Expression and function of myelin expression factor 2 in hepatocellular carcinoma

The ONCOMINE database (https://​www.​oncomine.​org/​resource/​login.​html) is currently the world's largest cancer gene chip database and integrated data mining platform with the most complete cancer mutation spectrum, gene expression data and related clinical information [11]. In this study, we searched for differentially expressed genes in HCC. We set the P value to less than 0.05, and the fold change to greater than 2. Combined with the existing literature retrieval platform, we searched for liver cancer biomarkers that have not been studied and reported.

HCCDB (http://​lifeome.​net/​database/​hccdb/​home.​html) is a web-based database of the HCC expression atlas that includes 15 public HCC gene expression datasets and a total of 3917 samples [12, 13]. We used HCCDB to further study MYEF2 expression in HCC.

THE HUMAN PROTEIN ATLAS (https://​www.​proteinatlas.​org/​) is a Swedish program launched in 2003 that provides the distribution of human proteins in tissues and cells. The distribution and expression of each protein in normal human tissues, tumour tissues, cell lines and blood were examined using immunohistochemistry [14, 15]. In the present study, we used THE HUMAN PROTEIN ATLAS database for protein expression profiling.

The Cancer Genome Atlas database (https://​tcga-data.​nci.​nih.​gov/​tcga/​) is the largest part of the International Cancer Genome Consortium (ICGC) research plan, which is mainly designed to obtain a comprehensive, multidimensional map for a variety of cancer genomes [16]. In this study, we obtained RNA-seq data and clinical data on MYEF2. Ethical approval was not needed because all data were publicly available.

The Kaplan–Meier plotter database (http://​kmplot.​com/​analysis/​) included studies on 54,675 genes and 18,674 cancer samples and evaluates the effects of 54,000 genes on survival rates in patients with 21 cancers [17]. In the present study, we analysed the correlation between the prognostic value of MYEF2 in determining the survival of patients with HCC, along with other factors.

From January 2008 to December 2016, 49 pairs of HCC tissues and corresponding normal tissues were selected from patients with HCC who underwent surgical treatment at Nantong Third Hospital Affiliated with Nantong University. The distance between the tumour tissue and normal tissue was greater than 2.5 cm. After in vitro experiments, the tumour tissues were immediately frozen at − 80 °C until use. In addition, 142 pairs of tissues were fixed with 10% neutral formalin buffer for 24 h, embedded in paraffin and prepared as paraffin sections for preservation. The diagnosis of all patients with HCC was confirmed according to the HCC guidelines for diagnosis and treatment of the European Association [18]. This study was approved by the Ethics Committee of Nantong Third Affiliated Hospital of Nantong University. All patients signed informed consent forms.

Normal human liver cells HL-7702 (L-02) and Hep 3B2.1-7, SK-HEP-1, HuH-7, and PLC/PRF/5 liver cancer cells were provided by the cell bank of the Chinese Academy of Sciences (Shanghai, China) and cultured at the Nantong Hepatology Research Institute. Hep 3B2.1-7, SK-HEP-1 and PLC/PRF/5 cells were cultured in MEM (NaHCO3 and sodium pyruvate were added) containing 10% foetal bovine serum (FBS; Cell Sciences, Canton, MA). L-02 cells were cultured in RPMI-1640 (Gibco, Thermo Fisher Scientific, Waltham, MA) containing 10% FBS. HuH-7 cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific) containing 10% FBS. All cells were cultured in a humidified incubator with 5% CO₂ at 37 °C.

The experimental scheme was the same as the previous research of our research group [19‐21]. After total RNA was isolated from tissues and cells by RNAiso Plus (Takara, Beijing, China), the concentration and purity of the isolated RNA were detected by UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Next, the isolated RNA was converted into complementary DNA (cDNA) using a PrimeScript RT Reagent kit (Perfect Real Time; Takara Biotechnology Co., Ltd.). Reaction conditions: 37 °C, 15 min; 85 °C, 5 s, cool to 4 °C. qPCR was performed using a SYBR-Green PCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China). qPCR was initially performed at 95 °C for 5 min, and then samples were subjected to 40 cycles of amplification at 95 °C for 10 s and at 60 °C for 30 s. β-actin was used as an internal control gene. The fold amplification for each gene was calculated using the 2^−ΔΔCq method. The primer sequences were as follows: MYEF2 forward 5′-GATTTTTATCGGGTCCAATGGG-3′, reverse 5′-ACAGCCTTTTGA CTTTCCATTC-3′; β-actin forward 5′-GGACTTCCGAGCAAGAGATGG-3′, reverse 5′-AGGAAGGAAGGCTGGAAGA-3′.

Liver cells were lysed with RIPA lysis buffer containing a phosphatase inhibitor cocktail (Beyotime Institute of Biotechnology, Shanghai, China). Then, the samples were centrifuged at 10,000×g for 10 min at 4 °C, and the supernatant was collected. A BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to measure the protein concentration. Equal amounts of protein were electrophoretically separated on 10% SDS–PAGE gels and transferred to nitrocellulose membranes at 100 V for 90 min. β-Actin (cat. no. ab8227; 1:2000; Abcam) was used as an internal control protein, and a specific primary antibody (cat. no. 16051-1-AP; 1:5000; Proteintech) was used to analyse the expression of MYEF2. After an overnight incubation at 4 °C, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (cat. no. KC-RB-035; 1:3000; Kangchen Biotech Co., Ltd., Shanghai, China) at room temperature for 1 h. The membranes were washed three times with Tris-buffered saline containing Tween-20 and visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Inc.). ImageJ software 1.46 (National Institutes of Health, Bethesda, MD, USA) was used to analyse the grey value of protein bands. Due to the impact of polyclonal antibodies, we cannot provide cleaner, smoother backgrounds and bands. To make the experimental results clearer and more aesthetically pleasing, our experimental results were cut and highly exposed by using the drawing software. We provided original images of full-length blots, which is included in the Additional file 1.

The paraffin sections were dewaxed with a series of xylene solutions and gradient of ethanol concentrations. The dewaxed tissue slices were dried in a microwave in citrate buffer (pH 6.0) for antigen retrieval. Then, we incubated the sections with 3% H₂O₂ for 15 min to block endogenous peroxidase activity. Slices were incubated with a rabbit anti-human MYEF2 antibody (cat. no. 16051-1-AP; 1:200; Proteintech) overnight at 4 °C. The next day, a horseradish peroxidase-conjugated anti-mouse/rabbit secondary antibody (cat. no. D-3004; Shanghai Changdao Biotechnology Co., Ltd., Shanghai, China) was incubated with the sections for 30 min at room temperature. After washes with PBS, 3,3′-diaminobenzidine (DAB, Maixin-Bio, Guangzhou, China) was used for staining at room temperature for 18 s, followed by haematoxylin staining. Finally, two researchers evaluated all stained sections in a blinded manner. The staining intensity was graded from 0 to 3:0 (no staining), 1 (weak staining), 2 (medium staining) and 3 (strong staining).

For MYEF2 knockdown, Genepharma (Suzhou City, Jiangsu Province, China) designed and constructed two siRNAs specifically targeting MYEF2 and a negative control (Si-NC). According to the instructions, siRNA (10 μM) was transfected into SK-HEP-1 and Hep 3B2.1-7 cells with Lipofectamine™ 2000 (Thermo Fisher Scientific). The transfection efficiency was verified by performing PCR and western blotting.

For MYEF2 overexpression, Genepharma designed and constructed a plasmid (pc-DNA3.1-MYEF2) for the specific overexpression of MYEF2 and an empty plasmid (pc-DNA3.1-NC). According to the instructions, the plasmid (2.5 μM) was transfected into PLC/PRF/5 cells using Lipofectamine™ 3000 (Thermo Fisher Scientific). The transfection efficiency was verified by performing qPCR and western blotting.

Cell migration was detected using wound healing assay. In brief, we seeded the transfected cells into 6-well plates. The cells were then scratched on the monolayer using a 200 μl suction head. After removing dead cells with PBS, the cells were cultured in serum-free medium. We photographed and recorded the scratch width at the specified time. GraphPad Prism 9.0 software was used to visualize the results.

Cell migration and invasion were detected using Transwell chambers coated without or with Matrigel (BD Biosciences, San Jose, CA, USA). Matrigel was coated on the basement membrane of the Transwell chamber in the invasion assay. A serum-free cell suspension containing 2 × 105 hepatoma cells was inoculated into the upper chamber (8 μm, Millipore). Medium containing 20% FBS was added to the bottom chamber as a chemoattractant. After a certain period of culture at 37 °C with 5% CO₂ and saturated humidity, the cells remaining in the upper chamber were removed, and the cells that migrated through the membrane were stained with crystal violet (Beyotime) after fixation with methanol. The number of cells that migrated or invaded was calculated from five randomly selected fields using an optical microscope (Olympus, Japan). Cell migration time: SK-HEP-1 cells, 20 h; PLC/PRF/5 cells, 14 d. Cell invasion time: SK-HEP-1 cells, 24 h; PLC/PRF/5 cells, 15 d.

We screened 423 studies to assess MYEF2 expression in different tumours from the ONCOMINE database. The results of 11 studies were statistically significant. Six studies reported high MYEF2 expression and 5 studies reported low MYEF2 expression. MYEF2 expression was upregulated in colorectal cancer, leukaemia, liver cancer, melanoma and ovarian cancer (Fig. 1A). Subsequently, we evaluated the MYEF2 expression level in several studies from the HCCDB database. The expression of MYEF2 in HCC tissues was generally upregulated compared with that in normal tissues (Fig. 1B). We obtained the expression of MYEF2 protein in 12 HCC tissues from THE HUMAN PROTEIN ATLAS, 8 cases of HCC tissues showed high expression, 2 cases showed moderate expression, 1 case showed low expression, and 1 case showed negative expression. MYEF2 was mainly expressed in the nucleus (Fig. 1C).

We searched the Kaplan–Meier Plotter database by setting conditions and identified 364 cases of HCC. The overall survival (OS) and disease-specific survival (DSS) of patients with high MYEF2 expression were significantly shorter than those of patients with low MYEF2 expression (P = 0.018 and P = 0.033, respectively) (Fig. 1D, E). Additionally, the progression-free survival (PFS) and recurrence-free survival (RFS) of patients with high MYEF2 expression were shorter than those of patients with low MYEF2 expression, although the results were not statistically significant (Fig. 1F, G). Taken together, MYEF2 was prognostically relevant with HCC and was a promising biomarker for predicting survival.

We downloaded MYEF2 mRNA expression data from The Cancer Genome Atlas. The total sample number was 424, including 374 HCC tissue samples and 50 normal tissue samples. The database provided the basic characteristics of the patients. MYEF2 expression was upregulated in HCC (Fig. 2A). A receiver operating characteristic (ROC) curve was established to explore the diagnostic value of MYEF2. The outcome showed an AUC of 0.641 with a sensitivity of 48.66% and a specificity of 88.00%. The ideal cut-off value was 0.062 (Fig. 2B). We further analysed the correlations between MYEF2 expression and different pathological features (Table 1). MYEF2 expression in patients with advanced HCC (stage 2–4) was significantly higher than that in patients with early HCC (stage 1). Higher MYEF2 expression was detected in patients with high-grade (poorly differentiated) HCC (G3–G4) than in patients with low-grade (well differentiated) HCC (G1–G2). Significantly higher MYEF2 expression was observed in patients with T2–4 HCC than in patients with T1 HCC. The differences were statistically significant. In addition, the expression of MYEF2 in HCC tissues was not significantly correlated with age, lymph node metastasis, distant metastasis, alpha fetoprotein (AFP) level, total bilirubin (TB) level, albumin (Alb) level, creatinine (Cre) level, prothrombin time (PT) or platelet (Plt).

The survival analysis showed that the average survival time of 273 patients with low MYEF2 expression was 2015.97 ± 129.66 days and that of 101 patients with high MYEF2 expression was 1516.36 ± 174.68 days. MYEF2 expression was associated with the survival of patients with HCC. The prognosis of patients with HCC presenting high MYEF2 expression was significantly poor (Fig. 2C). A univariate analysis of each clinicopathological parameter was performed to examine its correlation with the survival of patients with HCC and identify variables with potential prognostic significance. The MYEF2 expression level, tumour stage, T stage, lymph node metastasis and distant metastasis were risk factors affecting the prognosis of patients with HCC. The multivariate analysis showed that MYEF2 expression and distant metastasis were independent risk factors for the prognosis of patients with HCC (Table 2).

We examined MYEF2 expression in HCC and adjacent normal liver tissues using qPCR. The expression of the MYEF2 mRNA was upregulated in HCC compared with adjacent normal liver tissues (P < 0.05, Fig. 3A). In addition, immunohistochemical staining was performed to detect the expression and cellular localization of MYEF2 in HCC and adjacent normal liver tissues. MYEF2 was mainly expressed in the nucleus (Fig. 3B). In 142 pairs of HCC tissues and adjacent normal tissues, the expression of the MYEF2 protein in HCC tissues was significantly higher than that in adjacent normal liver tissues (P < 0.05, Fig. 3C). Subsequently, we detected the expression of MYEF2 in HCC cell lines and normal liver cell lines. qPCR showed that compared with normal liver cell lines, the MYEF2 mRNA was expressed at high levels in HCC cell lines (Fig. 3D).

According to the difference between the HCC tissue staining score and adjacent normal tissue staining score, all patients were divided into two groups. A difference > 0 was considered the high expression group, and a difference ≤ 0 was considered the low expression group. All patients were followed for 3–5 years (Table 3). The Kaplan–Meier curve showed that relatively high MYEF2 expression was an important prognostic factor indicating shorter overall survival of patients. The survival rate of 93 patients with high expression was significantly lower than that of 48 patients with low expression (P = 0.033, Fig. 3E).

Subsequently, we performed a univariate analysis on some pathological parameters to examine their correlations with the survival of patients. The hazard ratio and P values for each parameter were used to predict the prognosis of patients with HCC. The importance of each parameter was calculated by performing multivariate Cox proportional hazard analysis. The univariate analysis showed that MYEF2 expression and vascular invasion were important prognostic factors for patients with HCC. The multivariate analysis produced the same results as the univariate analysis (Table 4). Thus, MYEF2 expression was an independent risk factor affecting the prognosis of patients with HCC.

We successfully established in vitro HCC cell models with MYEF2 knockdown and overexpression and verified the transfection efficiency by performing qPCR and western blotting (Fig. 4).

We examined the effect of MYEF2 on the migration ability of HCC cells through wound healing assay. The results showed that the scratch width of the experimental group (SiRNA1, SiRNA2) was significantly larger than that of the control group (Si-NC). After overexpression of MYEF2, compared with the control group (pc-DNA3.1-NC), the scratch width of the experimental group (pc-DNA3.1-MYEF2) was significantly reduced. Thus, MYEF2 might promote the migration of HCC cells (Fig. 5).

In addition, the Transwell assays showed that when MYEF2 was knocked down, cell invasion and migration were significantly reduced compared with the negative control group. In contrast, compared with the negative control group, MYEF2 overexpression increased invasion and migration of HCC cells (Fig. 6). In conclusion, MYEF2 may be involved in the development of HCC by enhancing the invasion and migration of HCC cells.