摘要
目的探讨丝胶对PI3K/Akt/mTOR信号通路的调控及其对三阴性乳腺癌(TNBC)MDA-MB-468细胞自噬的诱导作用,并阐明作用靶点。方法将MDA-MB-468细胞随机分为对照组(不含药物)、实验组(8 mg·mL^(-1)丝胶)、Akt激活剂组(8 mg·mL^(-1)丝胶+10μmol·L^(-1) SC79),分别给予相应药物作用24 h后,采用RT-qPCR法检测各组细胞PI3K、Akt、mTOR、自噬效应蛋白(Beclin1)、自噬相关蛋白5(ATG5)、微管相关轻链蛋白3(LC3)、螯合体1(P62)mRNA表达水平;Western blotting法检测各组细胞PI3K、Akt、p-Akt、mTOR、p-mTOR、Beclin1、ATG5、LC3、P62蛋白表达水平;免疫荧光染色法检测各组细胞LC3、P62蛋白的定位及荧光强度。结果与对照组相比,实验组MDA-MB-468细胞PI3K、Akt、mTOR、P62 mRNA及PI3K、Akt、p-Akt、mTOR、p-mTOR、P62蛋白水平均显著降低(P<0.05),Beclin1、ATG5、LC3 mRNA水平及Beclin1、ATG5、LC3Ⅱ/LC3Ⅰ蛋白水平均显著升高(P<0.05);与实验组相比,Akt激活剂组MDA-MB-468细胞PI3K、Akt、mTOR、P62 mRNA及PI3K、Akt、p-Akt、mTOR、p-mTOR、P62蛋白水平均显著升高(P<0.05),Beclin1、ATG5、LC3 mRNA水平及Beclin1、ATG5、LC3Ⅱ/LC3Ⅰ蛋白水平均显著降低(P<0.05)。结论丝胶对MDA-MB-468细胞自噬的诱导作用可通过以Akt为靶点调控PI3K/Akt/mTOR信号通路的转导来实现。
Objective To investigate the induction of autophagyofMDA-MB-468 cells in triple-negative breast cancer(TNBC)by sericin through regulating thephosphatidylinositol 3-kinase(PI3K)/protein kinaseB(Akt)/mammalian target of rapamycin(mTOR)signaling pathway,and to clarify the target of action.Methods MDA-MB-468 cells were randomly divided into a control group(without drugs),an experimental group(8 mg·mL^(-1) sericin),and anAkt activator group(8 mg·mL^(-1) sericin+10μmol·L^(-1) SC79).After 24 hours treatment with corresponding drugs,the mRNA expression levels of PI3K,Akt,mTOR,autophagy effector protein(Beclin1),autophagy associated protein 5(ATG5),microtubule associated light chain protein3(LC3)and chelator 1(P62)in cells of each group were detected by RT qPCR.The protein expression levels of PI3K,Akt,p-Akt,mTOR,p-mTOR,Beclin1,ATG5,LC3 and P62 in cells of each group were detected by Western blotting.The localization and fluorescence intensity of LC3 and P62 proteins in cells of each group were detected by immunofluorescence staining.Results Compared with the control group,the mRNA levels of PI3K,Akt,mTOR,and P62 and the protein levels of PI3K,Akt,p-Akt,mTOR,p-mTOR,and P62 in MDA-MB-468 cells of the experimental group were significantly decreased(P<0.05),while the mRNA levels of Beclin1,ATG5,LC3 and the protein levels of Beclin1,ATG5,LC3Ⅱ/LC3Ⅰwere significantly increased(P<0.05).Compared with the experimental group,the mRNA levels of PI3K,Akt,mTOR,P62 and the protein levels of PI3K,Akt,p-Akt,mTOR,p-mTOR,P62 in MDA-MB-468 cells of theAkt activator group were significantly increased(P<0.05),while the mRNA levels of Beclin1,ATG5,LC3 and the protein levels of Beclin1,ATG5,LC3Ⅱ/LC3Ⅰwere significantly decreased(P<0.05).Conclusion The induction effect ofsericin on autophagy ofMDA-MB-468 cells can be achieved by targetingAkt to regulate the transduction of the PI3K/Akt/mTOR signaling pathway.
作者
李警耀
张睿
金美琪
陈志宏
LI Jingyao;ZHANG Rui;JIN Meiqi;CHEN Zhihong(School of Basic Medical Sciences,Chengde Medical University,Chengde,Hebei,067000,China)
出处
《承德医学院学报》
2025年第6期456-462,共7页
Journal of Chengde Medical University
基金
国家自然科学基金项目(81441133)
2025年承德医学院硕士在读研究生创新能力培养资助项目(CYCXZZ202501)。
