P53-R273H mutation enhances colorectal cancer stemness through regulating specific lncRNAs
P53-R273H mutation enhances colorectal cancer stemness through regulating specific lncRNAs
复制标题
P53-R273H突变通过调节特定lncRNA增强结直肠癌干性
DOI：
10.1186/s13046-019-1375-9
复制
发表时间：
2019-08-28
影响因子：
11.3
通讯作者：
Song, Wei
中科院分区：
文献类型：
--
作者：
Zhao, Yuechao;Li, Yiran;Song, Wei
背景TP53是所有癌症类型中最常突变的基因之一，TP53突变体在结直肠癌(CRC)中发生率超过60%。在所有突变体中，存在三个热点，包括p53-R175H、p53-R248W和p53-R273H。新的证据将癌症的发生归因于癌症干细胞（CSC）。长非编码 RNA (lncRNA) 在维持 CSC 的干性方面发挥着至关重要的作用。然而，尚不清楚突变的p53调控的lncRNA是否与CSC干性的维持有关。方法采用RNA测序（RNA-seq）和ChIP测序（ChIP-seq）来追踪体细胞敲入法产生的HCT116内源性p53点突变球状细胞中p53-R273H调控的lncRNA网络。 RT-qPCR用于检测lncRNA表达模式，验证生物信息学分析。通过Transwell、球体形成、荧光激活细胞分选仪（FACS）、异种移植裸鼠模型、极限稀释分析（ELDA）评估肿瘤频率、蛋白质印迹分析和化疗耐药分析来阐明lnc273–31和lnc273–34在癌症干细胞中的功能和可能机制。结果sp53-R273H比p53-R175H和p53-R175H表现出更多的CSC特征。 p53-R248W。 RNA-seq 分析鉴定出 37 个上调和 4 个下调的差异表达 lncRNA，受 p53-R273H 调节。结合 ChIP-seq 分析，我们进一步验证了两种 lncRNA（名为 lnc273-31 和 lnc273-34）对于维持 CSC 干性至关重要。进一步的研究表明，lnc273-31 或 lnc273-34 的缺失显着降低了结直肠癌的迁移、侵袭、癌症干细胞的自我更新和体外化疗耐药性。此外，lnc273-31 或 lnc273-34 的缺失显着延迟了体内癌症的发生和致瘤细胞频率。此外，lnc273-31 和 lnc273-34 对上皮间质转化 (EMT) 有影响。最后，与野生型 p53 相比，lnc273-31 和 lnc273-34 在 p53-R273H 突变的 CRC 组织中显着高表达。结论本研究揭示了结直肠 CSC 中 p53-R273H 特异性调节的一组高可信度 lncRNA。此外，我们证明其中两个 lnc273-31 和 lnc273-34 是结直肠 CSC 自我更新、肿瘤增殖和化疗耐药所必需的。此外，这两种 lncRNA 的表达在具有 p53-R273H 突变的结直肠癌患者样本中增强。这两种 lncRNA 可能作为 p53-R273H 突变患者的有希望的预测因子，并且对于化疗至关重要。
BackgroundTP53 is one of the most frequently mutated genes among all cancer types, and TP53 mutants occur more than 60% in colorectal cancer (CRC). Among all mutants, there are three hot spots, including p53-R175H, p53-R248W and p53-R273H. Emerging evidence attributes cancer carcinogenesis to cancer stem cells (CSCs). Long noncoding RNAs (lncRNAs) play crucial roles in maintaining the stemness of CSCs. However, it is unknown if mutant p53-regulated lncRNAs are implicated in the maintenance of CSC stemness.MethodsRNA-sequencing (RNA-seq) and ChIP-sequencing (ChIP-seq) were used to trace the lncRNA network regulated by p53-R273H in HCT116 endogenous p53 point mutant spheroid cells generated by the somatic cell knock-in method. RT-qPCR was used to detect lncRNA expression patterns, verifying the bioinformatics analysis. Transwell, spheroid formation, fluorescence activated cell sorter (FACS), xenograft nude mouse model, tumor frequency assessed by extreme limiting dilution analysis (ELDA), Western blot assays and chemoresistance analysis were performed to elucidate the functions and possible mechanism of lnc273–31 and lnc273–34 in cancer stem cells.Resultsp53-R273H exhibited more characteristics of CSC than p53-R175H and p53-R248W. RNA-seq profiling identified 37 up regulated and 4 down regulated differentially expressed lncRNAs regulated by p53-R273H. Combined with ChIP-seq profiling, we further verified two lncRNAs, named as lnc273–31 and lnc273–34, were essential in the maintenance of CSC stemness. Further investigation illustrated that lnc273–31 or lnc273–34 depletion dramatically diminished colorectal cancer migration, invasion, cancer stem cell self-renewal and chemoresistance in vitro. Moreover, the absence of lnc273–31 or lnc273–34 dramatically delayed cancer initiation and tumorigenic cell frequency in vivo. Also, lnc273–31 and lnc273–34 have an impact on epithelial-to mesenchymal transition (EMT). Finally, lnc273–31 and lnc273–34 were significantly highly expressed in CRC tissues with p53-R273H mutation compared to those with wildtype p53.ConclusionsThe present study unveiled a high-confidence set of lncRNAs regulated by p53-R273H specific in colorectal CSCs. Furthermore, we demonstrated that two of them, lnc273–31 and lnc273–34, were required for colorectal CSC self-renewal, tumor propagation and chemoresistance. Also, the expression of these two lncRNAs augmented in colorectal cancer patient samples with p53-R273H mutation. These two lncRNAs may serve as promising predictors for patients with p53-R273H mutation and are vital for chemotherapy.