RNF128 Promotes Malignant Behaviors via EGFR/MEK/ERK Pathway in Hepatocellular Carcinoma

Abstract

Background: The ubiquitin-proteasome system participates in the pathogenesis and progression of hepatocellular carcinoma (HCC). As an E3 ubiquitin ligase, RNF128 has been proved vital in carcinogenesis, whereas, little is known about the oncogenic mechanisms of RNF128 in HCC.

Materials and methods: Through tissue microarray from HCC patients, we analyzed RNF128 expression and its relationship with clinical outcomes in HCC. Western blot and quantitative realtime polymerase chain reaction (qRT-PCR) were performed to examine expression levels of RNF128 in HCC tissues and cell lines. Effects of RNF128 on HCC cellular biological functions and the potential mechanism were evaluated through knockdown and overexpression assays in vitro and in vivo methods.

Results: RNF128 expression was found to be remarkably elevated in HCC tissues compared with adjacent normal tissues. Furthermore, the overexpression of RNF128 enhanced hepatoma cells proliferation, colony formation, migration, invasion, and apoptotic resistance both in vitro and in vivo. Mechanistically, RNF128 activated EGFR/MEK/ERK signaling pathway and the EGFR inhibitor, gefitinib partially reversed RNF128-enhanced proliferation, invasion, and migration in hepatoma cells.

Conclusion: RNF128 promotes HCC progression by activating EGFR/MEK/ERK signaling pathway, which might function as a novel prognostic molecular signature with the potential to be a candidate therapeutic target for HCC patients.

Keywords: EGFR/MEK/ERK signaling pathway; RNF128; hepatocellular carcinoma; prognosis; ubiquitination.

Figure 1

RNF128 is highly expressed in HCC and signifies poor HCC prognosis: (A) RNF128 protein levels in 16 pairs of frozen HCC tissues and their corresponding peritumor tissues. (B) RNF128 mRNA levels were verified by qRT-PCR, shown as log (T/N). (C) Tissue microarray analysis of RNF128 expression in HCC and matched peritumor tissues. RNF128 expression was higher in HCC relative to peritumor tissue. (D) Representative images of TMA stained with H&E and anti-RNF128 IHC. Scale bars: 0×= 500μm; 200×= 100μm. (E) Kaplan–Meier analysis of overall survival and recurrence in 171 patients, which were divided into 2 groups by median optical density. ****p< 0.0001.

Figure 2

High levels of RNF128 promote HCC cells proliferation: (A) CCK8 assay was used to assess HCC cell proliferation. RNF128 knockdown significantly suppressed hepatoma cell proliferation, while overexpression of RNF128 increased their proliferation. (B) RNF128 knockdown significantly suppressed colony formation by hepatoma cells, while overexpression of RNF128 increased colony formation. **p<0.01; ***p<0.001, ****p< 0.0001.

Figure 3

High levels of RNF128 promote HCC cells migration and invasion: effects of RNF128 knockdown or overexpression on migration and invasion were measured by transwell assays (A) and wound-healing migration assays (B). Scale bar: 200×= 100μm. *p<0.05, **p<0.01, ***p<0.001.

Figure 4

High levels of RNF128 inhibit apoptosis of HCC cells: Apoptosis was evaluated in HepG2 cells, PLC/PRF/5 cells (A), MHCC97-L cells and MHCC97-H cells (B) upon RNF128 overexpression or inhibition using flow cytometry. *p<0.05, **p<0.01; ***p<0.001.

Figure 5

High levels of RNF128 promote HCC progression in nude mice: (A) Tumors derived from nude mice bearing MHCC97-L-control cells and MHCC97-L-shRNF128 cells, PLC/PRF/5-control cells and PLC/PRF/5-RNF128 cells (n=5). (B and C) Statistical analysis of tumor volume and weight in different groups to assess tumor growth. *p<0.05, **p<0.01.

Figure 6

EGFR/MEK/ERK pathway activation is involved in RNF128 enhanced HCC progression: (A) mRNA expression level correlation between RNF128 and EGFR/MEK/ERK pathway marker genes (EGFR, MAP2K1, MAPK1) in HCC cells. (B) Expression of EGFR/MEK/ERK pathway markers (p-MEK, MEK, p-ERK, ERK, EGFR, p-EGFR) in MHCC97-L-control/MHCC97-L-shRNF128 and HepG2-control/HepG2-RNF128 cells quantified by Western blotting. (C) RNF128 over-expressing cells were pretreated with gefitinib (1μM) for 24 hours, Western blot analysis of EGFR/MEK/ERK pathway markers (EGFR, p-EGFR, MEK, p-MEK, ERK, p-ERK) expression in the indicated cells. Gefitinib counteracted RNF128-enhanced p-EGFR, p-ERK, p-MEK expression in HepG2 cells. (D and E) RNF128-enhanced migration, invasion, and proliferation in HepG2 cells was eliminated by gefitinib. Scale bar: 200× = 100μm. *p<0.05, **p<0.01; ***p<0.001.