GSE196008
Deletion of miR-181ab1 reduces liver tumour progression via upregulation of CBX7
Purpose: miR-181 is upregulated in human liver cancer. The cancer genome atlas (TCGA) liver cancer data show that the iCluster1 subclass of hepatocellular carcinoma (HCC) has a higher expression of miR-181 than either the iCluster2 or iCluster3 subclass, suggesting it may be a driver of oncogenesis. However, the miR-181 family has also been reported to inhibit tumour progression. Thus, we aim to examine the function of miR-181 and its vital mediators in hepatocarcinogenesis. Methods: Primary liver tumours were induced with diethylnitrosamine (DEN) in global (miR-181ab1GKO), liver-specific (miR-181ab1LKO) and hematopoietic & endothelial lineage-specific (miR-181ab1vavKO) miR-181ab1 deletion mice. The molecular and cellular characterization was carried out using immunohistochemistry and whole-genome transcriptome analysis. Liver-specific miR-181a and CBX7 double knockout mice were used to investigate the role of CBX7 in mediating miR-181 effects on liver tumour progression. An AAV/Transposase Hybrid-Vector System used in this study gave a reliable long-term expression of CBX7 in vivo. Relevant gene expression profiles and patient outcomes were downloaded from TCGA database, and their correlations were analysed. The mRNA profiles of miR-181ab1 wild-type (WT) and knockout (KO) mice were generated by deep sequencing, in triplicate, using Illumina HiSeq. After quality control procedures, short reads were mapped to mouse genome assembly (mm10) by STAR version 2.7.3a. Mapped reads were quantified by featureCounts version 2.0.0 from Subread package. Ward's hierarchical clustering method was applied to group the samples based on the gene expression matrix. Differential gene expression analysis was then conducted using R package Limma. Gene set enrichment analysis was subsequently performed with R package clusterProfiler. Overlaps between differential expressed gene sets and gene sets in MSigDB were computed with Gene Set Enrichment Analysis (GSEA). Results: The number and size of liver tumours in 181ab1GKO mice were significantly reduced by 50% and 90%, respectively, compared to WT mice at 34-week post-DEN injection. The number of tumours in 181ab1LKO mice was no different from that of control mice, but the size of liver tumours was significantly reduced by 90%. The number and size of tumours were similar in miR-181ab1vavKO and WT mice. Consistent with the roles of miR-181 in the promotion of epithelial to mesenchymal transition (EMT), the TGF-beta signalling pathway was inhibited in 181ab1GKO tumours compared with WT tumours, and expression of E-Cadherin, an EMT marker, was significantly increased in miR-181ab1 KO tumours compared to WT tumours. CBX7 was reduced in WT liver tumours compared to non-tumours, and restored CBX7 expression inhibited the progression of liver tumours in WT mice. At the same time, CBX7 was upregulated in miR-181ab1 KO tumours compared to WT tumours, and CBX7 deletion turned the reduced progression of miR-181ab1 KO liver tumours back to WT liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iCluster 2 & 3 subclasses of human HCC compared to iCluster 1. Gene expression profiles were significantly overlapped between human low proliferative periportal-type HCCs and miR-181ab1 KO tumours. Conclusions: Primary liver tumour progression is inhibited by loss of miR-181ab1 via up-regulation of CBX7 expression at the level of the liver cancer cells themselves, but tumour induction requires both hepatic and non-hepatic expression. Tumours in miR-181ab1 KO mice resemble low-proliferative periportal-type human HCC and could represent a new model to study therapies for such difficult-to-treat tumours. Overall design: Liver tumour mRNA profiles of miR-181ab1 wild type (WT) and knockout (KO) mice. Diethylnitrosamine (DEN)-induced liver cancer models were made in multiple miR-181ab1 and/or CBX7 KO mice. CBX7 was over-expressed in the liver with AAV.