肝癌HepG2细胞株胰岛素样生长因子1和表皮生长因子受体基因沉默后放疗增敏及机制

Radiosensitivity and mechanisms of epidermal growth factor receptor and insulin-like growth factor-1 receptor blockaded by siRNA therapy for human hepatocellular cancer Hep G2

目的：探讨同时沉默胰岛素样生长因子1（IGFl）和表皮生长因子（EGF）二受体基因的抗瘤放疗增敏及机制。方法：分别构建靶向EGFR、IGFlR及两受体的小分子干扰RNA（siRNA-EGFR、siRNA-IGFIR、siRNA-EGFR＆IGFlR），构建与任何基因无同源的阴性对照质粒（siRNA-HK），分别转染48照射肝癌HepG2细胞株。成克隆实验测定细胞存活分数，采用单击多靶模型和线性二次函数模型拟合细胞存活曲线，求出放射生物学参数D0、Dq、N和cc、口、SFa值。流式细胞仪检测细胞周期和凋亡变化，细胞周期及相关蛋白、细胞凋亡及相关蛋白采用蛋白印迹。结果：同时沉默EGFR和IGFlR组与对照组及单基因干扰组相比抑制了细胞增殖、诱导细胞凋亡，阻滞细胞周期G1／S期的进程，bcl-2和cdk2表达明显下调，而p21和bax表达增加。且同时沉默EGFR和IGFlR组和对照组细胞的D。值分别为0．588、2．0704Gy；Dq值分别为0．5625、2．0307Gy；a值分别为0．515、0．0216Gy^-1。；SF2值分别为0．22、0．619。同时沉默EGFR和IGFIR组Do、Dq和SF2值均减小，a值增大。结论：同时沉默EGFR和IGFlR基因具有更有效地干扰肝癌HepG2细胞株增殖、诱导细胞凋亡，其机制可能与bcl-2和cdk2表达下调和p21与bax表达增加有关，结合干扰多个受体分子可能是一种十分有前途的新的肝癌治疗途经。

Objective： To investgate the radiosensitivity and mechanisms of inhibiting insulin-like growth factor-1 receptor （IGFIR） and epidermal growth factor receptor （EGFR） signaling in hepatocellular cancer （HCC） cells by siRNA simultaneous blockade o{ both IGFIR and EGFR. Methods： The growth, cell cycle, cellular apoptosis, and the related proteins incl.uding bcl-2, cdk2, bax and p21 were analyzed by flow cytometry and Western blotting. Clonogenic assay was performed to determine the survival fraction. The parameters Do, Dq and N for the single-hit multi-target model and the parameters a,13 and SF2 for the linear-quadratic model were calculated. Results： After transfection for 48 hours, both silencing EGFR and IGF-1R inhibited the transition of the cells from G1 to S phases, and up-regulated the expressions of bax and p21 and down-regulated expression of bcl-2 and cdk2. Both silencing EGFR and IGF-1R cells had a Do parameter of 0. 588 Gy, a Dq parameter of 0. 562 5Gy, a parameter of 0. 515 Gy 1 and a SF2 parameter of 0. 22, Control ceils had a Do parameter of 2. 070 CGy, a Dqpa- rameter of 2. 030 7Gv, a parameter of 0. 021 6 Gy-1 and a SF2 parameter of 0. 619. Compared with human control HCC cells, decreased Do, Dq, SF2 and an increased value were seen in HCC of both silencing EGFR and IGFR. Conclusion： siRNA simultaneous blockade of EGFR and IGFIR inhibit proliferation of HepGz by inhibiting their G1/S phase transition via upregulated bax and p21 and down-regulated cdk2 and bcl-2; on the other hand, it induces apoptosis in human HCC cell lines Hep G2 cells. Thus, combination of IGFIR and EGFR inhibition may be a promising novel treatment in HCC.