AbstractBackgroundColorectal cancer (CRC) is a fatal malignant tumor with poor prognosis. Cancer stem cells (CSCs) can cause metastasis, recurrence and drug resistance in CRC. This research aimed to analyze stemness‐related prognostic genes of CRC based on single‐cell RNA‐sequencing (scRNA‐seq) data.MethodsDESeq2 was applied to analyze the differentially expressed genes (DEGs). The mRNA stemness index (mRNAsi) was calculated by one‐class logistic regression (OCLR). The stemness‐related cells were analyzed based on scRNA‐seq dataset GSE166555. Monocle 2 algorithm was used for stemness‐related cells pseudotime trajectory analysis. The stemness‐related prognostic genes were analyzed by clusterProfiler and survival package. The stemness of CRC cells was detected by spheroid formation assay, and the expression of stemness‐related prognostic genes was verified by qRT‐PCR and Western blot.Results7916 DEGs between the CRC and normal tissues were obtained. The mRNAsi of the CRC tissues was shown to be significantly higher than that of the normal tissues. 7 and 8 cell types were annotated respectively in the normal and CRC tissues through analysis of the scRNA‐seq data. Cell–cell interactions (CCIs) in the tumor tissues were revealed to be significantly enhanced than that in the normal tissues. By calculating the ‘stemness score’, CSCs, epithelial cells (EPCs) and cancer‐associated fibroblasts (CAFs) were defined as stemness‐related cells. Through pseudotime trajectory analysis, 2111 genes were identified as state 2‐specific genes. Then, 41 genes were obtained by taking intersection of the up‐regulated genes with state 2‐specific genes and marker genes of CSCs, EPCs and CAFs. The univariate COX regression analysis revealed 5 stemness‐related prognostic genes (TIMP1, PGF, FSTL3, SNAI1 and FOXC1). Kaplan–Meier curve analysis indicated that the higher the expression of 5 genes, the lower the survival rate. In vitro cell experiment confirmed that the expression of TIMP1, PGF and SNAI1 was consistent with that revealed by bioinformatics analysis.ConclusionsTIMP1, PGF and SNAI1 were identified as stemness‐related prognostic genes of CRC, and possibly potential therapeutic targets for CRC.