【摘要】 目的研究亮氨酸重复序列免疫球蛋白样蛋白1(LRIG1)体外对U251细胞的放疗增敏作用及其机制。方法将pLenti-TO-LRIG1-V5及空质粒pLenti-TO-V5采用慢病毒法感染U251细胞,筛选出稳定感染细胞株后给予2Gy辐照。采用CCK-8法检测细胞增殖能力,平板克隆形成实验检测细胞克隆形成能力,Transwell法检测U251细胞侵袭能力,流式细胞术检测细胞凋亡情况,γ-H2AX免疫荧光法检测双链DNA损伤情况,Western blot检测相关蛋白的表达。结果U251-LRIG1组细胞接受辐照后较U251-Vector组细胞增殖、侵袭、克隆形成能力降低,细胞凋亡增加,γ-H2AX荧光染色焦点数增多(P<0.05)。U251细胞感染LRIG1慢病毒后,EGFR、N-Cadherin、Vimentin、DNA-PKcs、Beclin-1和LC3II/I表达水平下降,E-Cadherin表达水平增高(P<0.05)。结论在U251细胞中过表达LRIG1可以通过下调EGFR逆转上皮间充质转化,抑制DNA-PKcs及细胞自噬等多种途径抑制U251细胞的恶性生物学行为,增加放疗敏感性。更多还原

【Abstract】 Objective To explore the effect of leucine-rich repeats and immunoglobulin-like domain proteins(LRIG1)on the radiosensitivity of glioblastoma cell line U251 and its possible mechanisms in vitro.Methods The lentiviral vector expressing LRIG1 was transfected into U251 cells.The empty vector transfected U251 cells served as the control.The stably infected cells were screened and received 2 Gy radiation.Cell proliferation was detected using CCK-8assay,colony formation ability was assessed using plate colony forming assay,cell invasion ability was evaluated by transwell assay.Flow cytometry was adopted to detect the apoptosis rate.γ-H2 AX foci were counted on immunofluorescence pictures to evaluate double-stranded DNA damage.The related proteins were examined by Western blot.Results The cell proliferation,invasion and colony forming ability of LRIG1 overexpressing U251cells were decreased with increased apoptosis rate andγ-H2 AX foci(P<0.05).Compared to the control,U251 cells infected with LRIG1-lentivirus dramatically decreased the expressions of LRIG1,EGFR,N-Cadherin,Vimentin,DNA-PKcs,Beclin-1,and LC3II/I,but increased E-Cadherin expression(P<0.05).Conclusion LRIG1 overexpressing U251 cells can inhibit the malignant behavior of U251 and enhance its radiosensitivity by down-regulating EGFR,reversing EMT,suppressing DNA-PKcs expression and autophagy.更多还原