Ago-RIP Sequencing Identifies New MicroRNA-449a-5p Target Genes Increasing Sorafenib Efficacy in Hepatocellular Carcinoma

Thea Reinkens; Amelie Stalke; Nicole Huge; Beate Vajen; Marlies Eilers; Vera Schäffer; Oliver Dittrich-Breiholz; Brigitte Schlegelberger; Thomas Illig; Britta Skawran

doi:10.7150/jca.66016

Ago-RIP Sequencing Identifies New MicroRNA-449a-5p Target Genes Increasing Sorafenib Efficacy in Hepatocellular Carcinoma

J Cancer. 2022 Jan 1;13(1):62-75. doi: 10.7150/jca.66016. eCollection 2022.

Authors

Affiliations

¹ Department of Human Genetics, Hannover Medical School, Hannover, Germany.
² Research Core Unit Genomics, Hannover Medical School, Hannover, Germany.
³ Hannover Unified Biobank (HUB), Hannover Medical School, Hannover, Germany.

Abstract

BACKGROUND: Patients with hepatocellular carcinoma (HCC) have very limited treatment options. For the last fourteen years, the multi-tyrosine kinase inhibitor sorafenib has been used as standard-of-care therapeutic agent in advanced HCC. Unfortunately, drug resistance develops in many cases. Therefore, we aimed to find a way to mitigate drug resistance and to improve the sorafenib efficacy in HCC cells. MicroRNAs play a significant role in targeting genes involved in tumor control suggesting microRNA/sorafenib combination therapy as a promising treatment option in advanced HCC. METHODS: MiR-449a-5p target genes were identified by Ago-RIP sequencing and validated by luciferase reporter assays and expression analyses. Target gene expression and survival data were analyzed in public HCC datasets. Tumor-relevant functional effects of miR-449a-5p and its target genes as well as their impact on the effects of sorafenib were analyzed using in vitro assays. An indirect transwell co-culture system was used to survey anti-angiogenic effects of miR-449a-5p. RESULTS: PEA15, PPP1CA and TUFT1 were identified as direct target genes of miR-449a-5p. Overexpression of these genes correlated with a poor outcome of HCC patients. Transfection with miR-449a-5p and repression of miR-449a-5p target genes inhibited cell proliferation and angiogenesis, induced apoptosis and reduced AKT and ERK signaling in HLE and Huh7 cells. Importantly, miR-449a-5p potentiated the efficacy of sorafenib in HCC cells via downregulation of PEA15, PPP1CA and TUFT1. CONCLUSIONS: This study provides detailed insights into the targetome and regulatory network of miR-449a-5p. Our results demonstrate for the first time that targeting PEA15, PPP1CA and TUFT1 via miR-449a overexpression could have significant implications in counteracting sorafenib resistance suggesting miR-449a-5p as a promising candidate for a microRNA/sorafenib combination therapy.

Keywords: Ago-RIP sequencing; Liver cancer; drug resistance; microRNA combination therapy; microRNA target genes; multi-tyrosine kinase inhibitor.