MRPS23 is a novel prognostic biomarker and promotes glioma progression

The MRPS23 expression in human cancer

The TIMER [5], TCGA, GTEx database and UALCAN database were used to examine the expression of MRPS23 in pan-cancer tissue [6].

The prognosis and clinical information of MRPS23 in pan-cancer

The GEPIA databases and PrognoScan databases [7] were employed to analysis the OS, DSS and PFI of MRPS23 in pan-cancer. The MRPS23 gene mutation information was analyzed via cBioPortal [8].

The relationship between stem cell and RNA modification of MRPS23 in pan-cancer

We downloaded MRPS23 gene expression and clinical data from UCSC (https://xenabrowser.net/) [9]. We employed the GeneMANIA and STRING database to construct gene–protein interactions network of MRPS23.

Immunological functions analysis

The TIMER and XCELL tools were used to employ the MRPS23 immunological roles [10]. We adopted the TISIDB to detach the relationship between MRPS23 expression and immune modulator, obtained TMB, and MSI scores from TCGA.

Cell lines and cell culture

Cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM medium supplemented with 10% FBS. All the cells were cultured at 37° C with 5% CO₂. MRPS23 siRNA kits (si-MRPS23#1: 5’-GUGUAUGGGUCUGGUCAAATT-3’; si-MRPS23#2: 5’-GAAAUCCGAACACUUGAGUTT-3’) and negative control siRNAs (si-NC: 5’-UUCUCCGAACGUGUCACG UTT-3’) were purchased from GenePharma (China).

CCK8 assays

For CCK8 assay, U87 and U251 cells were cultured in a 96-well plate (2,000 cells per well) supplemented with 200 μL of RPMI-1640 medium containing 10% FBS for 24, 48, 72 and 96 h, and measured with a CCK8 kit (Promega, Madison, WI, USA).

MRPS23 expression in pan-cancer

First, we excavated TIMER database to examine the MRPS23 expression level in pan-cancers, indicating that MRPS23 was increased in diverse human cancer, especially in gliomas (Figure 1A). Next, we combined the TCGA and GTEx cancer database, and found that MRPS23 was up-regulated in glioma than in paired adjacent normal tissues (Figure 1B).

The prognosis values of MRPS23 in pan-cancer

We further explore the prognosis of MRPS23 in different tumor types. We found that increased MRPS23 expression correlated with poor OS for glioma, HNSC, KICH, LAML, LIHC, and oral squamous cell carcinoma (OSCC) (Figure 2A), poor DSS in glioma, HNSC, KICH, KIRP, and OSCC (Figure 2B), and poor PFI in adrenocortical carcinoma (ACC), glioma, HNSC, KICH, KIRP, LIHC, and OSCC (Figure 2C).

MRPS23 could act as a potential diagnostic biomarker in pan-cancer

The ability of ROC curves to serve as diagnostic markers has been previously reported in a large number of literatures. We continue to analyze the ROC curve values of MRPS23 in different tumor types. Results showed that MRPS23 (AUC>0.75) for diagnosing BLCA, CESC, CHOL, COAD, LAML, colon adenocarcinoma/rectum adenocarcinoma esophageal carcinoma (COADREAD), GBM, THCA, glioma, HNSC, KIRP, LIHC, THYM, OSCC, OV, SKCM, LUAD, STAD, READ, PAAD, SKCM, DLBC, TGCT, LUSC and UCS (Figure 3A–3F).

Gene mutation landscape of MRPS23

We analyzed the MRPS23 mutational data. In cBioPortal, the mutation frequency was higher BRCA, pleural mesothelioma, endometrial cancer, BLCA, HNSC, and mature B-Cell neoplasms than in other types of cancer (Figure 4A). In addition, amplification was the most frequent alteration type (Figure 4B). MRPS23 has different mutation types in different tumors (Figure 4C). Moreover, DNA methylation analysis indicated that MRPS23 expression may be regulated by DNA methylation in BLCA, BRCA, CHOL, HNSC, LIHC, PRAD, READ, LUAD, and THCA (Figure 4D). All these results indicate that MRPS23 genetic alterations affect MRPS23 prognostic ability. MRPS23 had a positive relationship with TMB in LUAD, BRCA, glioma, LGG, STES, SARC, KIPAN, STAD, KIRC, LUSC, THYM, and BLCA (Figure 5A). MRPS23 had positive relationship with MSI in CESC, STES, SARC, KIRP, UCEC, and KIRC, and had negative relationship with glioma, LUAD, COAD, COADREAD, PRAD, LUSC, and OV (Figure 5B). Collectively, these results confirmed that MRPS23 can influence antitumor immunity.

Immune cell infiltration of MRPS23

Immune cells have an important role in tumor development [11]. TIMER database was used to reveal the relationship between the infiltration levels and CCDC50 expression in 32 types of cancers. In 30 kinds of cancers, the expression of MRPS23 was highly correlated with 6 major immune cells (Figure 6A). MRPS23 expression level had high correlation with the stroma score in 25 types human cancer, the microenvironment in 27 types cancer, the 38 types of immune cells in 32 types cancer, and the immune score in 24 types cancer (Figure 6B).

Stem cell and RNA modification analysis of MRPS23

It has a strong correlation between stem cells and malignant progression of cancer [12]. Here, we explore the relationship between MRPS23 expression level and stem cell. We found MRPS23 expression was positively correlated with CESC, LAML, BRCA, COADREAD, LIHC, STES, SARC, READ, OV, TGCT, PCPG, KIPAN, SKCM, and BLCA (Figure 7A).

RNA modification plays critical role in normal development and tumorigenesis [13]. The correlation between RNA modification regulators and MRPS23 in cancer was assessed using the TCGA TARGET GTEx. We revealed the relationship between MRPS23 expression and RNA modification regulators of pan-cancer. MRPS23 expression was markedly correlated with m1A, m5C and m6A in many types of cancers (Figure 7B).

Interaction network of MRPS23 in pan-cancer

The MRPS23 gene-gene interaction network is analyzed via GeneMANIA (Figure 8A). Next, we conducted MRPS23 interaction-protein network of the seed gene using STRING. As expected, several nodes and edges were obtained in the PPI network (Figure 8B).

Clinical characteristics of MRPS23 in glioma

The relationship between MRPS23 expression level and glioma pathology was also explored. High MRPS23 expression level was correlated with adverse clinical features (Figure 9A and Table 1). Up-regulated MRPS23 had a worse OS in of glioma subgroups, including gender, IDH status (WT), 1p/19q codeletion (non-codel), histological type (astrocytoma), age (<=60), race (white) (Figure 9B). Cox regression analyses showed that WHO grade and MRPS23 expression level were associated with glioma prognosis (Tables 2–4). These confirmed that MRPS23 has an important role in glioma progression. A nomogram was contrasted to confirm MRPS23 as a glioma biomarker to predict OS, DSS, and PFI. As shown in (Figure 10A–10F), the calibration curves had predictions for the three nomograms for clinical outcomes (1-, 3-, and 5-years) (Figure 10A–10F). Therefore, it is expected to become a model to predict survival of glioma patients with MPRS23.

Biological functions of MRPS23 in glioma

To analyze GO and KEGG enrichment, we discovered the top 50 similar genes related to MRPS23 in glioma using the GEPIA database. GO analysis results confirmed that MRPS23 mainly participated in Cdc73/Paf1 complex, cellular protein complex localization, SMN-Sm protein complex, negative regulation of fibroblast proliferation, regulation of protein binding, structural constituent of ribosome, nuclear import, mitochondrion, and mitochondrial translational elongation (Figure 11A, 11B). In summary, MRPS23 plays a crucial role in regulating glioma malignant progression.

Down-regulation of MRPS23 suppresses the malignant phenotype of glioma

The biological function of MRPS23 in glioma progression has been further explored and verified. We found that compared with normal astrocytes (NHA), MRPS23 was upregulated in glioma cell lines and glioma tissues (Figure 12A and Supplementary Figure 1A). We used CGGA database analysis and found that high expression of MRPS23 was associated with adverse clinical characteristics and poor prognosis in glioma patients (Supplementary Figure 1B–1F). We further used siRNA of MRPS23 to down-regulate its expression in U87 and U251 cells. The knockdown efficacy was verified via qRT-PCR by using cell lines expressing a negative control (Figure 12B). As expected, MRPS23 knockdown inhibited the proliferation of U87 and U251 cells (Figure 12C, 12D). The migration ability of glioma cells was significantly inhibited after MRPS23 gene knockdown (Figure 12E, 12F). We also found that the expression level of MRPS23 was significantly positively correlated with the expression of m6A modification-related genes (Supplementary Figure 1G–1I). These results support an oncogenic role of MRPS23 in glioma.