背景:非小细胞肺癌(NSCLC)的进展受环状RNA(circularRNAs,circRNAs)的显著影响,尤其是在肿瘤缺氧微环境中。然而,NSCLC中circRNAs失调的确切功能和潜在机制仍未被研究.
方法:通过高通量RNA测序鉴定NSCLC组织中差异表达的circRNAs。通过Sanger测序严格确认了circ_0007386的特征,RNaseR处理和放线菌素D处理。使用CCK8,克隆形成试验研究了circ_0007386对增殖和凋亡的影响,TUNEL染色,和体外流式细胞术测定。在体内,异种移植肿瘤模型用于评估其对增殖的影响。机械上,通过双荧光素酶报告基因测定和拯救实验,检测了circ_0007386,miR-383-5p和CIRBP的调控关系.此外,我们通过RNA免疫沉淀法检测了EIF4A3与CRIM1pre-mRNA的结合,以及在低氧条件下通过免疫共沉淀法检测了YAP1与EIF4A3之间的相互作用.
结果:我们的调查揭示了一种新的circRNA,命名为circ_0007386,其在NSCLC组织和细胞系中上调。Circ_0007386在体外和体内调节NSCLC中的增殖和凋亡。功能上,circ_0007386充当miR-383-5p的海绵,TargetingCIRBP,通过PI3K/AKT信号通路影响NSCLC细胞增殖和凋亡。此外,在缺氧条件下,YAP1和EIF4A3之间的相互作用增强,导致EIF4A4与CRIM1前mRNA结合。这促进了CRIM1pre-mRNA的反向剪接,增加circ_0007386的形成。circ_0007386/miR-383-5p/CIRBP轴与NSCLC患者的临床特征和预后显著相关。
结论:Circ_0007386,在低氧条件下受YAP1-EIF4A3相互作用调节,通过miR-383-5p/CIRBP轴在NSCLC进展中发挥致癌作用。
BACKGROUND: The progression of non-small cell lung cancer (NSCLC) is significantly influenced by circular RNAs (circRNAs), especially in tumor hypoxia microenvironment. However, the precise functions and underlying mechanisms of dysregulated circRNAs in NSCLC remain largely unexplored.
METHODS: Differentially expressed circRNAs in NSCLC tissues were identified through high-throughput RNA sequencing. The characteristics of circ_0007386 were rigorously confirmed via Sanger sequencing, RNase R treatment and actinomycin D treatment. The effects of circ_0007386 on proliferation and apoptosis were investigated using CCK8, cloning formation assays, TUNEL staining, and flow cytometry assays in vitro. In vivo, xenograft tumor models were used to evaluate its impact on proliferation. Mechanistically, the regulatory relationships of circ_0007386, miR-383-5p and CIRBP were examined through dual luciferase reporter assays and rescue experiments. Additionally, we detected the binding of EIF4A3 to CRIM1 pre-mRNA by RNA immunoprecipitation and the interaction between YAP1 and EIF4A3 under hypoxic conditions by co-immunoprecipitation.
RESULTS: Our investigation revealed a novel circRNA, designated as circ_0007386, that was upregulated in NSCLC tissues and cell lines. Circ_0007386 modulated proliferation and apoptosis in NSCLC both in vitro and in vivo. Functionally, circ_0007386 acted as a sponge for miR-383-5p, targeting CIRBP, which influenced NSCLC cell proliferation and apoptosis via the PI3K/AKT signaling pathway. Furthermore, under hypoxic conditions, the interaction between YAP1 and EIF4A3 was enhanced, leading to the displacement of EIF4A4 from binding to CRIM1 pre-mRNA. This facilitated the back-splicing of CRIM1 pre-mRNA, increasing the formation of circ_0007386. The circ_0007386/miR-383-5p/CIRBP axis was significantly associated with the clinical features and prognosis of NSCLC patients.
CONCLUSIONS: Circ_0007386, regulated by YAP1-EIF4A3 interaction under hypoxia conditions, plays an oncogenic role in NSCLC progression via the miR-383-5p/CIRBP axis.