碱韭总黄酮通过m^(6)A-IGF2BP2依赖性途径促进宫颈癌细胞凋亡的机制研究  

Study on Mechanism of Total Flavonoids from Allium polyrhizum Promoting Apoptosis of Cervical Cancer Cells Through m^(6)A-IGF2BP2 Dependent Pathway

作　　者：赵彩权 赵鹏[1] 图雅[1] 白力格 ZHAO Cai-Quan;ZHAO Peng;TU Ya;BAI Li-Ge(College of Ecology and Environment,Baotou Teachers'College,Baotou 014030,China)

机构地区：[1]包头师范学院生态环境学院,包头014030

出　　处：《农业生物技术学报》2024年第10期2344-2360,共17页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32360148);内蒙古自治区自然科学基金(2021BS08004,2021BS08017);包头师范学院高层次人才引进科研启动基金(BTTCRCQD2020-004,BTTCRCQD2020-005)。

摘　　要：宫颈癌(cervical cancer,CC)是妇科恶性肿瘤,大部分黄酮类化合物具有抗宫颈癌活性,然而碱韭(Allium polyrhizum)总黄酮是否也具有抗宫颈癌活性鲜少报道。为揭示碱韭总黄酮对宫颈癌细胞的作用及机制,本研究利用CCK-8(cell counting kit-8)实验、划痕实验、流式细胞术、实时定量PCR、免疫印迹、斑点杂交、N6-甲基腺苷(N6-methyladenine,m^(6)A)定量、甲基化RNA免疫共沉淀(methylated RNA immunoprecipitation,MeRIP)和RNA免疫共沉淀(RNA immunoprecipitation,RIP)等方法,对碱韭总黄酮处理的宫颈癌细胞生长能力进行检测,发现碱韭总黄酮对HeLa、SiHa和CaSki 3种宫颈癌细胞有不同程度的抑制作用,可有效抑制其增殖和迁移能力,同时促进HeLa细胞凋亡。碱韭总黄酮处理HeLa细胞后显著上调其促凋亡蛋白B淋巴细胞瘤-2相关X蛋白(B-cell lymphoma-2 associated X protein,BAX)的表达,全基因组m^(6)A修饰水平显著上升,且与m^(6)A去甲基化酶脂肪量和肥胖相关蛋白(fat mass and obesityassociated protein,FTO)表达显著下调有关;此外,m^(6)A读取蛋白胰岛素样生长因子2 mRNA结合蛋白2(insulin like growth factor 2 mRNA binding protein 2,IGF2BP2)的表达显著上调。生物信息学预测发现,BAX mRNA上有1个极高可信度的m^(6)A修饰位点,而IGF2BP2与此位点结合;同时,m^(6)A抗体和IGF2BP2抗体均能够显著富集BAX mRNA。综上,本研究证明碱韭总黄酮具有良好的抗宫颈癌活性,且可能通过m^(6)A-IGF2BP2相关分子机制促进宫颈癌细胞凋亡而发挥抗癌作用。本研究结果可为黄酮类化合物抗肿瘤机制的相关研究提供理论基础,也为新型药物的开发与应用提供一定的理论参考。Cervical cancer(CC)is a gynecological malignancy,and most of the flavonoids have anti-cervical cancer activities.However,whether the total flavonoids of Allium polyrhizum also have anti-cervical cancer activity is rarely reported.Quantification of cell counting kit-8(CCK-8)assay,scratch assay,flow cytometry,real-time quantitative PCR,immunoblotting,spot hybridization,N6-methyladenine(m^(6)A),methylated RNA immunoprecipitation(MeRIP)and RNA immunoprecipitation(RIP)methods were used to detect the growth of cervical cancer cells treated with total flavonoids of A.polyrhizum,and found that total flavonoids of A.polyrhizum had a significant effect on HeLa,SiHa and CaSki cervical cancer cells,which could effectively inhibit their proliferation and migration,and promote the apoptosis of HeLa cells.After treatment of HeLa cells with A.polyrhizum total flavonoids,the expression of a pro-apoptotic protein of B-cell lymphoma-2 associated X protein(BAX)was significantly up-regulated,and the high level of global m^(6)A modification was related to significant downregulation of m^(6)A demethylase fat mass and obesity-associated protein(FTO)expression.In addition,the expression of the m^(6)A-reading protein insulin like growth factor 2 mRNA binding protein 2(IGF2BP2)was significantly increased.Bioinformatics prediction revealed that there was a high confidence m^(6)A modification site on BAX mRNA,and IGF2BP2 also bound to this site.Similarly,both the m^(6)A antibody and IGF2BP2 antibody could significantly enrich BAX mRNA.In conclusion,this study demonstrated that the total flavonoids of A.polyrhizum had good anti-cervical cancer activity and may played an anticancer role by promoting apoptosis of cervical cancer cells through the m^(6)A-IGF2BP2 related molecular mechanism.Finally,the results of this study could provide a theoretical basis for the related research on the anti-tumor mechanism of flavonoids,as well as some theoretical reference for the development and application of novel drugs.

关 键 词：碱韭 总黄酮 宫颈癌细胞 N6-甲基腺苷(m^(6)A) 细胞凋亡 

分 类 号：S857.4[农业科学—临床兽医学] S58[农业科学—兽医学]