ObjectiveTo explore the expression of genes related to hepatocellular carcinoma (HCC) stem cells and their prognostic correlation by using weighted gene co-expression network analysis (WGCNA).MethodsFirstly, the transcriptome sequencing (RNA-seq) and clinical data of HCC were downloaded from the public database the Cancer Genome Atlas (TCGA), and the mRNA expression-based stiffness index (mRNAsi) table of cancer stem cells was downloaded and sorted out to analyze the relationship between mRNAsi and pathological grade and prognosis of HCC. The mRNAsi of HCC was downloaded and the prognostic value of mRNAsi was discussed. Then we used WGCNA to screen the key modules related to liver cancer stem cells (LSCS). Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes were used for the functional and pathway enrichment analysis. The online database STRING was used to construct hub genes coding proteins interaction (PPI) network and screen key genes. Finally, the key genes were analyzed for expression differences and expression correlations. The online database Kaplan-Meier plotter was used for survival analysis and verified.ResultsmRNAsi was significantly upregulated in cancer tissues (P<0.001), and increased with the increase of pathological grade of HCC (P=0.001). The mortality rate of the higher mRNAsi group was higher than that of the lower mRNAsi group (P=0.006). GO analysis found that hub genes were mainly involved in biological processes, such as mitosis and DNA replication, and KEGG showed that hub genes were enriched in cell cycle, DNA mismatch repair, oocyte meiosis, and other signaling pathways. We screened 10 key genes (included CCNB1, CDC20, CDCA8, NDC80, KIF20A, TTK, CDC45, KIF15, MCM2, and NCAPG) related to mRNAsi of HCC based on WGCNA. The key genes were highly expressed in the tumor samples compared to the normal samples. In addition, there was a strong interaction between proteins of these key genes (P<0.05), a strong co-expression relationship at the transcriptional level, and all related to prognosis of HCC.ConclusionsmRNAsi plays an important role in the occurrence and development of HCC. Ten key genes related to LSCS were screened, which may act as therapeutic targets for inhibiting the stem cell characteristics of HCC.