overexpression and knockdown.The results showed that LAPTM4B could affect the expression of ATG3(autophagy related protein).Moreover,the expression of LAPTM4B was positively correlated with ATG3 in tumor tissues.5.LAPTM4B promotes autophagy through regulation of ATG3 in HCC.Silence of ATG3 abolished the promotion of autophagy induced by LAPTM4B upregulation,whereas overexpression of ATG3 rescued autophagy inhibition mediated by LAPTM4B silence.ConclusionLAPTM4B can not only increase the cell proliferation in HCC,but also induce autophagy of HCC.LAPTM4B promotes autophagy in an ATG3-dependent manner in HCC.更多还原