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Overexpression of MATN3 predicts poor prognosis of gastric adenocarcinoma: based on TCGA database

Background: Matrilin-3 (MATN3) was previously reported to in the cartilage extracellular matrix and had a role in the development and homeostasis of cartilage and bone. We evaluated the role of MATN3 from The Cancer Genome Atlas (TCGA) gastric adenocarcinoma dataset. Methods: The information of 406 patients was downloaded from the TCGA database. We analyzed the relationship between clinicopathologic variables and MATN3 with the Wilcoxon signed-rank test and logistic regression. The Kaplan-Meier and the Cox regression method was used to assess the association of clinicopathologic variables with overall survival (OS). The association between expression of MATN3 and biological pathway was performed using Gene Set Enrichment Analysis (GSEA). Results: MATN3 was overexpressed in gastric adenocarcinoma (GAC) than that in normal tissues (p﹤0.05). MATN3 overexpression was significantly associated with TNM stage (p= 0.012), and T stage (p﹤0.01). Kaplan-Meier survival analysis demonstrated that GAC with MATN3 high had a poorer prognosis compared with MATN3- low (p﹤0.01). The univariate analysis showed that MATN3- high was correlated with a statistically significant poor OS (HR: 1.06; 95% confidence interval [CI]: 1.02- 1.11; p = 0.014). The multivariate analysis showed that MATN3 was also independently associated with OS, with a HR of 1.08 (CI: 1.04- 1.13; p﹤0.01). GSEA showed that NOTCH, WNT, and MTOR signaling pathway were differentially enriched in MANT3 high expression phenotype. Conclusion: Overexpression of MATN3 mRNA level may be a potential non-favorable prognostic biomarker in GAC. Moreover, MATN3 may be a key molecular regulating NOTCH, WNT, and MTOR signaling pathways in GAC.


Introduction
Gastric adenocarcinoma (GAC) remains one of the most widespread tumors in the world. It was the fifth most frequently diagnosed cancer and the third leading cause of cancer death according to the data of epidemiological survey in 2018 [1]. Recurrence and metastasis is the main cause of death in patients with GAC, which seriously hinders the success of treatment [2]. GAC is a complicated multi-step disease. The pathogenesis of GAC involves genomic changes, including gene amplification, deletion, insertion or mutation, which ultimately trigger a series of epigenetic and genetic changes. Therefore, the key to the prognosis and treatment of gastric cancer is to identify key genes and understand their molecular mechanisms. The matrilin (MATNs) are a family of oligomeric extracellular matrix (ECM) proteins, which consist at least four related proteins (matrilin-1 to 4). This family is thought to take part in the formation of filamentous networks in the extracellular matrices of various tissues. Matrilin-3 (MATN3), which is composed of one vWFA domain, four EGF-like domains, and a C-terminal coiled-coil domain, is the simplest member of the matrilin family [3,4]. Previous studies have found that MATN3 protein is present in the cartilage extracellular matrix and plays important roles in the development and homeostasis of cartilage and bone [5]. Mutations in the gene result in multiple epiphyseal dysplasia [6,7].Until now, the expression level of MATN3 in malignant tumors, especially in GAC, remains unclear.
Thus, this study aimed to investigate the prognostic value of MATN3 expression in GAC based on the TCGA datasets. GSEA was performed to gain further insight into the biological pathways involved in GAC pathogenesis related MATN3 regulatory network.
Therefore, the purpose of this study was to evaluate the prognostic value of MATN3 expression in GAC based on TCGA, and further understand the GAC signaling pathway related to MATN3 regulation through GSEA.

Downloading mRNA data and analysis
The mRNA expression data and corresponding clinicopathologic data of the stomach projects were downloaded from TCGA website. We selected patients with pathological type of adenomas or adenocarcinomas. The expression differences for discrete variables were visualized using Boxplots [8]. Finally, The mRNA expression data of 406 samples with GAC and clinicopathologic data were analyzed (Table 1).

GSEA
In this study, the association between expression of MATN3 and biological pathway was performed using Gene Set Enrichment Analysis (GSEA v2.0) [9]. An ordered list of all genes according to their correlation with MATN3 expression was firstly generated, and samples from the TCGA datasets were divided into MATN3-high and -low groups. Default settings were used and permutation analysis (1000 times) was performed to determine thresholds for significance. False Discovery Rate (FDR) was calculated. When FDR is ﹤0.25, the gene set is considered significantly enriched.  were analyzed. METN3 in cancer tissues was significantly higher than that in normal tissues (Fig. 1A-B), and overexpression of MATN3 correlated significantly with the tumor clinical stage (p = 0.012) and T stage (p﹤0.01) (Fig. 1C-D). Logistic regression analysis revealed that when the samples were grouped to MATN3-high group and MATN3-low group by the median value (2.34), overexpression of MATN3 was associated with poor clinicopathologic variables ( Table  2).

Statistical analysis
Overexpression of MATN3 in GAC was also significantly associated with high expression was more likely to progress to more advanced stage than GAC with MATN3 low expression. metastasis were also associated with poor prognosis (Table 3).

Furthermore, multivariate analysis showed expression level of MATN3
was an independent prognostic factor, with a HR of 1.08 (CI: 1.04-1.13, p﹤0.01) ( Table 4). Table 3. Univariate analysis of the relationship between OS and clinicopathologic variables in TCGA patients MATN3, as a member of the matrilin protein family, is a noncollagenous extracellular matrix [10,11]. According to previous reports [5,11,12] [12]. Existing data [13,14]  Zhou et al. [15] found that six gene signatures, including MATN3, could be used to predict recurrence in stage III and IV patients after surgery plus chemoradiotherapy. Wu et al. [16] also found MATN3 overexpression in GAC and its prognostic significance in OS of patients. In this study, MATN3 levels were significantly increased in stage IV patients and T4 patients, and multivariate analysis suggested that MATN3 was an independent prognostic factor (p= 0.000). Together, all these findings suggested that high expression of MATN3 could indicate a poor prognosis for GAC patients, and MATN3 might be a pivotal target gene involved in the process of GAC cell growth and metastasis. However, the molecular mechanisms underlying differentiation remained unclear.
In our study, we observed that MATN3 high expression phenotype was associated with the following signaling pathways: NOTCH, MAPK, and MTOR signaling pathways. MATN3 low expression phenotype was associated with P53 signaling which has been identified as one of the key signaling pathways that inhibit gastric cancer. Notch receptor plays different roles in the genesis and development of tumor [17]. The activation of Notch 1 and Notch 2 not only promotes the progression of gastric cancer [18,19], but also inhibits the invasion of gastric cancer [20,21]. NOTHC 3 contributes to glandular differentiation of gastric cancer [22], Notch4 promotes GC growth through the activation of Wnt1/β-catenin signaling [23]. In addition, studies have confirmed that MTOR signaling pathway is activated in human gastric cancer and promotes cell proliferation. MTOR signaling pathway decreases after Notch inhibition, suggesting that MTOR is located downstream of Notch in gastric cancer cells [24]. In this paper, it is also proved that NOTCH, WNT and MTOR signaling pathways are active in gastric cancer cells with high expression of MATN3. However, the relationship between the expression of MATN3 and the above signaling pathways is reported for the first time, and its underlying regulatory mechanism needs to be further clarified.
However, using mRNA to predict protein expression was far from perfect [25]. This study did not evaluate the correlation between MATN3 mRNA expression and MATN3 protein expression, which was also the limitation.

Acknowledgments
The results shown here are in whole or part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/.

Funding
None.

Author contributions
Zheng GL collected the data, analyzed of data and wrote the manuscript; Zhao Y made critical revisions to the manuscript; Zheng ZC made substantial contributions to the conception, design, and coordination of the study and gave final approval of the version; all authors read and approved the final manuscript.

Ethics approval and consent to participate
Since this study only involves analysis of publically available database (TCGA) and does not contain any identifying patient information, the ethical approval of this study by Institutional review board (IRB) is not required. The TCGA data did not include the use of human subjects or personal identifying information. Thus, no informed consent was required for this part of the study.