ETV4 as a potential target gene for alleviating proliferation metastasis of HCC

Hepatocellular carcinoma (HCC) develops and progresses via the RAF/MERK/ERK signaling pathway. Sorafenib, a first-line therapy for advanced HCC, inhibits the RAF gene. However, RAF dimerization and ERK activation caused by RAF inhibition in HCC, and this paradoxical RAF/MERK/ERK activation is one of the reasons for Sorafenib resistance in liver cell carcinoma. ETV4 is an ERK downstream gene, hence inhibiting it may help to reduce treatment resistance in HCC. The human hepatoma cell line HepG2.2.15 was used as an HCC tumor cell model, while the HL-7702 cell line was used as a human normal hepatocytes cell model. In order to find out the function of ETV4, Small interfering RNA (siRNA) was used to knock down ETV4 gene. Experiments on cell phenotype and function were performed to test the effects of knocking down ETV4.

There were four groups in vitro: control group, siRNA-NC group, siRNA-ETV4 group and normal group. Cultured 5 × 10³/100ul cells in 96-plate-well and 2 × 10⁵/2 ml cells in 6-plate-well. Cell proliferation assays include small interfering RNA transfection, real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR), Western blot analysis, migration and invasion assays, flow cytometry, and chromatin immunoprecipitation (ChIP) assay. In vivo, nude mouse xenograft model was established by short hairpin RNA, including tumor volume measurement, western blotting, immunohistochemistry assay. One-way ANOVA was used to test for a statistically significant difference.

Silencing of ETV4 decreased RNA expression of ERK1, ERK2, MMP1, and MMP9, as well as the protein levels of p-ERK, MMP1, and MMP9. Cell proliferation, migration, and invasion were also reduced with low ETV4 expression. Furthermore, apoptosis of HCC cells increased in low expression of ETV4 group. HCC cells were obstructed in G2/M cell cycle phase and decreased quantity in the DNA preparation in the G0/G1 phase. Knockdown ETV4 decreased KI67 and Vimentin in the tumor tissues, while the protein level of E-cadherin was significantly increased. ETV4 enriched at the MMP1-EBS3 and MMP9-EBS4 regions in HCC.

ETV4 is a key gene connecting the RAF/MER/ERK pathway and the MMP family in tumor growth and development, especially in HCC. This study provides functional and mechanistic evidence suggesting that ETV4 knockdown limits the progression of HCC. This effect is mediated by the reduction of tumor cell proliferation and migration, which may occur, at least in part, through the modulation of the ERK/ETV4/MMPs axis.