Expression and function of myelin expression factor 2 in hepatocellular carcinoma

Abstract

Introduction: Hepatocellular carcinoma (HCC) is one of the most common malignant tumours in the world and has a high mortality rate. However, the pathogenesis of HCC remains unclear. This study aimed to investigate the potential biomarkers of HCC.

Methods: ONCOMINE, HCCDB and THE HUMAN PROTEIN ATLAS were used to identify myelin expression factor 2 (MYEF2) as a potential biomarker for HCC. The Cancer Genome Atlas database was used to further validate and analyse the value of MYEF2. Kaplan-Meier Plotter was used for the prognostic analysis. The COX regression model and Kaplan-Meier method were used to investigate the clinical value of MYEF2 in the prognosis of HCC by reviewing the survival status of patients. Fluorescent quantitative polymerase chain reaction (qPCR) and immunohistochemistry were used to detect the expressions of the MYEF2 mRNA and protein in HCC tissues and cell lines. qPCR and Western blotting were used to validate the efficiency of MYEF2 knockout and overexpression in HCC cells. The invasion and migration abilities regulated by MYEF2 were detected by performing transwell and wound healing assays.

Results: MYEF2 is significantly upregulated in HCC and is mainly located in the nucleus of HCC cells. MYEF2 expression is significantly associated with the tumour stage, histological grade and TNM stage. High MYEF2 expression is an independent prognostic factor for patients with HCC. Functionally, elevated MYEF2 facilitated cell migration and invasion in vitro. In contrast, decreased MYEF2 inhibited cell migration and invasion.

Conclusions: MYEF2 may be a novel biomarker with potential diagnosis and prognosis values and as a potential therapeutic target for HCC.

Keywords: Hepatocellular carcinoma; Invasion; MYEF2; Migration; Prognosis.

Fig. 1

Preliminary screen of HCC biomarkers. A, B Dysregulated expression of MYEF2 in datasets containing different tumour tissues compared with normal tissues. C Expression of the MYEF2 protein in HCC from THE HUMAN PROTEIN ATLAS. D OS curve from the Kaplan–Meier Plotter database showing the prognostic difference between patients with high expression of MYEF2 and patients with low expression of MYEF2. E DSS curve from the Kaplan–Meier Plotter database showing the difference in the prognosis between patients with high MYEF2 expression and patients with low MYEF2 expression. F PFS curve from the Kaplan–Meier Plotter database showing the difference in the prognosis between patients with high MYEF2 expression and patients with low MYEF2 expression. G The RFS curve from the Kaplan–Meier Plotter database showed differences in the prognosis between patients with high MYEF2 expression and patients with low MYEF2 expression. MYEF2, myelin expression factor 2

Fig. 2

Analysis of data from The Cancer Genome Atlas database. A Images show the expression of the MYEF2 mRNA in HCC and adjacent tissues. B ROC curve for patients with HCC based on MYEF2 expression. C The prognostic value of MYEF2 expression in HCC. ***P < 0.001. MYEF2, myelin expression factor 2; ROC curve, receiver operating characteristic curve

Fig. 3

MYEF2 expression in HCC tissue and cell lines. A Fluorescent quantitative polymerase chain reaction was performed to detect MYEF2 mRNA expression in HCC (n = 49) and adjacent tissues (n = 79). B Representative images of immunohistochemical staining for MYEF2 in HCC and adjacent tissues. C Immunohistochemical score for MYEF2 expression in HCC and adjacent tissues. D Detection of MYEF2 mRNA expression using fluorescent quantitative polymerase chain reaction in HCC cell lines, including Hep 3B2.1-7, SK-HEP-1, HuH-7, and PLC/PRF/5, and in normal human liver HL-7702 (L-02) cells, which served as the control. E Kaplan–Meier plots of the overall survival of 276 patients with HCC stratified by MYEF2 expression. *P < 0.05, **P < 0.01, and ****P < 0.0001

Fig. 4

Validation of hepatocellular carcinoma cell model with MYEF2 knockdown and overexpression. A, B qPCR and Western blot were used to detect the knockdown efficiency of MYEF2. C, D qPCR and Western blot were used to detect the overexpression efficiency of MYEF2. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 5

Effect of MYEF2 on migration of HCC cells by wound healing assay. A The effect of MYEF2 knock-down on SK-HEP-1 cell migration was analyzed by scratch test. B The scratch test analyzed the effect of MYEF2 overexpression on the migration ability of PLC/PRF/5 cells. **P < 0.01, and ****P < 0.0001

Fig. 6

MYEF2 enhances invasion and migration of HCC cells. A Transwell assay was used to detect the effect of knock-down MYEF2 on the invasion and migration of SK-HEP-1 cells. B Transwell assay was used to detect the effect of overexpression of MYEF2 on the invasion and migration of PLC/PRF/5 cells. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001