E2F5调控自噬在α粒子辐射诱发人支气管上皮细胞恶性转化中的作用OA
E2F5 mediated autophagy in α-particle radiation-induced malignant transformation of human bronchial epithelial cells
目的:探讨E2F转录因子5(E2F5)通过调控细胞自噬在α粒子辐射诱发人支气管上皮细胞恶性转化中的作用.方法:以永生化人支气管上皮BEP2D细胞、α粒子辐射后第24代早期转化RH24细胞及恶性转化BERP35T-1细胞为研究对象.采用Western blot法检测3种细胞内E2F5蛋白的表达水平.选择恶性程度最高的BERP35T-1细胞,将其分为5组,分别为空白对照组、阴性对照组、雷帕霉素(Rapa)组、E2F5沉默组和E2F5沉默联合Rapa组.其中E2F5沉默组和E2F5沉默联合Rapa组细胞采用终浓度10 nmol/L的E2F5 siRNA转染24 h,含Rapa组加入终浓度25 pmol/L自噬激活剂Rapa作用24 h,阴性对照组转染等浓度无义序列.采用Western blot法检测自噬标志蛋白LC3B表达水平;CCK-8法、细胞划痕实验及Transwell侵袭实验分别检测细胞活力、划痕闭合率与侵袭细胞数量.结果:与BEP2D细胞相比,RH24和BERP35T-1细胞中E2F5蛋白表达均显著上调(P<0.01).沉默E2F5后,BERP35T-1细胞内LC3B-Ⅱ/Ⅰ比值由1.00±0.04降至0.51±0.04,细胞活力由(97.01±1.99)%降至(34.96±1.26)%,划痕后24h划痕闭合率由(19.18±0.17)%降至(5.61±2.16)%、划痕后48h由(54.17±2.72)%降至(13.14±3.20)%,穿膜细胞数由(225.67±8.14)个降至(42.67±0.58)个(P<0.01);而Rapa干预可逆转上述现象,使LC3B-Ⅱ/Ⅰ比值由0.69±0.02升至1.43±0.05,细胞活力由(69.44±4.45)%升至(100.73±4.28)%,划痕后24h划痕闭合率由(7.36±1.51)%升至(13.82±1.02)%、划痕后48h划痕闭合率由(22.84±0.13)%升至(39.11±0.61)%,穿膜细胞数由(90.67±7.77)个升至(241.67±44.86)个(P<0.05或P<0.01).结论:在α粒子辐射诱发BEP2D细胞恶性转化过程中,E2F5蛋白表达显著上调;其高表达可能通过激活细胞自噬,增强细胞的增殖、迁移和侵袭能力,进而促进α粒子辐射诱发的细胞恶性转化进程.
OBJECTIVE:To investigate the role of E2F transcription factor 5(E2F5)in regulating autophagy during α-particle radiation-induced malignant transformation of human bronchial epithelial cells.METHODS:Immortalized human bronchial epithelial BEP2D cells,early transformed RH24 cells(the 24 th generation after α-particle irradiation),and malignant transformed BERP35T-1 cells were used.Western blot was performed to detect E2F5 protein expression in the three cell lines.BERP35T-1 cells with the highest malignancy were selected and divided into five groups:blank control,negative control,rapamycin(Rapa),E2F5 silencing,and E2F5 silencing combined with Rapa.The E2F5 silencing and combined groups were transfected with E2F5 siRNA at a final concentration of 10 nmol/L for 24 h;the negative control group was transfected with an equal concentration of negative control sequence;and the Rapa groups were treated with 25 pmol/L autophagy activator rapamycin for 24 h.Western blot was used to detect the autophagy marker protein LC3B expression.Cell viability,wound healing rate,and the number of invasive cells were detected by CCK-8,wound healing,and Transwell invasion assays,respectively.RESULTS:Compared with BEP2D cells,E2F5 protein expression was significantly upregulated in both RH24 and BERP35T-1 cells(P<0.01).After E2F5 silencing in BERP35T-1 cells,the LC3B-Ⅱ/Ⅰ ratio decreased from 1.00±0.04 to 0.51±0.04,cell viability dropped from(97.01±1.99)%to(34.96±1.26)%,the wound healing rate at 24 h decreased from(19.18±0.17)%to(5.61±2.16)%,at 48 h from(54.17±2.72)%to(13.14±3.20)%,and the number of invaded cells decreased from 225.67±8.14 to 42.67±0.58(all P<0.01).Rapamycin intervention reversed these effects,as evidenced by an increase in LC3B-Ⅱ/Ⅰ ratio from 0.69±0.02 to 1.43±0.05,cell viability from(69.44±4.45)%to(100.73±4.28)%,wound healing rate at 24 h from(7.36±1.51)%to(13.82±1.02)%,at 48 h from(22.84±0.13)%to(39.11±0.61)%,and the number of invaded cells from 90.67±7.77 to 241.67±44.86(P<0.05 or P<0.01).CONCLUSION:During α-particle radiation-induced malignant transformation of BEP2D cells,E2F5 protein expression was significantly upregulated.Increased E2F5 activated autophagy,thereby enhancing cell viability,migration,and invasion,and consequently promoting the process of α-particle radiation-induced malignant transformation.
杨佳文;邵帅;李辰;王成芳;周密;曲功霖;苟巧
中国疾病预防控制中心辐射防护与核安全医学所,辐射防护与核应急中国疾病预防控制中心重点实验室,北京 100088中国疾病预防控制中心辐射防护与核安全医学所,辐射防护与核应急中国疾病预防控制中心重点实验室,北京 100088中国疾病预防控制中心辐射防护与核安全医学所,辐射防护与核应急中国疾病预防控制中心重点实验室,北京 100088中国疾病预防控制中心辐射防护与核安全医学所,辐射防护与核应急中国疾病预防控制中心重点实验室,北京 100088中国疾病预防控制中心辐射防护与核安全医学所,辐射防护与核应急中国疾病预防控制中心重点实验室,北京 100088中国疾病预防控制中心辐射防护与核安全医学所,辐射防护与核应急中国疾病预防控制中心重点实验室,北京 100088中国疾病预防控制中心辐射防护与核安全医学所,辐射防护与核应急中国疾病预防控制中心重点实验室,北京 100088
医药卫生
E2F转录因子5自噬α粒子人支气管上皮细胞恶性转化肺癌
E2F5autophagyalpha particlehuman bronchial epithelial cellmalignant transformationlung cancer
《癌变·畸变·突变》 2026 (4)
261-269,9
国家自然科学基金(81000862)
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