The prognostic value of major facilitator superfamily domain-containing protein 2A in patients with hepatocellular carcinoma

Introduction: We aimed to characterize the expression of major facilitator superfamily domain-containing protein 2A (MFSD2A) in hepatocellular carcinoma (HCC) patients and analyze its prognostic value. Results: Immunohistochemistry revealed that low expression of MFSD2A was present in 37 of 79 cases (46.84%), which was significantly correlated with poor histological differentiation (P = 0.012). The plasma MFSD2A level in HCC patients was significantly lower than in healthy controls (P = 0.0079) and controls with chronic hepatitis B virus (HBV) infection (P = 0.0430). Moreover, patients with lower MFSD2A expression had shorter survival than higher expression (P = 0.021). Multivariate analysis revealed that MFSD2A was an independent prognostic predictor for HCC patients (P = 0.027). Conclusion: The current study indicate MFSD2A may be an optimal diagnostic and prognostic biomarker for HCC. Methods: First, we examined MFSD2A expression in 24 paired HCC and nontumorous tissues by real-time quantitative PCR (RT-qPCR). Second, the protein levels of MFSD2A in 11 paired HCC and nontumorous tissues were investigated by western blotting (WB). Moreover, MFSD2A protein expression was evaluated by immunohistochemistry in 79 HCC patients. In addition, we detected the plasma level of MFSD2A in HCC patients and healthy individuals and investigated the relationship between MFSD2A expression and clinicopathological parameters or prognosis of HCC patients.


INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most common cancer types and the third leading cause of cancer-related deaths worldwide, affecting one million individuals annually [1]. More importantly, the incidence of HCC is still increasing worldwide [2]. Despite improvements in treatment strategies, the mortality rate remains high because of the lack of sensitive and specific markers for HCC. Alpha-fetoprotein (AFP) has been widely used as a biomarker for HCC, not only in diagnosis but also in surveillance [3][4][5]. However, one study reported that the sensitivity of AFP in elderly HCC patients was approximately 41.0%, while another study demonstrated it to be 60.5% [6,7]. Previous studies have shown that the efficiency for predicting the prognosis of HCC using serum AFP levels remains unsatisfactory (30-62%). In addition, the dynamic AFP levels range varies from one patient to another [8]. Therefore, the application of AFP is still limited in practice. The discovery of a marker for early diagnosis, as well as prognostic prediction, in HCC is urgent.
Major facilitator superfamily domain-containing protein 2A (MFSD2A) is a membrane protein for transporting various substrates, such as sugars, polyols, drugs, and neurotransmitters, although most members of this protein family are substrate-specific [9,10]. MFSD2A expression has been reported in many tissues, especially the liver, and is previously known to maintain blood-brain barrier function [11]. Recently, MFSD2A has been reported as a novel tumor suppressor gene that plays an important role in modulating the cell cycle and matrix attachment [12]. Another study also showed that MFSD2A+ hepatocytes repopulate the liver during injury and regeneration [13]. In China, most HCC cases result from chronic hepatitis B and are often associated with injury and regeneration of hepatocytes. Therefore, we hypothesize that MFSD2A may be another biomarker for the diagnosis and progression of HCC. However, there is no relative study of MFSD2A in HCC. Here, we investigated the expression of MFSD2A in HCC patients and analyzed its correlation with survival.

Low expression of MFSD2A in HCC tissues
The expression of MFSD2A in HCC and normal liver tissues was first analyzed in the TCGA and GTEx databases using the GEPIA web server [14]. The results showed significantly lower expression of MFSD2A in HCC tissues than in normal liver tissues (P < 0.01, Figure 1A). To confirm the expression of MFSD2A in public databases, we collected a total of 35 paired HCC and matched adjacent noncancerous tissues. Fresh tissue samples were frozen in liquid nitrogen and stored at -80°C. The transcript levels of MFSD2A were determined by RT-qPCR in 35 paired HCC and matched adjacent noncancerous tissues. The mRNA level of MFSD2A was significantly lower in 19 cancerous tissues (79%) than in the matched adjacent noncancerous tissues (P = 0.016, Figure 1B). We also tested MFSD2A levels in HCC samples by western blotting. The expression of MFSD2A was significantly decreased in cancerous tissues relative to that in the matched adjacent normal tissues (P = 0.0472, Figure  1C, 1D), which was consistent with the RT-qPCR and public database results.
In addition, we performed RT-qPCR and western blotting in several HCC cell lines and one immortalized hepatic cell line LO2. The results showed that MFSD2A expression was significantly lower in HCC cells than in LO2 cells (P < 0.05, Figure 1E, 1F).

Downregulation of MFSD2A was correlated with poor differentiation in HCC
Paraffin-embedded samples were obtained from 79 HCC patients after surgical resection. The patients had not received anticancer treatment prior to surgery (Table 1). We performed immunohistochemical staining on 79 paraffin-embedded HCC tissues. Overall, MFSD2A was positively and negatively expressed in 71 (89.9%) and 8 (10.1%) of the 79 HCC patients, respectively ( Figure  2A-2C, total score ≥ 1; Figure 2D, total score = 0). In addition, 37 of 79 (46.8%) cases had low MFSD2A expression, whereas 42 (53.1%) cases had high expression (Figure 2A and 2B, total score ≥ 4; Figure 2C and 2D, total score < 4). As listed in Table 2, the low expression of MFSD2A was significantly correlated with poor histological differentiation (P = 0.012), but not with age, sex, tumor size, live cirrhosis, lymph node metastasis, recurrence, serum AFP level, or HBsAg status. Furthermore, we mapped the expression of MFSD2A in the UALCAN database and found that low expression of MFSD2A was significantly correlated with tumor grade and stage (P<0.05, Figure 2E, 2F).

MFSD2A expression was an independent predictor of poor prognosis in HCC
To identify the variables of potential prognostic significance in all patients with HCC, we carried out univariate and multivariate analyses. Univariate analysis showed that MFSD2A expression, sex, and differentiation grade were significantly correlated with prognosis. MFSD2A expression was also an independent predictive factor for prognosis (P = 0.027) and differentiation grade (P = 0.013) based on multivariate analysis. The hazard ratio in patients with high levels of MFSD2A was 0.549 compared to that in patients with low expression (Table 3).
In addition, the prognostic value of MFSD2A expression on HCC patient survival was evaluated between patients with high and low MFSD2A expression. The five-year overall survival in the high MFSD2A expression group was significantly better than AGING  that in the low MFSD2A expression group (P =0.021) ( Figure 2G).  Figure 2H, 2I.

DISCUSSION
HCC is the fifth most common cancer worldwide, and it has a poor prognosis despite improved diagnosis and combined therapy [15]. One of the most promising treatments for HCC is targeted therapy, which may play an important role in tumor growth [16]. Therefore, it is important to identify reliable biomarkers. These biomarkers may provide us with useful prognostic information; more importantly, these biomarkers may be targets for HCC treatment.
MFSD2A is a 532-amino acid protein with 11 putative membrane-spanning hydrophobic domains and N-and C-terminal ends, with both terminals predicted to be intracellular. MFSD2A has been previously identified as a putative receptor for Syncytin-2 and to act as a transporter in humans; its encoding gene in located at chromosomal position 1q34.2; and it belongs to the major facilitator superfamily [17]. The transporter function of MFSD2A has been shown to play a tumorsuppressive function in non-small lung cancer [18].
To the best of our knowledge, there has been no research on the relation between the expression of MFSD2A and the diagnosis and prognosis of HCC. In the current study, we detected the expression of MFSD2A in plasma and tissue samples and analyzed its value for diagnosis and prognosis.
In this study, the detection of MFSD2A mRNA and protein expression levels in paired HCC and adjacent normal tissues indicated that both mRNA and protein levels of MFSD2A were significantly lower in cancerous tissues than in the adjacent normal tissues, which was consistent with the findings of Spinola M in non-small lung cancer. AGING

Clinical data collection
All of the research data collected included age, sex, AFP level, metastatic lymph nodes, tumor number, tumor size, and TNM stage. The tumor stage was determined according to the AJCC 7th TNM staging system [15].

Blood samples
Blood samples were obtained from 21 HCC patients, 16 chronic HBV-infected controls and 22 healthy controls (HCs). After blood coagulation, the supernatant, represented serum, was centrifuged after balancing (usually 3000 rpm, centrifuge for 5-10 minutes). The supernatant was carefully aspirated (with attention not to aspirate the cell components) and stored at -80°C for preservation.

Western blotting
Tissue samples were lysed in RIPA lysis buffer and harvested by centrifugation (12,000 rpm, 30 minutes) at 4°C. The collected protein was separated by electrophoresis in a 15% sodium dodecyl sulfate polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane. The membrane was placed in 5% nonfat milk for 1 h to block nonspecific binding sites and was then incubated with a goat antihuman MFSD2A antibody (ProSci incorporated; Cat.No.:6025, dilution 1:1,000) at 4°C overnight. After washing four times in Tris-buffered saline with Tween-20, the membrane was probed with a horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG antibody (1:5,000, Proteintech Group) at 37°C for 60 minutes. After four washes, bands were detected with an enhanced chemiluminescence reagent. Band density was measured with ImageJ software (National Institutes of Health) and was standardized to the density of GAPDH (mouse anti-human GAPDH monoclonal antibody, Shanghai Kangchen).

Detection of plasma MFSD2A
We moved the required number of wells from the kit (Signalway Antibody LLC, EK3646, USA) from the refrigerator 20 minutes before the experiment to equilibrate them at room temperature (20-25°C) and returned the remaining sealed wells to 4°C. A standard curve was established before adding samples: 8 wells were set as standard wells; 100 µl of sample dilution solution was added to each well; 100 µl of standard was added to one well, mixed thoroughly; and 100 µl was aspirated with a pipet and moved to the second well. Therefore, the mixture was repeatedly diluted to the seventh well, and 100 µl was removed from the seventh well and discarded so that the volume remained 100 µl. The eighth hole was the blank control; 100 µl of the sample was tested in each well of the test object. The reaction plate was incubated at 37°C for 120 minutes, washed thoroughly with washing solution 4-6 times, and dried on a filter paper; 50 µl antibody was added to the working solution per well; the resultant solution was mixed thoroughly, and the reaction plate was allowed to stand at 37°C for 60 minutes; the reaction plate was washed thoroughly with the washing solution 4-6 times, and dried on a filter paper; 100 μl of enzyme-labeled antibody working solution was added per well, and the reaction plate was incubated at 37°C for 60 minutes.
The plate was washed again before adding 100 µl of the substrate solution to each well, and the reaction was continued for 5-10 minutes in the dark at 37°C. Finally, 50 µl of stop solution was added to each well and mixed before measuring the absorbance at 450 nm.

Statistical analysis
Overall survival (OS) was defined from the time between the date of diagnosis to death or censoring of data. Differences in mRNA expression between HCC and noncancerous tissues were evaluated with pairedsamples t tests, whereas protein expression levels were compared with paired Student's t tests. The Kaplan-Meier method was used to compare survival using with the log-rank test. Univariate and multivariate analyses were performed to explore the effect of MFSD2A expression on patient survival. A two-sided P value < 0.05 was considered statistically significant. All statistical analyses were performed with SPSS software (version 16.0; SPSS Inc., Chicago, IL, USA).

Ethics approval
The current study was approved by the Institutional Review Board of Sun Yat-sen University Cancer Center, and all procedures met the basic standards of the Declaration of Helsinki. Ethics, consent, and permissions: All patients were clearly informed of the potential risks and benefits before undergoing the procedure. All patients stated their understanding and signed the informed consent form voluntarily.
The authenticity of this article has been validated by uploading the key raw data onto the Research Data Deposit public platform, with the approval RDD number as RDDA2019001051 The datasets used and/or analyzed during the current study are also available from the corresponding author on reasonable request.