MiR-22-3p targets the G2/M checkpoint inhibitor Wee1 and represents a possible biomarker of TACE response in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a hypervascular tumor and derive most of its blood supply from the hepatic artery. The locoregional treatment transarterial chemoembolization (TACE) represents the gold standard for HCC patients at intermediated stage, with survival rates ranging from 20 to 36 months. Despite the proven effectiveness of TACE, the identification of biomarkers predictive of response to the first or subsequent TACE cycle remains an unmet clinical need. MicroRNAs are pivotal players in drug resistance in HCC and show ideal characteristics as circulating biomarkers. MiR-22-3p is a tumor suppressor gene in several cancers, including HCC. Here, we aimed at identifying novel miR-22-3p targets involved in TACE resistance and at exploring circulating miR-22 as a candidate of TACE response. Serum and tissue miR-22-3p and WEE1 levels were quantified by qPCR and digital droplets PCR in HCC patients at early and intermediate stages, as well as in HCC cell lines. Functional analysis and luciferase reporter assay assessed WEE1 targeting by miR-22-3p in HCC cell lines and xenograft mice. Flow cytometric analysis evaluated cell cycle modulation after miR-22 overexpression or silencing in HCC cells. Live imaging and WB analyses evaluated cell growth and apoptotic cell death in miR-22 modulated HCC cells subjected to hypoxia and doxorubicin treatment. Statistical analysis was performed to investigate clinicopathological associations in TACE-treated patients. An inverse correlation between serum and tissue miR-22 levels was detected in surgically resected HCC patients. Functional analysis and luciferase reporter assay demonstrated WEE1 targeting by miR-22-3p in HCC cell lines and xenograft mice. Cell cycle and BrdU analyses showed a downregulation of G2/M in miR-22-overexpressing cells, and an upregulation in miR-22 stably silenced cells. An increase of cell growth and an inhibition of apoptotic markers was shown in miR-22 silenced cells undergoing hypoxia and doxorubicin treatment. Increased miR-22 serum levels were observed after each TACE cycle with respect to pre-treatment levels. Higher miR-22 levels associated with TACE resistance at 3 and 6 months of follow-up when assessed two days after treatment. Post-treatment miR-22 levels associated with alfa-fetoprotein and tumor size in TACE-treated patients. The cell cycle checkpoint inhibitor WEE1 is a novel miR-22-3p target in HCC and contributes to TACE resistance in low miR-22-expressing HCC cells. If validated in larger cohorts, miR-22-3p represents a promising circulating biomarker of TACE response when analyzed few days after treatment. [ABSTRACT FROM AUTHOR]