Abstract
Background. The clinical response rate for molecularly targeted medications is limited despite significant advancements in molecularly targeted therapy for hepatocellular carcinoma (HCC). Therefore, it is necessary to find new and robust therapeutic targets for the treatment of HCC. Recent research has shown that mesoderm/mesenchyme homeobox gene 1 (Meox1) is closely associated with cancer progression.
Objectives. The aim of this study was to evaluate the clinical relevance as well as biological function of Meox1 in HCC.
Materials and methods. Meox1 protein expression level was identified through immunohistochemistry (IHC) examination of pathological tissues from 25 HCC patients. The aim of the analysis was to investigate the relationship between clinicopathological traits and Meox1 expression. Biological function assays of Meox1 in HCC, including proliferation, colony formation, migration, and invasion, were performed with Huh7 and Hep3B cells.
Results. In this study, Meox1 expression in HCC tissues was significantly higher (p < 0.05) compared to paracancerous tissues. Especially in HCC tissues of patients with cirrhosis, the level of Meox1 expression was significantly elevated when compared to HCC tissues of patients without cirrhosis (p < 0.05). High Meox1 expression was significantly associated with tumor-node-metastasis (TNM) stage (p < 0.05) and the Barcelona Clinic Liver Cancer (BCLC) stage (p < 0.05). Moreover, Meox1 silencing suppressed the proliferation, colony formation, migration, and invasion of Huh7 and Hep3B cells.
Conclusions. Our data reveal that Meox1 may play a crucial role in the development of HCC, and given the function of Meox1 in proliferation and metastasis, targeting Meox1 may offer a promising approach for combined and adjuvant therapeutics of HCC.
Key words: progression, proliferation, hepatocellular carcinoma, metastasis, mesoderm/mesenchyme homeobox gene l
Background
Hepatocellular carcinoma (HCC), which accounts for about 75–85% of primary liver cancer cases in China, is the leading cause of cancer-related deaths.1, 2 The main therapeutic strategies for HCC patients with early-stage disease are surgery and radiofrequency ablation.3, 4, 5 However, most HCC patients are at an advanced stage at the first diagnosis and are treated with transcatheter arterial chemoembolization (TACE).5 Molecularly targeted therapy includes various kinase inhibitors,6 such as sorafenib,7 lenvatinib8 and apatinib,9 and immune checkpoint inhibitors, such as nivolumab10 and pembrolizumab.11 Despite significant advancements in molecularly targeted therapy for HCC, these medications have a limited clinical response rate,12 which emphasizes the need to explore novel and effective therapeutic targets for HCC treatment.
A key transcription factor called mesoderm/mesenchyme homeobox l (Meox1) is necessary for cell proliferation and differentiation, and organ formation throughout embryonic development.13, 14 To date, many studies linked aberrant expression of Meox1 to the development of cancer. The Meox1 protein was markedly upregulated in human non-small cell lung cancer (NSCLC) tissues and linked to unfavorable prognosis. The inhibition of Meox1 expression suppressed lung cancer cell proliferation and mammosphere formation in vitro.15 The Meox1 protein was also abnormally expressed in ovarian cancer, which promotes cell growth through interaction with PBX1.16 Moreover, Meox1 was crucial in breast cancer stem cell (CSC) maintenance and epithelial–mesenchymal transition, and it was linked to unfavorable survival outcomes, breast cancer stage and lymph node metastasis in trastuzumab-resistant PTEN-deficient breast cancer.17 In addition, Meox1 knockdown inhibited the proliferation of triple-negative breast cancer cells in vitro and tumor growth in vivo.18 However, the clinical significance and biological role of Meox1 in HCC have not been investigated.
During our research, we not only investigated the expression level of Meox1 in human HCC tissues and evaluated the relationship between Meox1 expression and poor progression in patients with HCC, but also elucidated the role of Meox1 in HCC cell malignancy in vitro for the first time.
Objectives
The aim of this study was to assess the clinical significance and biological role of Meox1 in HCC.
Materials and methods
Collection of clinical samples
Pathological specimens were collected from 25 patients with HCC who underwent surgical operations at the 980th Hospital of People’s Liberation Army (PLA) Joint Logistics Support Force (Shijiazhuang, China) between January 2018 and November 2022. The clinicopathological parameters, including age, gender, pathogeny, cirrhosis, tumor size, tumor number, vascular invasion, lymph node metastasis, tumor-node-metastasis (TNM) stage,19 the Barcelona Clinic Liver Cancer (BCLC) stage system,20 and results of biochemical tests, including alpha-fetoprotein (AFP), carcinoma embryonic antigen (CEA), prothrombin time (PT), albumin, as well as total bilirubin, were collected. Radiotherapy, chemotherapy or other biological therapies were not administered to any of the patients. Furthermore, no occurrences of malignant tumors, cardiovascular or cerebrovascular ailments, diabetes, pulmonary fibrosis, or kidney disease were observed among the patients. The Ethics Committee of the 980th Hospital of the PLA Joint Logistics Support Force gave its approval to this study (approval No. 2022-KY-127). All patients signed written informed consent.
Immunohistochemistry analysis
The protein level of Meox1 was analyzed using immunohistochemistry (IHC) staining. Briefly, after deparaffinization in xylene and in various concentrations of alcohol, antigen retrieval was conducted using ethylenediaminetetraacetic acid (EDTA) buffer (pH 9.0), followed by treatment with 3% hydrogen peroxide for 25 min in the dark at room temperature. After that, the specimens were incubated with primary antibodies against Meox1 (1:500; cat. No. ab105349; Abcam, Cambridge, UK) overnight at 4°C after being blocked with 3% bovine serum albumin (BSA) for 30 min.
A semiquantitative scoring system was used to assess Meox1 expression in accordance with the percentage of positive cells and staining intensity. Pathologists scored the expression in a blinded manner.15, 21 Four categories were established for the percentage of positive cells: 0 (0~5%), 1 (6~25%), 2 (26~ivided into 4 levels: 0 (no staining), 1 (faint staining), 2 (moderate staining), and 3 (strong staining). The multiplication of both factors determined a positive grade that could be negative (0), weakly positive (from 1 to 4), moderately positive (from 5 to 8), or strongly positive (from 9 to 12). Meox1 expression was classified into 2 categories: low expression (negative and weakly positive) or high expression (moderately and strongly positive).
Cell lines and culture
The Huh7 and Hep3B cell lines were stored at the Hebei Key Lab of Laboratory Animal Science of Hebei Medical University (Shijiazhuang, China) and cultured as previously described.21, 22
RNA interference and stable-knockdown cell screening
Recombinant lentivirus carrying specific short hairpin RNA (shRNA) for Meox1 or a negative control was packaged by GenePharma Co., Ltd. (Shanghai, China). Two different Meox1 shRNAs were used to perform the experiment. The sequence for Meox1 shRNA 1 was 5’-GAA ATC ATC CAG GCG GAG AAA-3’; Meox1 shRNA 2 was as follows: 5’-CTG CCA ATG AGA CAG AGA A-3’;23 The negative control shRNA sequence was as follows: 5’-TTC TCC GAA CGT GTC ACG T-3’.
To conduct Meox1 inhibition experiments, we infected HCC cells (Huh7 and Hep3B) with recombinant lentivirus carrying its specific shRNA (sh-Meox1) and negative control shRNA (sh-NC). Stable cell clones with Meox1 knockdown were established via 0.6 μg/mL puromycin selection for Huh7 cells and 0.3 μg/mL puromycin for Hep3B cells.
Western blotting
Radioimmunoprecipitation (RIPA) lysis buffer (Solarbio, Beijing, China) containing freshly added phenylmethyl sulphonyl fluoride (PMSF; Solarbio) was used to extract total protein from cells based on the manufacturer’s instructions. A bicinchoninic acid assay (BCA) protein assay kit (Solarbio) was used to test the concentrations of protein according to the manufacturer’s guidelines. The protein samples underwent separation utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then moved onto a nitrocellulose membrane (MilliporeSigma, St. Louis, USA), followed by blocking with 5% non-fat dry milk. Primary antibodies, including anti-Meox1 (1:1,000; cat. No. ab105349; Abcam) and anti-GAPDH (1:10,000; cat. No. ab181602; Abcam), were used to conduct the experiments. The secondary antibody used was horseradish peroxidase (HRP)-conjugated Affinipure goat anti-rabbit immunoglobulin G (IgG) (H+L) (1:5,000; cat. No. SA00001-2; Proteintech, San Diego, USA). The immunoblots were detected as previously described.22
Cell proliferation assay
Cell Counting Kit-8 (CCK-8; Dojindo Lab, Kumamoto, Japan) was employed to assess cell proliferation. The analysis was conducted according to the previously described methods.22
Colony formation assay
Six-well plates containing 500 cells/well were used to seed cell lines, which were then routinely cultivated for 2 weeks. The plates were discarded after a visible cell colony formed, rinsed with phosphate-buffered saline (PBS) and treated with methyl alcohol for 30 min, followed by staining with 0.5% crystal violet for 20 min to count the cell number. The colony formation efficiency was calculated using the following formula: colony number divided by seeded cell number multiplied by 100%.
Cell migration and invasion assay
Eight-micrometer Transwell® chambers containing polycarbonate filters (Corning Company; Corning, USA) were used to conduct the cell invasion and migration assays. For the invasion assay, the chambers were pre-coated with Matrigel (BD Biosciences, Franklin Lakes, USA), and the migration assay was carried out without Matrigel. The analysis was conducted as previously described.22
Statistical analyses
Statistical analysis was performed using IBM SPSS 26.0 software (IBM Corp., Armonk, USA). Pathological specimens were collected from 25 patients with HCC undergoing surgical operations at the 980th Hospital of PLA Joint Logistics Support Force. Three separate independent runs of each cell function experiment (cell proliferation, colony formation, cell invasion and migration assay) were conducted. The frequency or medians (interquartile range (IQR)) was used to represent the values. The Fisher’s exact test or non-parametric Mann–Whitney U test or Kruskal–Wallis H test were utilized for examining the data, as appropriate. The Spearman’s correlation analysis was utilized for examining the relationship between the 2 variables. Survival curves for HCC were produced using the Kaplan–Meier method and the log-rank test. All comparisons were 2-tailed, and a p < 0.05 denoted statistical significance.
Results
High expression of Meox1 in HCC tissues
To evaluate the expression level of Meox1 in human HCC tissues, we conducted an IHC analysis. The results revealed that Meox1 was primarily localized in the nucleus (Figure 1A). In the 25 HCC tissues, the Meox1 showed negative expression in 2, while weak, moderate and strong positive expression in 6, 14 and 3, respectively (Table 1). Among the 25 paracancerous tissues, 4 were negative and 13 and 8 were weakly and moderately positive, respectively, and there was no strongly positive expression (Table 1). Meox1 expression was significantly higher in HCC tissues compared to paracancerous tissues (p < 0.05, Mann–Whitney U test). Furthermore, we compared the percentage of positive cells expressing Meox1 in hepatocellular carcinoma (HCC) and paracancerous tissues (Figure 1B). The median (IQR) of positive cells percentage of Meox1 in HCC was 0.7318 (0.2904–0.8486), while in paracancerous tissue it was 0.3607 (0.0932–0.663); a statistically significant distinction existed (p = 0.048, Mann–Whitney U test). The result indicate that Meox1 is highly expressed in HCC tissues.
High expression of Meox1 in HCC patients with cirrhosis
We further observed the expression level of Meox1 in HCC tissues of patients with and without cirrhosis. The results indicated that among 18 HCC patients with cirrhosis, Meox1 expression was low in 3 HCC tissues and high in 15. In the following 7 patients without cirrhosis, Meox1 expression was low in 5 HCC tissues and high in 2 (Table 2). The expression of Meox1 was markedly elevated in HCC tissues from patients with cirrhosis compared to patients without cirrhosis (p < 0.05, Fisher’s exact test).
We also observed the expression level of Meox1 in paracancerous tissues of patients with and without cirrhosis (Table 3). In 18 HCC patients with cirrhosis, 10 patients had low Meox1 expression while 8 had high expression, respectively. However, in 7 HCC without cirrhosis, Meox1 was expressed at low levels in 7 patients, and there was no high expression. The results show that there is no significant difference in Meox1 expression between paracancerous tissues of HCC with cirrhosis and HCC without cirrhosis (p = 0.057, Fisher’s exact test).
Meox1 expression level linked to poor progression in HCC
Statistical analysis revealed a significant relationship between Meox1 expression and BCLC stage (p < 0.05, Fisher’s exact test) as well as TNM stage (p < 0.05, Fisher’s exact test). However, Meox1 expression did not show any statistically significant relationships with other clinicopathological traits such as age, sex, tumor size, tumor number, vascular invasion, and lymph node metastasis (Table 4). In addition, Meox1 expression and clinical parameters (Table 5), such as alpha-fetoprotein (AFP), carcinoma embryonic antigen (CEA), CA125, CA199, prothrombin time (PT), albumin, as well as total bilirubin, did not significantly correlate, according to Spearman’s analysis. These results showed that abnormal expression of Meox1 may be correlated with the progression of HCC.
Gene expression of Meox1 in HCC
We further investigated the gene expression of Meox1 in HCC using the Gene Expression Profiling Interactive Analysis (GEPIA) database. The analysis revealed that the expression of the Meox1 gene was notably increased in 369 HCC tissues than in 160 normal tissues (Figure 2A). However, the survival analysis suggested that high Meox1 expression may not be significantly correlated with overall survival (OS) in HCC (Figure 2B).
Meox1 silencing suppressed HCC cell malignancy in vitro
To explore the role of Meox1 in HCC cell malignancy, Meox1 expression was reduced by infecting HCC cells (Huh7 and Hep3B cells) with recombinant lentiviruses containing its particular shRNA (sh-Meox1). As shown in Figure 3A and 3B, HCC cell lines with stable Meox1 knockdown were successfully constructed, and the knockdown efficiency was confirmed. Initially, we observed the function of Meox1 in cell proliferation using CCK-8 assay. The results indicated that the growth rate of Huh7 cells with knockdown using 2 sh-Meox1 was significantly decreased compared to those in sh-NC and the blank control at 72 h, 96 h and 120 h (Figure 3C, p < 0.05, respectively, Kruskal–Wallis H test). The same phenomenon was observed in Hep3B cells (Figure 3D, p < 0.05, Kruskal–Wallis H test), indicating that Meox1 silencing suppressed the proliferation of HCC cells. In addition, colony formation assays revealed that the size and the number of colonies were markedly decreased in both Huh7 and Hep3B cells with Meox1 knockdown compared to those in the sh-NC and blank control groups (Figure 4, p < 0.05, Kruskal–Wallis H-test). The data suggested that Meox1 silencing inhibited colony formation of HCC cells and may exert a negative effect on cell self-renewal function.
Cell migration and invasion are important characteristics of malignancy.24, 25 Therefore, we assessed the contribution of Meox1 in HCC cell migration and invasion using a transwell (An experimental method used for the study of biological processes such as cell migration and invasion) assay (Figure 5). The findings demonstrate that the migration of cells significantly decreased when Meox1 was knocked down in both Huh7 and Hep3B cells, compared to the sh-NC and blank control groups. In Huh7 and Hep3B cells with Meox1 knockdown, the number of invading cells notably decreased, indicating that Meox1 regulates both migration and invasion properties in HCC cells. Based on the data, silencing Meox1 reduced the malignancy of HCC cells and may be critical for HCC development.
Discussion
The discovery of novel and effective therapeutic targets for HCC treatment is crucial for clinical application. Accumulated evidence has shown that Meox1 is closely related to the progression of several cancers, including lung,15 breast17, 18 and ovarian cancer.16 Studies have confirmed that Meox1 expression in tumors was increased. This study aimed to evaluate Meox1 expression in HCC and investigate how it affected the biological functions of HCC cells.
We first assessed the expression level of Meox1 in HCC through IHC and bioinformatics analysis. The findings demonstrated a notable upregulation of Meox1 in HCC tissues, aligning with previous studies conducted on lung, breast and ovarian cancer. Importantly, our observations indicated a notable increase in Meox1 expression levels in HCC tissues from patients with cirrhosis compared to those without cirrhosis. Advanced liver fibrosis and cirrhosis are principle risk factors for HCC, and up to 90% of cases are based on this background.26, 27 The tumor microenvironment (TME) has a vital function in progression of HCC. Liver fibrosis can promote HCC progression by regulating the TME, including cytokine secretion, immune surveillance, tumor angiogenesis, and extracellular matrix synthesis.28, 29 According to numerous research, Meox1 is crucial in organ fibrosis.23, 30, 31, 32, 33 More recently, 1 study found that Meox1 is a central regulator in the transformation of fibroblasts to profibrotic myofibroblasts and is necessary for tumor growth factor beta (TGFβ)-induced fibroblast activation.33 In addition, Meox1 may promote hepatic stellate cell (HSC) activation, which is a crucial event during liver fibrosis.33 We speculated that abnormal Meox1 expression might be closely linked to liver fibrosis as well as HCC and involved in the TME, but this hypothesis needs to be further explored.
Previous data from breast cancer have shown that elevated Meox1 is linked to an advanced tumor stage and poor OS.17 Another study on lung cancer also indicated that increased Meox1 promoted tumor progression and contributed to shorter OS, increased lymph node metastasis, and advanced stage of HCC, and was an independent poor prognosis predictive factor identified with Cox multivariate regression analysis.15 We further evaluated the potential implication of Meox1 in HCC, and clinical relevance analysis showed that high Meox1 expression was positively correlated with advanced tumor stage. However, the survival analysis from GEPIA databases indicated that high Meox1 expression may not be significantly correlated with OS in HCC. We speculate that this variation may be brought about by Meox1’s various tumor-specific actions.
Meox1 is considered an important transcription factor that promotes tumor cell growth in ovarian cancer.16 Inhibition of Meox1 expression also effectively suppressed the proliferation of lung cancer15 and breast cancer cells.17, 18 We constructed Meox1 stable knockdown HCC cell lines to examine Meox1’s role in HCC. It was found that inhibiting Meox1 expression significantly suppressed the proliferation and colony formation of 2 different HCC cell lines. These results imply that Meox1 may be crucial in regulating cell proliferation. Moreover, experiments with shRNA knockdown showed that downregulation of Meox1 expression reduced migration and invasion in 2 different HCC cell lines, indicating that Meox1 could regulate HCC cell metastasis. These results suggest that targeting Meox1 not only decreases the rapid proliferative behavior, but also suppresses the aggressive metastatic potential to reduce the malignancy of HCC cells, and in consideration of the significant correlation between the Meox1 and vascular invasion and advanced tumor stage in HCC, it is plausible that abnormal expression of Meox1 may accelarate the progression of HCC. Future investigations should involve HCC clinical research with large samples, and HCC animal experiments are warranted to verify the role of Meox1 in HCC. The detailed molecular mechanism of the role of Meox1 in HCC progression deserves further research.
Limitations
The study may have been conducted in a specific HCC model or population, which means that the results may not be applicable to other types of cancers or populations.
Conclusions
Our studies found that Meox1 was highly expressed in HCC tissues, especially in HCC with cirrhosis, and was closely correlated with advanced stage of HCC. Moreover, Meox1 silencing suppressed the proliferation, colony formation, migration, and invasion of HCC cells. Given the physiological significance of Meox1 in proliferation and metastatic features and the implications of these findings for the progression of HCC, targeting Meox1 may offer a possible strategy for adjuvant and combination therapies of HCC.
Supplementary data
The Supplementary materials are available at https://doi.org/10.5281/zenodo.10090681. The package includes the following files:
Supplementary Fig. 1 Kruskal-Wallis H test was utilized for examining the data from colony formation of Huh7 cells.
Supplementary Fig. 2 Kruskal-Wallis H test was utilized for examining the data from colony formation of Hep3B cells.
Supplementary Fig. 3. Kruskal-Wallis H test was utilized for examining the data from migration of Huh7 cells.
Supplementary Fig. 4. Kruskal-Wallis H test was utilized for examining the data from invasion of Huh7 cells.
Supplementary Fig. 5. Kruskal-Wallis H test was utilized for examining the data from migration of Hep3B cells.
Supplementary Fig. 6. Kruskal-Wallis H testwas utilized for examining the data from invasion of Hep3B cells.
Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Consent for publication
Not applicable.
