背景:胃癌(GC)是一种常见的恶性肿瘤,RNA结合蛋白聚嘧啶束结合蛋白1(PTBP1)已被确定为各种肿瘤类型的关键因素。此外,异常的自噬水平已被证明显著影响肿瘤发生和进展。尽管如此,PTBP1在GC自噬调控中的精确调控机制尚不清楚。
方法:为了评估PTBP1在GC中的表达,我们采用了一种利用蛋白质印迹的综合方法,实时定量聚合酶链反应(RT-qPCR),和生物信息学分析。为了进一步鉴定GC细胞中与PTBP1结合的下游靶基因,我们利用RNA免疫沉淀联合测序(si-PTBP1RNA-seq).评估PTBP1对胃癌发生的影响,我们进行了CCK-8检测,集落形成试验,和GC异种移植小鼠模型测定。此外,我们用透射电子显微镜,免疫荧光,流式细胞术,westernblot,RT-qPCR,和GC异种移植小鼠模型实验,以阐明PTBP1调节GC自噬的具体机制。
结果:我们的发现表明,与邻近的正常组织相比,PTBP1在GC组织中明显过表达。沉默PTBP1导致自噬体的异常积累,从而在体外和体内抑制GC细胞活力。机械上,干扰PTBP1促进硫氧还蛋白相互作用蛋白(TXNIP)mRNA的稳定性,导致TXNIP介导的氧化应激增加。因此,溶酶体功能受损,最终导致自噬通量的阻塞。此外,我们的结果表明,干扰PTBP1增强了氯喹的抗肿瘤作用,在体外和体内。
结论:PTBP1敲低通过直接结合TXNIPmRNA并促进其表达而损害GC进展。基于这些结果,PTBP1成为GC的有希望的治疗靶标。
BACKGROUND: Gastric cancer (GC) is a prevalent malignant tumor, and the RNA-binding protein polypyrimidine tract-binding protein 1 (PTBP1) has been identified as a crucial factor in various tumor types. Moreover, abnormal autophagy levels have been shown to significantly impact tumorigenesis and progression. Despite this, the precise regulatory mechanism of PTBP1 in autophagy regulation in GC remains poorly understood.
METHODS: To assess the expression of PTBP1 in GC, we employed a comprehensive approach utilizing western blot, real-time quantitative polymerase chain reaction (RT-qPCR), and bioinformatics analysis. To further identify the downstream target genes that bind to PTBP1 in GC cells, we utilized RNA immunoprecipitation coupled with sequencing (si-PTBP1 RNA-seq). To evaluate the impact of PTBP1 on gastric carcinogenesis, we conducted CCK-8 assays, colony formation assays, and GC xenograft mouse model assays. Additionally, we utilized a transmission electron microscope, immunofluorescence, flow cytometry, western blot, RT-qPCR, and GC xenograft mouse model experiments to elucidate the specific mechanism underlying PTBP1\'s regulation of autophagy in GC.
RESULTS: Our findings indicated that PTBP1 was significantly overexpressed in GC tissues compared with adjacent normal tissues. Silencing PTBP1 resulted in abnormal accumulation of autophagosomes, thereby inhibiting GC cell viability both in vitro and in vivo. Mechanistically, interference with PTBP1 promoted the stability of thioredoxin-interacting protein (TXNIP) mRNA, leading to increased TXNIP-mediated oxidative stress. Consequently, this impaired lysosomal function, ultimately resulting in blockage of autophagic flux. Furthermore, our results suggested that interference with PTBP1 enhanced the antitumor effects of
chloroquine, both in vitro and in vivo.
CONCLUSIONS: PTBP1 knockdown impairs GC progression by directly binding to TXNIP mRNA and promoting its expression. Based on these results, PTBP1 emerges as a promising therapeutic target for GC.