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01.12.2019 | Research | Ausgabe 1/2019 Open Access

Journal of Experimental & Clinical Cancer Research 1/2019

P53-R273H mutation enhances colorectal cancer stemness through regulating specific lncRNAs

Zeitschrift:
Journal of Experimental & Clinical Cancer Research > Ausgabe 1/2019
Autoren:
Yuechao Zhao, Yiran Li, Jie Sheng, Fan Wu, Kai Li, Rong Huang, Xiaojuan Wang, Tao Jiao, Xin Guan, Yan Lu, Xiao Chen, Zhiwen Luo, Yanchi Zhou, Hanjie Hu, Wenjie Liu, Boyu Du, Shiying Miao, Jianqiang Cai, Linfang Wang, Hong Zhao, Jianming Ying, Xinyu Bi, Wei Song
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s13046-019-1375-9) contains supplementary material, which is available to authorized users.
Yuechao Zhao and Yiran Li contributed equally to this work.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.
Zusatzmaterial
Additional file 1: Figure S1A. Western blot analysis of HCT116 p53−/− over-expression p53 point mutants. Figure S2A. Schematic diagram of constructing HCT116 endogenous p53 point mutant (PM) cells. Figure S2B. Sanger sequencing of p53-R273H point mutant (R273H PM) representative cells. Figure S3. Global profiling and identification of p53-R273H-regulated lncRNAs. Figure S3A. Different types of transcripts. Figure S3B. Different types of noncoding transcripts. Figure S3C. A two-dimensional heatmap of 1957 lncRNAs. Figure S3D. Principal components analysis (PCA) for three independent replicates. The principal component accounted of p53-R273H and p53-ctrl were 85.7 and 69.6% respectively. Figure S4A. Validation of differentially expressed 41 lncRNAs by RT-qPCR. Figure S5. Genome-wide analysis of p53-R273H-regulated protein coding genes in CSC state. Figure S5A. A two-dimensional heatmap of 307 mRNAs. Figure S5B. Principal components analysis (PCA) for three independent replicates. Figure S5C. Hierarchical clustering for three independent replicates. Figure S5D. Signaling pathway based on KEGG enrichment analysis of p53-R273H-regulated coding genes in CSC state. Figure S5E. GO biological processes enrichment analysis of p53-R273H-regulated coding genes in CSC state. Figure S5F. Regulatory network construction of TFs (dark blue), lncRNAs (red) and mRNAs (light green). The average degree of lncRNAs was 46.39, higher than 35.39, the average degree of protein coding genes. Figure S6A. ChIP-qPCR for validating of the binding of p53 and the promotor of lnc273–31 or lnc273–34. Figure S6B. The expression levels in ALDH positive and ALDH negative cells sorted by FACS. Figure S6C. Subcellular localization of lnc273–31 and lnc273–34 was analyzed by RT-qPCR upon biochemical fractionation of p53-R273H speroid cells. Figure S7A. Quantitative real-time PCR analyzed the expression of stemness-related genes in HCT116 p53 PM cells. Figure S7B. Western blot analysis of ZEB1 and snail in lnc273–31 or lnc273–34 depletion cells. Figure S8. The association of age (S8A), gender (S8B), smoking (S8C), alcohol abuse (S8D), family history (S8E), lymphatic vessel (S8F), TNM stage (S8G-I) and the expression levels of lncRNAs in 25 colorectal cancer patients with or without p53-R273H mutation. Table S1. Primers for qPT-PCR. Table S2. Purchased ASO pool sequences. Table S3. Primers for ChIP-qPCR. (DOCX 2058 kb)
Literatur
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