PAK基因沉默提高CD133+肝癌肿瘤干细胞放疗敏感性的作用及机制研究

Hepatcellular carcinoma(HCC)is a leading cause of global cancer death.Curative therapy is not an option for most patients,often because of underlying liver disease.Experience in radiation therapy for HCC is rapidly increasing.The cancer stem cells(CSC) have gained intense interest as key tumor initiating cells that may also play an intergral role in recurrence following chemo- and rediotherapy. In our previous study,we demonstrated that distribution of CD133+cells in G2/M cell cycle arrest was lower than that of CD133-cells,and the anti-apoptotic activity of CD133+cells was significantly higher than that of CD133-cells postirradiation.The in vivo study with a xenograft model showed increased tumor formation in irradiated CD133+ cell-injected nude mice compared to the CD133- group, suggesting that CD133 contributes to radioresistance in HCC.Microarray analysis showed that the genes involved in cell proliferation were upregulated while the ones inducing apoptosis were down regulated in CD133+ cells,including cell cycle related gene PAK2 and MAPK signaling pathway. In this study,we will use the Si-RNA interference technique to inhibiting PAK2 genes in the CD133+ and CD133-cells,aim to investigate the relationship between PAK2 and cell cycle、cell proliferation、apoptosis after irradiation,revealing the effect of PAK2 in radioresistance on CD133+ HCC cells.Furthermore,we will use of protein array to investigate the machanism of radioresistance in CD133+ HCC cells. This study mey be useful to improve therapeutic effects of radiotherapy for HCC,and useful for the development of novel therapeutic strategies for targeting CSCs in HCC.

原发性肝癌是我国常见恶性肿瘤之一，死亡率高,大部分患者不能接受手术治疗。随着放疗技术的发展，放射治疗在肝癌综合治疗中的应用日益广泛。肿瘤干细胞是恶性肿瘤的起源,与肿瘤的生长、复发、放化疗抵抗有密切相关。P21激活激酶在肿瘤的发展及转移、药物抵抗中起重要作用。在前期研究中我们发现，肝癌细胞Huh7中CD133＋细胞较CD133-呈现高致瘤性及对放疗有抵抗性。进一步基因芯片研究发现，经放射线处理后，CD133+细胞内PAK2基因表达及MAPK信号通路激活高于CD133-细胞组。本研究将进一步通过SiRNA抑制CD133+与CD133- 细胞内PAK2表达，研究对放射线照射诱导细胞周期、细胞增殖、细胞凋亡的影响，明确肝癌肿瘤干细胞中PAK2与CD133+肝癌干细胞放射线抵抗中的作用，并利用蛋白芯片技术，阐明其中重要信号传导通路，为提高肝癌放疗效果及开发肝癌干细胞靶向治疗新技术奠定理论基础。

P21激活激酶2（PAK2）在肿瘤发生、发展、转移中起重要作用。我们在前期研究工作中发现，肝癌细胞株Huh7 CD133+细胞较CD133-细胞对放射线处理具有抵抗性，PAK2-RNA在细胞中表达明显高于CD133-细胞群，在经放射线处理后PAK2 RNA在CD133+细胞群中表达升高，而在CD133-细胞群中表达下降。本研究目的是通过抑制PAK2表达对放射线处理CD133+肝癌肿瘤干细胞的细胞凋亡、细胞周期、细胞增殖的调控作用及对信号传导通路激活的调控作用，证明PAK2基因在CD133+细胞放射线抵抗中的作用及其作用机制。研究中，我们首先用RNA干扰技术制作了永久抑制PAK2表达的Huh-7 ShPAK2细胞系。进一步研究发现，抑制PAK2表达的CD133+细胞经放射线照射处理48小时后生存率较对照干扰组生存率明显下降。细胞周期研究中发现，抑制PAK2表达导致抑制PAK2表达的CD133+细胞内G2/M阻滞，subG1期细胞比例无明显变化，经放射线处理24小时后subG1期细胞比例（16%）较对照组（5%）明显增加。另抑制PAK2表达的CD133+细胞经放射线照射24小时后诱导细胞凋亡为8.17%，较对照组（3.32%）显著升高。进一步我们用蛋白电泳方法测定了ERK的表达，发现ERK蛋白高表达于放射线处理的ShPAK2 CD133+ 细胞组。本研究证实了降低PAK2基因可提高肝癌细胞系Huh-7 CD133+肿瘤干细胞的细胞周期、细胞凋亡，可提高放射线诱导的CD133+细胞的细胞凋亡，增加对放射线的敏感性，其机制可能是通过ERK信号通路的激活。PAK2可能成为肝癌肿瘤干细胞靶向治疗新靶点,为肝癌放射治疗及综合治疗的发展奠定了一定的理论基础。