Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, 100005, China.
Department of Hepatobiliary Surgery, State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100021, China.
J Exp Clin Cancer Res. 2019 Aug 28;38(1):379. doi: 10.1186/s13046-019-1375-9.
TP53 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.
RNA-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.
p53-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.
The 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.
TP53 是所有癌症类型中突变最频繁的基因之一，在结直肠癌（CRC）中，TP53 突变超过 60%。在所有突变中，有三个热点，包括 p53-R175H、p53-R248W 和 p53-R273H。新出现的证据将癌症发生归因于癌症干细胞（CSC）。长链非编码 RNA（lncRNA）在维持 CSC 的干性方面起着至关重要的作用。然而，目前尚不清楚突变型 p53 调节的 lncRNA 是否参与 CSC 干性的维持。
使用 RNA 测序（RNA-seq）和染色质免疫沉淀测序（ChIP-seq）来追踪由体细胞敲入方法生成的内源性 p53 点突变球体细胞中 p53-R273H 调节的 lncRNA 网络。使用 RT-qPCR 检测 lncRNA 表达模式，验证生物信息学分析。进行 Transwell、球体形成、荧光激活细胞分选（FACS）、异种移植裸鼠模型、通过极端极限稀释分析（ELDA）评估肿瘤频率、Western blot 检测和化疗耐药性分析，以阐明 lnc273-31 和 lnc273-34 在癌症干细胞中的功能和可能机制。
p53-R273H 比 p53-R175H 和 p53-R248W 表现出更多的 CSC 特征。RNA-seq 分析鉴定出 37 个上调和 4 个下调的差异表达的受 p53-R273H 调节的 lncRNA。结合 ChIP-seq 分析，我们进一步验证了两个 lncRNA，命名为 lnc273-31 和 lnc273-34，对 CSC 干性的维持至关重要。进一步的研究表明，lnc273-31 或 lnc273-34 的耗竭显著减少了体外结直肠癌细胞迁移、侵袭、癌症干细胞自我更新和化疗耐药性。此外，lnc273-31 或 lnc273-34 的缺失显著延迟了体内癌症起始和肿瘤发生细胞频率。此外，lnc273-31 和 lnc273-34 对上皮-间充质转化（EMT）有影响。最后，lnc273-31 和 lnc273-34 在结直肠癌组织中表达明显高于野生型 p53 的 p53-R273H 突变。
本研究揭示了一组受 p53-R273H 特异性结直肠 CSC 调节的高可信度 lncRNA。此外，我们证明了其中的两个，lnc273-31 和 lnc273-34，对于结直肠 CSC 的自我更新、肿瘤增殖和化疗耐药性是必需的。此外，在结直肠癌患者样本中，这些两个 lncRNA 的表达在 p53-R273H 突变中增加。这两个 lncRNA 可能作为 p53-R273H 突变患者的有前途的预测因子，并对化疗至关重要。
