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Glucose deprivation enhances translation of long non-coding RNA SNHG15-encoded micropeptide that sustains colorectal cancer cell viability

포도당 결핍 조건에서 SNHG15 유래 마이크로펩타이드 번역과 이에 따른 대장암 세포 생존의 관한 연구

초록/요약 도움말

Solid tumors, including colorectal cancer （CRC）, are exposed to a glucose-deprived microenvironment due to rapid proliferation and insufficient vascularization. Under these conditions, most cancer cells undergo cell death, whereas a subset adapts to metabolic stress to sustain survival. However, the molecular mechanisms underlying this adaptive response remain incompletely understood. Long non-coding RNAs （lncRNAs） are known to exhibit dynamic expression changes under stress conditions and have been implicated in cellular adaptation. However, it remains unclear whether these functions are mediated by the RNA molecules themselves or by micropeptides encoded within lncRNAs. In this study, I identified lncRNAs that are commonly upregulated under glucose deprivation through integrative RNA-seq analysis across glioblastoma, lung, renal, pancreatic, and colorectal cancer cell lines. Among these, SNHG15 and LINC00662 were selected as candidate lncRNAs based on transcript features associated with translation, including 5′ cap and poly（A） tail structures, cytoplasmic localization, and ribosome association. To investigate the mechanism underlying their induction under glucose deprivation, ATF4 ChIP-qPCR analysis was performed and revealed that the increased expression of SNHG15 and LINC00662 is mediated by ATF4 binding to their promoter regions. Reanalysis of publicly available mass spectrometry datasets identified peptide evidence for SNHG15 ORF2, indicating that SNHG15 encodes a micropeptide. Subsequent Ribo-seq analysis demonstrated increased translation efficiency of SNHG15 ORF2 under glucose deprivation. This translation was further experimentally validated using a FLAG-tagged SNHG15 ORF2 expression construct and shown to be regulated in a 5′UTR-dependent manner. Furthermore, overexpression of the SNHG15-derived micropeptide increased cell viability in HCT116 cells under glucose starvation conditions. RNA-seq analysis revealed that overexpression of the SNHG15-derived micropeptide was associated with reduced expression of apoptosis-related genes. In addition, scRNA-seq analysis （GSE132465） of colorectal cancer patient tumors showed that tumor cells with high SNHG15 expression exhibited transcriptional features associated with metabolic stress, suggesting a potential association between SNHG15 expression and metabolic stress-related states in colorectal tumors. Collectively, this study suggests that the SNHG15-derived micropeptide may function as a potential regulator involved in the adaptation and survival of colorectal cancer cells under glucose deprivation conditions.

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목차 도움말

1. Introduction 1
2. Material and Methods 4
2.1 Cell cultures 4
2.2 RNA isolation and RT-qPCR 4
2.3 Western blot analysis 5
2.4 Chromatin immunoprecipitation （ChIP） 5
2.5 immunofluorescence （IF） 6
2.6 RNA-seq and bioinformatic analyses 7
2.7 Polysome profiling 8
2.8 Ribo-seq and bioinformatic analyses 9
2.9 Cell survival analysis using IncuCyte live-cell imaging 10
2.10 MS/MS-reanalysis 11
2.11 ChIP-seq and bioinformatic analyses 11
2.12 Sequence conservation analysis 12
2.13 Subcellular fractionation 12
2.14 Single-cell RNA-seq analysis 13
2.15 Site-Directed Mutagenesis 14
2.16 Statistical analysis 15
3. Results 16
3.1 Identification of lncRNAs with translational potential under glucose starvation 16
3.2 ATF4-mediated transcriptional regulation of SNHG15 and LINC00662 under glucose starvation 20
3.3 Identification of a translated ORF2 within SNHG15 under glucose starvation 24
3.4 Experimental validation of SNHG15 ORF2 translation and its 5′UTR-dependent regulation under glucose starvation 28
3.5 Characterization of SNHG15-high tumor epithelial cells through single-cell analysis 32
3.6 SNHG15-derived micropeptide enhances cell survival under glucose starvation 36
4. Discussion 40
5. Reference 46

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