背景:我们先前的研究首次表明,结合ATR的长链非编码RNA(lncRNA)对于ATR功能是必需的,并促进了癌症抗性。然而,在ATR激活中的特定lncRNAs仍不清楚,这限制了我们对这一关键生物过程的全面理解。
方法:使用RNA免疫沉淀(RIP),然后进行RNA测序来鉴定ATR结合lncRNAs,使用RIP-qPCR测定进一步验证。免疫荧光染色和免疫印迹法检测DNA损伤修复因子的激活。在确定了scaRNA2对DNA损伤试剂的细胞敏感性后,在患者来源的类器官和异种移植临床前模型中研究了scaRNA2对放疗的影响.在从直肠癌患者分离的组织中也验证了scaRNA2的临床相关性。
结果:ScaRNA2被鉴定为最富集的ATR结合lncRNA,并且被发现对于同源重组(HR)介导的DNA损伤修复至关重要。此外,scaRNA2敲低消除了对DNA损伤的ATR及其底物的募集。机械上,观察到scaRNA2对于Exo1介导的DNA末端切除是必需的,并且将MRN复合物与ATR激活桥接。下调scaRNA2可有效提高癌细胞对多种DNA损伤相关放化疗的敏感性。临床前,scaRNA2的敲减改善了放疗对患者来源的类器官和异种移植模型的影响.最后,在对放疗耐药的临床患者中,与ATR共定位的scaRNA2也增加.
结论:ScaRNA2被鉴定为与ATR结合的最丰富的lncRNA,并被证明可以将DNA末端切除与ATR激活桥接;因此,它可以作为癌症联合放化疗治疗的有效靶点。
BACKGROUND: Our previous study first showed that ATR-binding long noncoding RNA (lncRNA) is necessary for ATR function and promotes cancer resistance. However, the specific lncRNAs instrumental in ATR activation remain largely unclear, which limits our comprehensive understanding of this critical biological process.
METHODS: RNA immunoprecipitation (RIP) followed by RNA sequencing was employed to identify ATR-binding lncRNAs, which were further validated using RIP-qPCR assays. Immunofluorescence staining and Western blotting were applied to detect the activation of DNA damage repair factors. After the effect of scaRNA2 on cellular sensitivity to DNA-damaging reagents was determined, the effects of scaRNA2 on radiotherapy were investigated in patient-derived organoids and xenograft preclinical models. The clinical relevance of scaRNA2 was also validated in tissues isolated from rectal cancer patients.
RESULTS: ScaRNA2 was identified as the most enriched ATR-binding lncRNA and was found to be essential for homologous recombination (HR) mediated DNA damage repair. Furthermore, scaRNA2 knockdown abrogated the recruitment of ATR and its substrates in response to DNA damage. Mechanistically, scaRNA2 was observed to be necessary for Exo1-mediated DNA end resection and bridged the MRN complex to ATR activation. Knockdown of scaRNA2 effectively increased the sensitivity of cancer cells to multiple kinds of DNA damage-related chemoradiotherapy. Preclinically, knockdown of scaRNA2 improved the effects of radiotherapy on patient-derived organoids and xenograft models. Finally, an increase in scaRNA2 colocalized with ATR was also found in clinical patients who were resistant to radiotherapy.
CONCLUSIONS: ScaRNA2 was identified as the most abundant lncRNA bound to ATR and was demonstrated to bridge DNA end resection to ATR activation; thus, it could be applied as a potent target for combined cancer treatments with chemoradiotherapy.