目的:采用RNA干扰和皮下成瘤技术,研究苯并(α)芘(BaP)通过热休克蛋白90(HSP90)和低氧诱导因子-1(HIF-1)对分子伴侣介导的自噬(CMA)的影响。
方法:40只裸鼠前肢腋下接种沉默的HSP90A549细胞系,分为4组,用1.80mg/kg/dBaP-玉米油溶液灌胃60d(对照组除外),记录裸鼠和移植瘤的生长曲线。通过小动物成像技术观察肿瘤的大小和形态变化。qPCR,免疫印迹法和免疫组化法检测HSP90的表达,HSC70和Lamp-2A。A549细胞用0.1μmol/L处理,1μmol/L和10μmol/LBaP持续24小时,ELISA法和Westernblot法检测EPO、HIF-1α浓度和HIF-1α蛋白表达;用10μmol/LBaP和HIF-1α抑制剂处理A549细胞24h,qPCR,免疫印迹法和免疫荧光法检测HSP90α的表达,HSC70和Lamp-2A。
结果:BaP降低了沉默HSP90的裸鼠和移植瘤的重量;BaP降低了沉默HSP90的移植瘤组织中的HSC70,Lamp-2AmRNA和蛋白质;BaP降低了沉默HSP90的移植瘤的生物发光光子总数。10μmol/LBaP可使A549细胞中EPO和HIF-1α的浓度及HIF-1α的蛋白表达增加;HIF-1α抑制剂处理后,HSP90ā,A549细胞HSC70、Lamp-2AmRNA和蛋白表达及HSP90α荧光强度降低。
结论:BaP促进裸鼠移植瘤的生长,当HSP90α沉默时被抑制。BaP促进CMA的发生,可能通过促进HSP90α和HIF-1α的表达,是BaP激活CMA的重要调控基因。
OBJECTIVE: The effects of benzo (α) pyrene (BaP) on chaperone mediated autophagy (CMA) through heat shock protein 90 (HSP90) and hypoxia- inducible factor-1 (HIF-1) are studied by RNA interference and subcutaneous tumor formation technique in nude mice.
METHODS: 40 nude mice that were inoculated with the silenced HSP90ɑ A549 cells line under the armpits of the forelimbs were divided into 4 groups, and were intragastrically administered with 1.80 mg/kg/d BaP-corn oil solutionfor for 60d (except the Control group), and the growth curves of nude mice and transplanted tumors were recorded. The size and morphological changes of tumors were observed by small animal imaging technique. qPCR, Western blot and Immunohistochemistry were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A. A549 cells were treated with 0.1 μmol/L, 1 μmol/L and 10 μmol/L BaP for 24 h, EPO and HIF-1ɑ concentration and HIF-1ɑ protein expression were detected by Elisa and Western blot; A549 cells were treated with 10 μmol/L BaP and HIF-1ɑ inhibitor for 24 h, qPCR, Western blot and Immunofluorescence methods were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A.
RESULTS: The weight of nude mice and transplanted tumors silenced HSP90ɑ was reduced by BaP; the expression of HSP90ɑ, HSC70, Lamp-2A mRNA and proteins in transplanted tumor tissues silenced HSP90ɑ were reduced by BaP; the total number of bioluminescence photons of transplanted tumors silenced HSP90ɑ were reduced by BaP. The concentration of EPO and HIF-1ɑ and the expression of HIF-1ɑ protein in A549 cells was increased by 10 μmol/L BaP; with HIF-1ɑ inhibitors treated, HSP90ɑ, HSC70, Lamp-2A mRNA and proteins expression and the fluorescence intensity of HSP90ɑ were decreased of A549 cells.
CONCLUSIONS: The growth of transplanted tumor in nude mice is promoted by BaP, and is inhibited when HSP90ɑ was silenced. BaP promotes the occurrence of CMA by promoting the expression of HSP90ɑ and HIF-1ɑ, which are vital regulatory genes of BaP activation of CMA.