Fig 2: MYH10 exerts its tumour‐suppressive role through inactivation of the EGFR pathway. (A) Knockdown of MYH10 triggers the activation of the EGFR pathway. The immunobands of EGFR, p‐EGFR (Tyr1068), ERK1/2, p‐ERK1/2 (Thr202/Tyr204), AKT and p‐AKT (Ser473) were detected by immunoblotting assays in L‐02, HepG2 and SMMC7721 cells. (B) Overexpression of MYH10 inhibits EGFR pathway in HCCLM3 and MHCC97H cells. (C) Knockdown of MYH10 has no pro‐proliferation effect on HepG2 (top) and SMMC7721 (bottom) cells with or without Gefitinib treatment (10 µM). (D) Gefitinib treatment (10 µM) abolishes the pro‐migratory effects of MYH10 depletion on HepG2 (top) and SMMC7721 (bottom) cells. (E) Gefitinib treatment (10 µM) eliminates the activation of MYH10 depletion on EGFR pathway in HepG2 (left) and SMMC7721 (right) cells. *** p < 0.001 (Student's t test). n.s., not significant

Fig 3: Genomic loss at chromosome 17p13.1 correlates with down‐regulation of MYH10. (A) The characteristics of genomic loss at chromosome 17p13.1. The distribution of depletions (in blue) and amplifications (in red) of chromosome 17p13.1 from individual HCC on the basis of the copy number alteration (CNA) dataset of the Cancer Genome Atlas (TCGA)‐liver hepatocellular carcinoma (LIHC) cohort (Materials and Methods). The 17p13.1 cytoband is emphasized by a red line. (B) Left, the rank of Pearson correlations between the mRNA levels of each protein‐coding gene (n = 47) within the focally deleted region at 17p13.1 and the copy numbers of 17p13.1. Nineteen (in red) of 47 genes were identified as candidates with significant correlations (R > 0.2, p < 0.05). Right, the correlations between the mRNA levels of MYH10 and the copy numbers of 17p13.1. RPKM, reads per kilobase per million mapped reads. (C) Dysregulation of the 19 cis‐regulated genes by 17p13.1 deletions in multiple gene expression datasets of HCC cohorts from the HCCDB database (http://lifeome.net/database/hccdb). The genes with p < 0.05 (Wilcoxon rank‐sum test) and log2(fold‐change) <−0.2 were considered to be significantly down‐regulated in HCC tissues compared to adjacent non‐tumour liver tissues (ANTLs). (D) Down‐regulation of MYH10 in 11 independent cohorts of HCC patients (including TCGA‐LIHC, ICGC‐LIRI‐JP, GSE22058, GSE63898, GSE76427, GSE10143, GSE25097, GSE14520, GSE46444, GSE54236 and GSE64041). (E) Genomic alteration frequencies of MYH10 in 33 types of cancer from the TCGA database. (F) The correlations between the mRNA levels and the copy numbers of MYH10 in 33 types of cancer from the TCGA database, significant correlation with both R > 0.2 and p < 0.05; n.s., not significant. The abbreviations of cancer names are described on the TCGA (https://cancergenome.nih.gov/) database

Fig 4: Depletion of MYH10 induced activation of EGFR pathway. (A and B) The heatmap (A) and volcano plot (B) of the differential expressed genes (DGEs) in MYH10 knockdown HepG2 cells compared to the control cells. Significantly up‐regulated genes are indicated in red; down‐regulated genes are shown in blue. (C) KEGG pathway that was significantly enriched by DEGs in (B). Adjusted p (false discovery rate [FDR]) <0.05 was considered to be statistically significant. (D) The enrichment map of the GSEA results based on the gene expression profiles of MYH10‐depleted and control HepG2 cells. Nodes represent significant gene sets (p < 0.05), and node size represents the number of genes in the gene set. Edges represent the similarity between gene sets. Edge width represents the number of genes that overlap between a pair of gene sets. (E) GSEA plots of gene sets involved in negative regulation of EGF response/MAPKs subnetworks in (D). NES, normalized enrichment score. (F) The heatmap of dysregulated core genes (negative regulators of EGFR/MAPKs) in the gene sets in (E). (G) Relative mRNA expression levels of the negative regulators of EGFR or MAPKs were confirmed by RT‐qPCR assays in MYH10‐depleted HepG2 cells. The quantitative data are mean ± standard deviation (SD) of three independent experiments. *** p < 0.001 (Student's t test). (H) Pearson correlation analyses of the mRNA levels of MYH10 and the negative regulators of EGFR or MAPKs in the TCGA‐LIHC cohort. TPM, transcripts per million

Fig 5: MYH10 reduces HCC cell migration and invasion in vitro. (A) Stable knockdown of MYH10 by two independent shRNAs. (B) CCK‐8 assays revealed that knockdown of MYH10 has no pro‐growth effect on L‐02, HepG2 and SMMC7721 cells. (C) Knockdown of MYH10 does not affect the plate colony formation abilities of L‐02, HepG2 and SMMC7721 cells. (D) Transwell assays revealed that knockdown of MYH10 promotes migration and invasion of L‐02, HepG2 and SMMC7721 cells. (E) Construction of HCCLM3 and MHCC97H cells stably expressing exogenous MYH10. (F) CCK‐8 assays revealed that overexpression of MYH10 has no proliferative effect on HCCLM3 and MHCC97H cells. (G) Overexpression of MYH10 does not affect the plate colony formation abilities of HCCLM3 and MHCC97H cells. (H) Transwell assays revealed that overexpression of MYH10 decreases migration and invasion of HCCLM3 and MHCC97H cells. All quantification data are mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student's t test). n.s., not significant