GPX2 is a potential therapeutic target to induce cell apoptosis in lenvatinib against hepatocellular carcinoma.

INTRODUCTION: Lenvatinib has recently become available as the first-line therapy for advanced hepatocellular carcinoma (HCC), but its molecular mechanism in HCC remains largely unknown. OBJECTIVES: The current study aims to identify the molecular mechanisms of lenvatinib in HCC. METHODS: Gene expression microarrays, flow cytometry, western blot, qRT-PCR, immunohistochemistry and immunofluorescence were used to study the response of HCC cells to lenvatinib. Xenograft tumor of Huh7 cells was also established to detect the effect of lenvatinib in vivo. RESULTS: Herein, we found that lenvatinib could induce apoptosis via increasing reactive oxygen species (ROS) levels in HCC cells. Then, microarray analysis and qRT-PCR results confirmed that GPX2 was a vital target for lenvatinib against HCC. Loss and gain function of experiment showed that regulating GPX2 levels markedly affected the lenvatinib-induced ROS levels and apoptosis in HCC cells. In addition, analyses of The Cancer Genome Atlas database and the qRT-PCR results in our cohort both showed that GPX2 markedly overexpressed in tumor tissues and correlated with poor overall survival in HCC. Mechanistically, our findings further demonstrated that GPX2 was a downstream gene regulated by ß-catenin, while lenvatinib could prevent nuclear translocation of ß-catenin and further inhibit GPX2 expression in HCC cells. More importantly, the correlation of GPX2 expression with lenvatinib response was further analyzed in 22 HCC patients who received lenvatinib therapy, and the results showed that the objective response rate (ORR) in patients with low GPX2 expression was 44.4% (4/9), while the ORR in patients with high GPX2 levels was only 7.7% (1/13). CONCLUSION: Our findings indicated that GPX2 plays an important role in lenvatinib-induced HCC cell apoptosis, which might serve as a biomarker for instruction of lenvatinib therapy in HCC patients.

CircLIFR suppresses hepatocellular carcinoma progression by sponging miR-624-5p and inactivating the GSK-3ß/ß-catenin signaling pathway.

Circular RNAs have been reported to play essential roles in the tumorigenesis and progression of various cancers. However, the biological processes and mechanisms involved in hepatocellular carcinoma (HCC) remain unclear. Initial RNA-sequencing data and qRT-PCR results in our cohort showed that hsa_circ_0072309 (also called circLIFR) was markedly downregulated in HCC tissues. Kaplan-Meier analysis indicated that higher levels of circLIFR in HCC patients correlated with favorable overall survival and recurrence-free survival rates. Both in vitro and in vivo experiments indicated that circLIFR inhibited the proliferation and invasion abilities of HCC cells. We therefore conducted related experiments to explore the mechanism of circLIFR in HCC. Fluorescence in situ hybridization results revealed that circLIFR was mainly located in the cytoplasm, and RNA immunoprecipitation assays indicated that circLIFR was significantly enriched by Ago2 protein. These results suggested that circLIFR may function as a sponge of miRNAs to regulate HCC progression. We further conducted bioinformatics prediction as well as dual-luciferase reporter assays, and the results of which showed that circLIFR could sponge miR-624-5p to stabilize glycogen synthase kinase 3ß (GSK-3ß) expression. Loss and gain of function experiments demonstrated that regulation of the expression of miR-624-5p or GSK-3ß markedly affected HCC progression induced by circLIFR. Importantly, we also proved that circLIFR could facilitate the degradation of ß-catenin and prevent its translocation to the nucleus in HCC cells. Overall, our study demonstrated that circLIFR acts as a tumor suppressor in HCC by regulating miR-624-5p and inactivating the GSK-3ß/ß-catenin signaling pathway.

miR-126-3p contributes to sorafenib resistance in hepatocellular carcinoma via downregulating SPRED1.

BACKGROUND: Sorafenib can prolong the survival of patients with advanced hepatocellular carcinoma (HCC). However, drug resistance remains the main obstacle to improving its efficiency. This study aimed to explore the likely molecular mechanism of sorafenib resistance. METHODS: Differentially expressed microRNAs (miRNAs) related to sorafenib response were analyzed with the Limma package in R software. The expression levels of miR-126-3p and sprouty-related EVH1 domain-containing protein 1 (SPRED1) in HCC cells were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell viability and proliferation were detected with Cell Counting Kit-8 (CCK-8), EdU proliferation, and clone formation assays. Transwell assays were performed to measure cell migration and invasion. TargetScan, MicroRNA Target Prediction Database (miRDB), and StarBase v2.0 were used to predict the targets of miR-126-3p. SPRED1 was confirmed as a target gene of miR-126-3p by dual-luciferase reporter assay and Western blotting. Finally, the in vivo anti-tumor effect of LV-miR-126-3p inhibitor combined with sorafenib was evaluated via subcutaneous tumor models. RESULTS: HCC cells with high expression of miR-126-3p exhibited increased resistance to sorafenib. The results of bioinformatics analysis and the dual-luciferase reporter assay showed that miR-126-3p directly targeted SPRED1. The sensitivity of HCC cells to sorafenib was markedly enhanced by SPRED1 upregulation. Gain- and loss-of function experiments verified that miR-126-3p induced sorafenib resistance in HCC through downregulating SPRED1. Furthermore, the inhibition of miR-126-3p markedly increased the effectiveness of sorafenib against HCC in vivo. Mechanistically, our results suggested that miR-126-3p promoted sorafenib resistance via targeting SPRED1 and activating the ERK signaling pathway. CONCLUSIONS: Our study demonstrates that regulating the miR-126-3p/SPRED1 axis might be a promising strategy for enhancing the antitumor effect of sorafenib in the treatment of HCC.