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

Research Paper

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

Thea Reinkens1, Amelie Stalke1, Nicole Huge1, Beate Vajen1, Marlies Eilers1, Vera Schäffer1, Oliver Dittrich-Breiholz2, Brigitte Schlegelberger1, Thomas Illig1,3, Britta Skawran1 ✉

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.

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Citation:
Reinkens T, Stalke A, Huge N, Vajen B, Eilers M, Schäffer V, Dittrich-Breiholz O, Schlegelberger B, Illig T, Skawran B. Ago-RIP Sequencing Identifies New MicroRNA-449a-5p Target Genes Increasing Sorafenib Efficacy in Hepatocellular Carcinoma. J Cancer 2022; 13(1):62-75. doi:10.7150/jca.66016. Available from https://www.jcancer.org/v13p0062.htm

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Abstract

Graphic 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: Liver cancer, microRNA combination therapy, drug resistance, microRNA target genes, Ago-RIP sequencing, multi-tyrosine kinase inhibitor