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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。
Objective To investigate the radiosensitizing effect of leucine-repeat immunoglobulin-like protein 1 (LRIG1) on U251 cells in vitro and its mechanism. Methods U251 cells were infected with pLenti-TO-LRIG1-V5 and empty vector pLenti-TO-V5 by lentivirus, and 2Gy irradiated cells were selected after stable infection. Cell proliferation was detected by CCK-8 assay, cell clone formation assay was performed by plate clone formation assay, invasion ability of U251 cells was detected by Transwell assay, apoptosis was detected by flow cytometry, and DNA damage was detected by γ-H2AX immunofluorescence assay Western blot was used to detect the expression of related proteins. Results After U251-LRIG1 cells were irradiated, the proliferation, invasion and colony formation ability of U251-Vector cells were decreased, apoptosis was increased, and the focal point of γ-H2AX fluorescence staining was increased (P <0.05). The expression of EGFR, N-Cadherin, Vimentin, DNA-PKcs, Beclin-1 and LC3II / I decreased and the expression of E-Cadherin increased in U251 cells infected with LRIG1 lentivirus (P <0.05). Conclusion Overexpression of LRIG1 in U251 cells can inhibit the malignant biological behavior of U251 cells by down-regulating the transfection of EGFR, inhibiting DNA-PKcs and autophagy and increasing the radiosensitivity.