Abstract
Background/Aim: Long non-coding RNAs (lncRNAs) establish gene regulatory networks in different human cancers and are involved in tumorigenesis. lncRNA LINC00152 is over-expressed in several malignant tumors and involved in tumorigenesis; however, its underlying regulatory mechanisms remain unclear. Mesothelioma, a cancer originating from mesothelial cells, is highly aggressive with a poor prognosis. Therefore, identification of new therapeutic targets is necessary for mesothelioma treatment. Materials and Methods: Here, we conducted bioinformatics analyses of LINC00152 and enhancer of zeste homolog 2 (EZH2) expression levels and their correlation with the prognosis of patients with mesothelioma. Small interfering RNAs targeting LINC00152 and EZH2 were transfected into mesothelioma cell lines to analyze their biological functions and regulatory mechanisms. Results: High LINC00152 expression was associated with a poor prognosis of patients with mesothelioma. LINC00152 knockdown inhibited the proliferation, migration, and invasion of mesothelioma cell lines. These results suggest that LINC00152 is a tumor-promoting factor in mesothelioma. EZH2 is highly expressed in mesothelioma and other malignancies. Direct interaction between LINC00152 and EZH2 is associated with cancer development and progression. When EZH2 expression was suppressed, LINC00152 knockdown did not suppress the proliferation, migration, and invasion of mesothelioma cells. Therefore, the tumor-promoting effect of LINC00152 in mesothelioma was dependent on EZH2 expression. Conclusion: LINC00152 promotes mesothelioma cell proliferation, migration, and invasion in cooperation with EZH2, highlighting its potential as an effective therapeutic target for mesothelioma.
Mesothelioma is a malignancy that arises from mesothelial cells on the surface of serous membranes pleura, peritoneum, pericardium, and tunica vaginalis; it is highly aggressive with a poor prognosis (1). Occupational or environmental exposure to asbestos is a major cause of mesothelioma; however, exposure to low asbestos concentrations has also been reported to cause mesothelioma (2, 3), warranting further investigation of its genetic dysregulation mechanism. Asbestos is widely used as a building material and industrial product owing to its heat-resistant and insulating properties. The latency period between asbestos exposure and mesothelioma development can extend for several decades (4, 5). Mesothelioma is treated with multimodal therapy involving surgery, chemotherapy, and radiation therapy; however, the prognosis of patients remains poor worldwide (6). Understanding the genetic mechanisms is essential to develop effective therapies for mesothelioma. In recent years, the functions of non-coding RNAs (ncRNAs), which are not translated into proteins, in cancer have been elucidated and their involvement in the regulation of gene expression at various stages has attracted considerable attention (7, 8). However, the research on mesotheliomas is insufficient compared with that on other malignant tumors.
Long ncRNAs (lncRNAs) are ncRNAs longer than 200 nucleotides that are associated with various biological processes, such as cell proliferation, differentiation, drug resistance, and epigenetic regulation (9). lncRNAs are aberrantly expressed in many malignancies and involved in the regulation of gene expression at the transcriptional and post-transcriptional levels, attracting attention as potential therapeutic targets and biomarkers (10). Specifically, PVT1, MALAT1, and GAS5 lncRNAs play crucial roles in various biological functions, such as cell proliferation and invasion, in mesothelioma (11-13). lncRNAs are classified based on their location relative to the protein-coding gene regions (14). Although many lncRNAs share sequences with coding gene regions, long intergenic non-coding RNAs have independent transcription units that do not overlap with the coding gene region (14, 15).
lncRNA LINC00152 (also known as CYTOR) is a tumor promoter that regulates gene expression in many human cancers (16). LINC00152 expression is significantly upregulated in tumor tissues and promotes tumor progression in gastric (17), esophageal (18), pancreatic (19), and lung (20) cancers. A comprehensive molecular characterization study reported an association between LINC00152 expression and poor prognosis of patients with mesothelioma (21). However, the functional mechanisms of LINC00152 in mesothelioma remain unknown. To the best of our knowledge, this is the first study to analyze the biological functions and elucidate the molecular mechanisms of LINC00152 in mesothelioma.
Enhancer of zeste homolog 2 (EZH2), a member of the polycomb-group family, functions as a histone methyltransferase that represses the transcription of target genes (22). EZH2 is over-expressed in many human cancers and acts as a promising therapeutic target (23). For example, EZH2 expression is significantly up-regulated in tumor tissues and promotes tumor progression in breast (24), gastric (25), colon (26), and prostate (27) cancers. Moreover, EZH2 is a positive marker of mesothelioma that distinguishes it from reactive mesothelial hyperplasia, and its over-expression indicates a poor prognosis (28, 29). EZH2 directly binds to LINC00152 and regulates its downstream gene expression to promote tumorigenesis (30). Here, we used mesothelioma cells to validate the effects of EZH2 suppression on the biological functions of LINC00152.
In this study, we evaluated the prognostic value of LINC00152 in patients with mesothelioma. First, we knocked down LINC00152 in mesothelioma cell lines to evaluate its biological functions. We further evaluated whether the biological functions of LINC00152 were dependent on EZH2.
Materials and Methods
Bioinformatics analysis. Expression levels of LINC00152 and EZH2 in multiple tumors, including mesothelioma, were analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA) database. Survival analysis according to LINC00152 and EZH2 expression levels in mesothelioma cells was conducted using the Encyclopedia of RNA Interactomes (ENCORI) database.
Mesothelioma cell lines and tissues. Two mesothelioma cell lines, ACC-MESO1 (RIKEN BioResource Research Center, Tsukuba, Japan) and CRL-5915 (American Type Culture Collection, Manassas, VA, USA), were used in this study. The medium and culture conditions were as previously described (31).
Transient transfection of mesothelioma cells. In this study, small interfering RNAs (siRNAs) were used to suppress the expression of specific genes. siRNAs were transfected into mesothelioma cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The following siRNAs were used: Silencer Select LINC00152 siRNA (s195584), Silencer Select EZH2 siRNA (s4916), and Silencer Select negative control #1 siRNA (NC; Cat# 4390843). All samples were purchased from Thermo Fisher Scientific.
Proliferation assay. Mesothelioma cells were transfected with siRNAs, seeded in a 96-well plate, and cell proliferation was evaluated over time. Cell proliferation counts were determined every 24 h based on the cellular ATP levels using the Cell Titer Glo 2.0 reagent (Promega, Madison, WI, USA), as previously described (31).
Transwell migration assay. siRNA-transfected mesothelioma cells were seeded into BD FluoroBlok culture inserts (BD Biosciences, Franklin Lakes, NJ, USA) with 8 μm pores, and the number of migrated cells was counted after 72 h. The migrated mesothelioma cells were stained and photographed under a fluorescence microscope as previously described (32).
Transwell invasion assay. A specific number of siRNA-transfected mesothelioma cells were seeded into BD FluoroBlok culture inserts (BD Biosciences) with 8 μm pores, and the number of invaded cells was counted after 72 h. Culture inserts were coated with the Cultrex Reduced Growth Factor Basement Membrane Extract (Cat# 3433; R&D Systems, Minneapolis, MN, USA). The invaded mesothelioma cells were stained and photographed under a fluorescence microscope as previously described (32).
Real-time reverse transcription-polymerase chain reaction. Next, RNA extraction, reverse transcription, and polymerase amplification were performed as previously described (31). Briefly, siRNA-transfected mesothelioma cells were cultured for 72 h before RNA extraction, and subsequent procedures were performed according to the manufacturers’ instructions. The following primers were used in this study: LINC00152 forward: 5′-GGACCTGATAACGGGAACCA-3′ and LINC00152 reverse: 5′-GGGCTGAGTCGTGATTTTCG-3′; EZH2 forward: 5′-AATCAGAGTACATGCGACTGAGA-3′ and EZH2 reverse: 5′-GCTGTATCCTTCGCTGTTTCC-3′; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward: 5′-ACAACTTTGGTATCGTGGA AGG-3′ and GAPDH reverse: 5′-GCCATCACGCCACAGTTTC-3′
Western blotting analysis. Protein extraction, electrophoresis, transcription, blocking, antibody reaction, and chemiluminescence detection were performed as previously described (31) using siRNA-transfected mesothelioma cells were cultured for 72 h before protein extraction. Anti-EZH2 (1:2,000; rabbit monoclonal; Cat# 5246; Cell Signaling Technology, Danvers, MA, USA) and anti-GAPDH (1:5,000; rabbit monoclonal; Cat# 2118; Cell Signaling Technology) primary antibodies were used in this study. Anti-rabbit IgG-HRP antibody (1:2,000; Cat# 7074; Cell Signaling Technology) was used as the secondary antibody.
Statistical analyses. All experiments were conducted in triplicate. Experimental data are presented as the mean±standard deviation. An unpaired Student’s t-test was used for statistical comparisons between groups. Statistical significance was set at p<0.05.
Results
LINC00152 is a poor prognostic factor for patients with mesothelioma. LINC00152 is a tumor-promoting factor in various human malignancies. Bioinformatics analysis of GEPIA data revealed high LINC00152 expression levels in various human cancers, including mesothelioma (Figure 1A). ENCORI data analysis revealed that high LINC00152 expression in mesothelioma was associated with poor prognosis (Figure 1B). These results suggest LINC00152 as a prognostic marker for patients with mesothelioma.
Expression and survival analyses of long non-coding RNA LINC00152. LINC00152 is highly expressed in many malignancies and acts as a poor prognostic factor for mesothelioma. (A) Gene expression profiles of LINC00152 in paired normal and tumor tissue samples (detailed cancer abbreviations are available at: https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/tcga-study-abbreviations). Red and green dots indicate the tumor and normal tissue samples, respectively. (B) Survival analysis of LINC00152 in mesothelioma. Overall survival of patients with mesothelioma was significantly different between the high and low LINC00152 expression groups.
LINC00152 promotes mesothelioma cell proliferation, migration, and invasion. LINC00152 was knocked-down in mesothelioma cell lines for biological function analysis (Figure 2). LINC00152 siRNA was transfected into mesothelioma cells to achieve the suppression of LINC00152 expression. LINC00152 mRNA expression was suppressed by 94.9 and 98.7% in the ACC-MESO1 and CRL-5915 mesothelioma cell lines, respectively. Biological function analysis revealed that LINC00152 knockdown significantly suppressed the proliferation, migration, and invasion of mesothelioma cells. These results suggest that LINC00152 promotes tumor progression in mesothelioma.
Functional analysis of LINC00152. Compared to the negative control (NC) small interfering RNA (siRNA), transfection with LINC00152 siRNA suppressed LINC00152 expression levels in the ACC-MESO1 and CRL-5915 mesothelioma cell lines. (A) Validation of LINC00152 knockdown. LINC00152 mRNA expression was determined via reverse transcription-polymerase chain reaction. LINC00152 functions as a cancer-promoting factor. (B) Proliferation assay. Transfection with LINC00152 siRNA significantly suppressed mesothelioma cell proliferation compared to transfection with NC siRNA. Vertical axis represents the relative luminescence level, which reflects the number of viable cells. Relative numbers of viable cells at 0, 24, 48, and 72 h are shown. (C) Transwell migration assay. LINC00152 knockdown inhibited mesothelioma cell migration more than transfection with NC siRNA. The number of cells that passed through the 8 μm pores in the culture insert was measured 72 h after transfection. (D) Transwell invasion assay. LINC00152 knockdown inhibited mesothelioma cell invasion more than transfection with NC siRNA. The number of cells that passed through the 8 μm pores of the matrigel-coated culture insert was measured 72 h after transfection. *p<0.05.
EZH2 is a poor prognostic factor for patients with mesothelioma. EZH2 acts as a tumor-promoting factor in various human malignancies. Bioinformatics analysis of GEPIA data revealed high EZH2 expression levels in various human cancers, including mesothelioma (Figure 3A). ENCORI data analysis revealed that high EZH2 expression in mesothelioma was associated with poor prognosis (Figure 3B). These results suggest EZH2 as a prognostic marker for patients with mesothelioma.
Expression and survival analyses of enhancer of zeste homolog 2 (EZH2). EZH2 is highly expressed in many malignancies and acts as a poor prognostic factor for mesothelioma. (A) Gene expression profiles of EZH2 in paired normal and tumor tissue samples (detailed cancer abbreviations are available at: https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/tcga-study-abbreviations). Red and green dots indicate the tumor and normal tissue samples, respectively. (B) Survival analysis of EZH2 in mesothelioma. Overall survival of patients with mesothelioma was significantly different between the high and low EZH2 expression groups.
Promotion of mesothelioma cell proliferation, migration, and invasion by LINC00152 is dependent on EZH2. As LINC00152 directly binds to EZH2 and regulates the expression of its target genes, we examined whether LINC00152 exerts its tumor-promoting effects when EZH2 expression is suppressed (Figure 4). EZH2 siRNA was transfected into mesothelioma cells to suppress EZH2 expression. EZH2 mRNA expression was suppressed by 76.0 and 66.8% in the ACC-MESO1 and CRL-5915 mesothelioma cell lines, respectively. Similarly, EZH2 expression was reduced at the protein level in both cell lines. As expected, EZH2 expression was not altered by LINC00152 knockdown. This suggests that LINC00152 and EZH2 bind to each other but do not affect their expression levels. Next, LINC00152 siRNA was transfected into mesothelioma cells with EZH2 suppression and biological functional analysis was performed. No significant effects of LINC00152 knockdown were observed on mesothelioma cell proliferation, migration, and invasion. These results suggest that EZH2 is necessary for the tumor-promoting effects of LINC00152.
Functional analyses of EZH2 and LINC00152 knockdown. Compared to transfection with NC siRNA, transfection with EZH2 siRNA suppressed EZH2 expression in the ACC-MESO1 and CRL-5915 mesothelioma cell lines. (A) mRNA validation of EZH2 knockdown. EZH2 mRNA expression was confirmed via reverse transcription-polymerase chain reaction. (B) Protein validation of EZH2 knockdown. EZH2 protein expression was confirmed via western blotting. LINC00152 requires EZH2 to promote cancer progression. (C) Proliferation assay. When EZH2 expression was suppressed, LINC00152 siRNA transfection did not significantly affect the mesothelioma cell proliferation. Vertical axis represents the relative luminescence level, which reflects the number of viable cells. Numbers of viable cells at 0, 24, 48, and 72 h are shown. (D) Transwell migration assay. When EZH2 expression was suppressed, LINC00152 siRNA transfection did not significantly affect the mesothelioma cell migration. The number of cells that passed through the 8 μm pores in the culture insert was measured 72 h after transfection with each siRNA. (E) Transwell invasion assay. When EZH2 expression was suppressed, LINC00152 siRNA transfection did not significantly affect the mesothelioma cell invasion. The number of cells that passed through the 8-μm pores of the matrigel-coated culture inserts was measured 72 h after transfection with each siRNA. NC: Negative control. NS: Not significant. *p<0.05.
Discussion
In this study, we showed that LINC00152 acts as a tumor-promoting factor in mesothelioma, and its function is dependent on EZH2. Although the number of patients with mesothelioma is expected to increase worldwide, especially in developing countries, in the future, the efficacy of current therapeutics is inadequate and only few treatment options are available; hence, mesothelioma remains a lethal malignancy (33, 34). Although the molecular understanding of mesothelioma is essential for more accurate and rapid diagnosis and effective treatment, research on mesothelioma has been limited compared to that on other malignant tumors, necessitating the elucidation of molecular mechanisms underlying mesothelioma pathogenesis and progression in future studies. In particular, research on lncRNAs, a popular topic in oncology, has high beneficial value (35, 36). To the best of our knowledge, this is the first study to investigate lncRNA LINC00152 and its relationship with EZH2 in mesothelioma, which is a promising biomarker for mesothelioma.
In this study, high expression of LINC00152 was associated with the poor prognosis of patients with mesothelioma. Moreover, LINC00152 knockdown inhibited the proliferation, migration, and invasion of mesothelioma cell lines. Our results suggest LINC00152 as a tumor-promoting factor in mesothelioma. LINC00152 over-expression has been shown to promote tumorigenesis in various human malignancies (16, 37). This is consistent with the findings of this study on mesothelioma. ncRNAs, previously considered as “junk”, have various tumor-promoting or -suppressing roles and construct complex gene expression regulatory networks (38-40). In particular, lncRNAs, major components of ncRNAs, participate in cancer development and progression by binding to various proteins, mRNAs, and DNA via regulatory interaction sites and may be used in novel therapeutic strategies (41).
LINC00152 knockdown did not significantly suppress the proliferation, migration, and invasion of EZH2 knocked-down mesothelioma cell lines. These results indicate that the tumor-promoting effects of LINC00152 depend on EZH2. Several lncRNAs directly bind to EZH2 and down-regulate the expression levels of tumor suppressor genes, thereby affecting cancer functions (42). For example, in esophageal cancer, LINC00152 interacts with EZH2 to regulate the epithelial–mesenchymal transition and drug resistance by regulating ZEB1 expression (43). LINC00152 interacts with EZH2 to inhibit IL24 transcription in lung adenocarcinoma (44). EZH2 is highly expressed in mesothelioma, and its role in differentiating mesothelioma from reactive mesothelial hyperplasia has attracted attention (28, 45). EZH2 inhibitors are promising treatment options for mesothelioma (29). LINC00152 may be useful in identifying patients with mesothelioma who will benefit the most from EZH2 inhibitor treatment. Therefore, LINC00152 is not only a potential therapeutic target but also a potential biomarker for predicting prognosis and therapeutic efficacy of mesothelioma.
Conclusion
In conclusion, we found that LINC00152 acted as a prognostic factor and promoted cell proliferation, migration, and invasion in mesothelioma. Furthermore, co-expression of LINC00152 with EZH2 was found to be important for tumor progression. This study provides new insights into the roles of lncRNAs in the gene regulatory networks of mesothelioma carcinogenesis and progression. Moreover, our findings indicate the biological functions of LINC00152 in mesothelioma progression and suggest its potential as a therapeutic target for this disease.
Acknowledgements
The Authors are grateful to Yukari Go and Naomi Fukuhara for their technical assistance and administrative support. The Authors would also like to thank Editage (www.editage.com) for English language editing.
Footnotes
Authors’ Contributions
Ihiro Endo designed the study, performed the experiments, analyzed the data, interpreted the results, and prepared the manuscript. Vishwa Jeet Amatya designed and supervised the study and interpreted the results. Yukio Takeshima designed and supervised the study. Kei Kushitani, Tetsuya Nakagiri, and Kohei Aoe collected the data and samples. All Authors have approved the final manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest related to this study.
Funding
This work was supported by the JSPS KAKENHI Grant 22K06980.
- Received October 19, 2023.
- Revision received November 13, 2023.
- Accepted November 14, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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