背景:越来越多的研究证明了环状RNA(circularRNAs,circRNAs)与各种疾病的病理过程以及它们参与多种癌症的发作和进展的关联。然而,circRNAs在胃癌自噬调控中的功能作用和潜在机制尚未完全阐明。
方法:我们使用透射电子显微镜和mRFP-GFP-LC3双荧光自噬指示剂来研究自噬调节。细胞计数试剂盒-8测定,集落形成试验,5-乙炔基-2'-脱氧尿苷掺入测定,Transwell分析,并进行蛋白质印迹分析以确认circPTPN22对GC进展的影响。双重荧光素酶报告基因试验验证了circPTPN22和miR-6788-5p之间的结合,以及miR-6788-5p和p21激活的激酶-1(PAK1)。功能拯救实验评估circPTPN22是否通过竞争性结合miR-6788-5p调节PAK1表达,影响GC细胞的自噬和其他生物学过程。我们使用裸鼠异种移植模型研究了circPTPN22对体内GC肿瘤的影响。生物信息学工具预测了circPTPN22的上游调节转录因子和结合蛋白,而染色质免疫沉淀和核糖核蛋白免疫沉淀测定证实了结合状态。
结果:在GC中上调circPTPN22已被证明可以抑制自噬并促进细胞增殖,迁移,和入侵。机械上,circPTPN22直接结合miR-6788-5p,随后调节PAK1的表达,从而激活蛋白激酶B(Akt)和细胞外信号调节激酶(Erk)的磷酸化。这种调节最终影响GC细胞中的自噬水平。此外,runt相关转录因子1(RUNX1)负调控circPTPN22表达,而RNA结合蛋白如FUS(融合在肉瘤中)和ELAVL1(重组ELAV样蛋白1)正调节其表达。抑制自噬途径可增加FUS表达,进一步上调GC细胞中的circPTPN22,从而加剧GC的进展。
结论:在转录因子RUNX1和RNA结合蛋白FUS和ELAVL1的调控下,circPTPN22通过miR-6788-5p/PAK1轴激活Akt和Erk的磷酸化,从而调节GC细胞中的自噬。抑制自噬增加FUS,进而在PTPN22周围上调,形成正反馈回路,最终加速GC的进展。
BACKGROUND: An increasing number of studies have demonstrated the association of circular RNAs (circRNAs) with the pathological processes of various diseases and their involvement in the onset and progression of multiple cancers. Nevertheless, the functional roles and underlying mechanisms of circRNAs in the autophagy regulation of gastric cancer (GC) have not been fully elucidated.
METHODS: We used transmission electron microscopy and the mRFP-GFP-LC3 dual fluorescent autophagy indicator to investigate autophagy regulation. The cell counting kit-8 assay, colony formation assay, 5-ethynyl-2\'-deoxyuridine incorporation assay, Transwell assay, and Western blot assay were conducted to confirm circPTPN22\'s influence on GC progression. Dual luciferase reporter assays validated the binding between circPTPN22 and miR-6788-5p, as well as miR-6788-5p and p21-activated kinase-1 (PAK1). Functional rescue experiments assessed whether circPTPN22 modulates PAK1 expression by competitively binding miR-6788-5p, affecting autophagy and other biological processes in GC cells. We investigated the impact of circPTPN22 on in vivo GC tumors using a nude mouse xenograft model. Bioinformatics tools predicted upstream regulatory transcription factors and binding proteins of circPTPN22, while chromatin immunoprecipitation and ribonucleoprotein immunoprecipitation assays confirmed the binding status.
RESULTS: Upregulation of circPTPN22 in GC has been shown to inhibit autophagy and promote cell proliferation, migration, and invasion. Mechanistically, circPTPN22 directly binds to miR-6788-5p, subsequently regulating the expression of PAK1, which activates protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) phosphorylation. This modulation ultimately affects autophagy levels in GC cells. Additionally, runt-related transcription factor 1 (RUNX1) negatively regulates circPTPN22 expression, while RNA-binding proteins such as FUS (fused in sarcoma) and ELAVL1 (recombinant ELAV-like protein 1) positively regulate its expression. Inhibition of the autophagy pathway can increase FUS expression, further upregulating circPTPN22 in GC cells, thereby exacerbating the progression of GC.
CONCLUSIONS: Under the regulation of the transcription factor RUNX1 and RNA-binding proteins FUS and ELAVL1, circPTPN22 activates the phosphorylation of Akt and Erk through the miR-6788-5p/PAK1 axis, thereby modulating autophagy in GC cells. Inhibition of autophagy increases FUS, which in turn upregulates circPTPN22, forming a positive feedback loop that ultimately accelerates the progression of GC.