High PRMT6 expression Associated With Prognosis and Immune Inltration in Glioma

Glioma is characterised by easy invasion of surrounding tissues, high mortality and poor prognosis. Moreover, the prognosis of glioma is getting worse and worse with the increase of grade, which is not optimistic. Therefore, biological markers for glioma are needed in clinical to detect and evaluate the situation and prognosis of patients with glioma. In many studies, we have found that the protein arginine methyltransferase 6 (PRMT6) expression is elevated in various tumors, which is associated with prognosis of patient. However, there has been no report or study on the role of PRMT6 in glioma. Stomach adenocarcinoma (STAD), Testicular Germ Cell Tumors (TGCT), Thyroid carcinoma (THCA), Uterine corpus endometrial carcinoma (UCEC), Uterine Carcinosarcoma (UCS). There were also some negatively correlated tumors: Kidney renal clear cell carcinoma (KIRC), Acute Myeloid Leukemia (LAML), Kidney Chromophobe (cid:0) KICH (cid:0) , Kidney renal papillary cell carcinoma (KIRP). calculated the C-index, which was 0.84 (95%CI:0.865-0.815) in the nomogram of TCGA, and 0.772 (95%CI:0.801-0.743) in the nomogram of CGGA. The calibration curves of the 1-year and 3-year survival rates of the two models were drawn respectively, and the results showed that the two models had better validation performance.


Abstract Background
Glioma is characterised by easy invasion of surrounding tissues, high mortality and poor prognosis. Moreover, the prognosis of glioma is getting worse and worse with the increase of grade, which is not optimistic. Therefore, biological markers for glioma are needed in clinical to detect and evaluate the situation and prognosis of patients with glioma. In many studies, we have found that the protein arginine methyltransferase 6 (PRMT6) expression is elevated in various tumors, which is associated with prognosis of patient.
However, there has been no report or study on the role of PRMT6 in glioma.

Methods
In this study, we used various tumor-related databases to analyze the mechanism of PRMT6 in tumors, especially gliomas, from bioinformatics, and carried out relevant experimental veri cation with tumor tissues extracted from patients during surgery. Besides, we analyzed the relationship between PRMT6 expression and immune in ltration and immune-related cells, and discussed the possible mechanisms. We also discussed the role of PRMT6 expression in glioma from mutation, clinical indicators, enrichment analysis, and immunohistochemical results.

Results
PRMT6 is signi cantly differentially expressed in multiple tumors, which is associated with survival and prognosis. Especially in gliomas, the PRMT6 expression gradually increased with the grade increasing. Besides, PRMT6 can be used as an independent prognostic risk factor in the nomogram and has been veri ed in various databases.

Conclusions
Our results indicate that high PRMT6 expression is a potential biomarker for predicting prognosis and progression of glioma.

Background
Glioma is the most common and aggressive tumors in the central nervous system, accounting for 70-80% among primary malignancy tumors [1][2][3]. Low grade glioma (LGG) is a kind of glioma with slow growth at the initial stage [4], while glioblastoma multiforme (GBM) is easy to invade with high recurrence rate [5]. Currently, treatments of glioma are mainly surgery and chemoradiotherapy, and the relationship between surgical resection range and prognosis is controversial. However, glioma is more likely to recrudesce and respond less well to treatment, leading to a risk of postoperative seizures [6,7]. At the molecular level, the molecular markers of glioma mainly include IDH mutation, 1p/19q co-deletion [8], MGMT promoter methylation [9] and TP53 mutation.
However, the pathogenesis and molecular of glioma are still poorly understood and need a lot of research. It originates out of considerable signi cance and the urgent need to study tumor markers at the molecular level. Recently, more and more attention has been paid to the important role of immune cell in ltration in glioma. Some studies have indicated that in ltration of immune cells in gliomas can promote the invasion and progression of gliomas [10,11]. If macrophages have a high proportion in tumor tissue, the interaction of tumour-in ltrating T cells and multiple signalling pathways remains to be developed, such as the inhibitory effect of PD-1 on in ammatory response [12]. Even though with these immune mechanisms, immunotherapy for gliomas remains to be further explored.
In the nucleus, protein arginine methylation is a post-translational modi cation involved in signal transduction [13], which is catalyzed by the protein arginine methyltransferase (PRMT). There are eleven known types of PRMT, which can fall into three categories [14].
PRMT6 produces asymmetric dimethylarginine with unique characteristics of self-methylation [15], responsible for the methylation of histone H3R2 and wide expression in various tissues of the body [16]. As a result, PRMT6 had been studied in many tumors. According to PRMT6's unique biological role, many studies have concluded that it can promote the development of cancer through different signaling pathways or in ammatory cells [17]. Recent studies had reported that PRMT6 promotes the proliferation of endometrial cancer cells through the AKT/mTOR pathway, further promoting carcinogenesis [18]. Additionally, there were also related studies on lung cancer, hepatocellular carcinoma, colon cancer and prostate cancer [19]. Whereas, the mechanism of PRMT6 in gliomas is not precise, with insu cient immune-related studies.
At present, there have no studies being conducted on the role of PRMT6 in glioma. Based on multiple tumor-related public databases, we explored the potential mechanism and role of PRMT6 in gliomas in terms of immune mechanism, epigenetics and clinical prognosis. Our analysis revealed that PRMT6 is a potential tumor marker for glioma, providing important evidence for its epigenetic and immunological correlation.

Materials And Methods
Analysis on gene expression level Genotype-Tissue Expression (GTEx) database mainly includes gene expression in healthy human tissues and organs [20]. We obtained the PRMT6 expression in various normal tissues from this database. In Figure 1a, the X-axis lists 31 kinds of tissues and the Y-axis represents the PRMT6 expression calculated by Log2 (TPM+1).
The Cancer Genome Atlas (TCGA) database is a comprehensive database, containing not only the molecular level data of dozens of primary tumors, but also the pertinent clinical data. First, we selected the data set from the TCGA database with the number of normal samples greater than equal 5, with a differential analysis among the 18 tumors (Figure 1b) performed. Then, we combined the normal samples from the GTEx database with those from the TCGA database for further difference analysis of PRMT6. 24 tumor samples were taken as X-axis, and the PRMT6 expression calculated by Log2 (TPM+1) as Y-axis ( Figure 1c).
Cancer Cell Line Encyclopedia (CCLE) database mainly records the genetic characteristics of cell lines, sequencing more than 900 cancer cells from more than 30 human tissues. In this study, the data from multiple tumor cell lines were downloaded from the CCLE database, compared with PRMT6 expression levels in 21 tissues (Additional le 1: Figure S1). 1019 samples and 21 tissues were taken as X-axis, and the PRMT6 expression calculated by Log2(TPM+1) as Y-axis.

Analysis on gene expression and survival prognosis
In our study, the relationship between PRMT6 expression and clinical prognostic indicators, including overall survival (OS), diseasespeci c survival (DSS) and progression-free survival (PFS), was analyzed with gene expression pro les from the TCGA database in 33 tumors. We used Kaplan-Meier survival analysis curve and forest map respectively to visualize the relationship between them ( Figure   2).

Analysis on gene expression and immune estimation
CIBERSORT is a software to analyze immune cell in ltration [21]. We downloaded markers for gene expression of 22 immune cells from the website. In this study, scores of immune in ltration were collected and the correlation between the PRMT6 expression in these tumors and the immune cell in ltration (Additional le 2: Figure S2). The immune cell in ltrate scores of CIBERSORT were taken as the X-axis, and the PRMT6 expression calculated by Log2(TPM+1) as Y-axis ( Figure 3a).
We performed a comprehensive analysis of the total number of immune and stomatal cells in each type of tumor using the estimation package in R software (Additional le 3: Figure S3). The corresponding scores was taken as X-axis, and the PRMT6 expression calculated by Log2 (TPM+1) as Y-axis, with the distribution of each score described by a density curve (Figure 3c-f).
We obtained the information of more than 40 immune checkpoint genes and investigated the relationship between the PRMT6 expression and the immune checkpoint gene expression ( Figure 3b).
TISIDB database is an open online tool to integrate various tumor immunology resources [22], which we used to analysis the relationship between PRMT6 expression in various tumors and immune or molecular subtypes (Additional le 4: Figure S4).

Analysis on gene expression and mutation
The number of gene mutations in each tumor was obtained from the TCGA database, which was corrected by comparing the total length of exons. We tested the correlation between PRMT6 expression and tumor mutational burden (TMB) and visualized the relationship between them through radar map (Figure 4a).
Microsatellite instability (MSI) is a change in the length of a microsatellite due to insertion or deletion of duplicate units in the tumor compared to normal tissues. We visualized the correlation between the PRMT6 expression in tumors and MSI by radar map ( Figure   4b).

Analysis on gene expression and clinical indicators
Chinese Glioma Genome Atlas (CGGA) is a complete genome sequencing database for patients with glioma in China. We obtained the clinical information and PRMT6 gene expression information of 325 patients with glioma from the CGGA database [23,24]. Then, we analyzed the clinical information of CGGA and TCGA databases respectively, and the PRMT6 expression and its correlation (Figure 5aj). Kruskal−Wallis test and Mann−Whitney test were used to determine whether gene expression was different among various clinical indicators. On the X axis is the classi cation of various clinical indicators, and on the Y axis is the level of gene expression.

Construct and verify the nomogram
We used the high and low PRMT6 expression as an independent prognostic risk factor and constructed a nomenclature diagram combined with other common risk factors (Figure 6c). Risk factors analyzed in this study included age, gender, tumor grade, IDH mutation status, 1p19q co-deletion status, chemotherapy status, and PRMT6 expression level. To verify whether PRMT6 can act as an independent prognostic risk factor, we rst plotted the survival analysis curve with Kaplan-Meier method (Figure 6a-b). Then, we applied univariate Cox and least absolute shrinkage and selection operator (LASSO) regression model to screen all the variables. We took TCGA database as the training set and CGGA database as the veri cation set, and consistency index (C-index) and calibration curve to evaluate the nomogram (Figure 6d-e).

Gene Set Enrichment Analysis(GSEA)
GSEA is a computational method to determine whether a prede ned data set has signi cant consistent differences between two biological states [25], which can be downloaded from the website. The expression dataset was the PRMT6 expression, the experimental group and the control group were set to high PRMT6 expression and low PRMT6 expression, and the number of permutations was set to 1000. In the results obtained, NOM p-val and FDR q-VAL value less than 0.05 were considered to be signi cantly different.

Immunohistochemical analysis
The specimens used in this study were collected from 32 glioma tissues and 4 glioma paracancerous tissues surgically from the First PRMT6 antibody used in this study is rabbit anti-human polyclonal antibody from Novus, USA (No. NB 110-40713). SP hypersensitive immunohistochemical kit was used for the secondary antibody, which was purchased from Fuzhou Maixin Biotechnology Development Limited Liability Company, China.
The experimental procedure was strictly carried out in accordance with the instructions of the immunohistochemical kit. Firstly, the para n sections were dehydrated and then microwaved to repair the antigen. The primary antibody (dilution concentration of PRMT6 was 1:500) was added for incubation for 1 hour, and then the secondary antibody was dropped to incubate DAB for color development. The blank control group was set to replace primary antibody with phosphate buffer saline. Through the identi cation of pathologists in our hospital, the staining intensity and positive cell rate of each section were graded by semi-quantitative integration method, and then the positive intensity was obtained by the product of two fractions (Table 1). Immunohistochemical scores: 0-1 is negative expression, 2-4 is low expression, and 6-9 is high expression.

PRMT6 expression in different human tissues and cancers
To elucidate the differences between PRMT6 in healthy tissues and tumor tissues, we performed a visual analysis of the PRMT6 expression in healthy tissues. As observed in Figure 1a, PRMT6 is expressed at low levels in healthy brain tissues. Then we also analyzed the

Analysis of survival prognosis
We used clinical information from the TCGA database to investigate the correlation between PRMT6 and survival prognosis. Forest maps of OS, DSS, and PFI showed that the PRMT6 expression is a risk factor in BLCA, LGG and UCEC, as well as a protection factor in BRCA. In addition, PRMT6 expression was divided into high and low groups, with survival analysis curves. In OS analysis, the high and low PRMT6 expression was signi cant in LGG and UCEC. In DSS analysis, the high and low PRMT6 expression was signi cant in LGG, LUAD and UCEC. In PFI analysis, the high and low PRMT6 expression was signi cant in LGG, COAD and LUAD. These evidence suggest that PRMT6 has important reference for prognosis in tumors.

Multidimensional immune correlation analysis
In order to investigate the effect of PRMT6 on immune level, we analyzed its interaction with different immune in ltrating cells, immune microenvironment, immune subtype and immune checkpoint genes from different perspectives. As we can see from Additional File 1 Figure S1, Also, we analyzed the expression relationship between PRMT6 and 47 common immune checkpoint genes. From Figure 3b, we can see that the PRMT6 expression in various tumours is strongly correlated with immune checkpoint genes.
Finally, we used the TISIDB online tool to analyze the relationship between gene expression and immune or molecular subtypes (Additional le 3 Figure S3). The results show that the expression of LGG is signi cantly higher than others'.

Mutation correlation analysis
We calculated TMB in each cancer tumor, and we can see that from the radar map in ACC

Various clinical indicators correlation analysis
To further elucidate the potential clinical value of PRMT6 in glioma, we analyzed various clinically common indicators including age, tumor grade, IDH mutation status, 1p19q co-deletion status, chemotherapy status. As can be seen from Figure 5, the PRMT6 expression level in TCGA and CGGA databases was signi cantly correlated with various clinically common risk factors.

Independent prognostic risk factor analysis
We considered high and low PRMT6 expression as an independent risk factor. First, survival analysis curves were performed based on TCGA and CGGA data, showing that the high and low PRMT6 expression signi cantly affected the prognosis of patients. Then, we constructed the nomogram by screening for various independent risk factors. We used TCGA database as the training set and CGGA database as the validation set for external validation. As from the results of the nomogram, PMRT6 is potentially valuable as an independent prognostic risk factor. In order to test the accuracy of the nomogram model, we calculated the C-index, which was 0.84 (95%CI:0.865-0.815) in the nomogram of TCGA, and 0.772 (95%CI:0.801-0.743) in the nomogram of CGGA. The calibration curves of the 1-year and 3-year survival rates of the two models were drawn respectively, and the results showed that the two models had better validation performance.

Gene enrichment results
According to the results of KEGG and GO enrichment analysis, the role of PRMT6 in gliomas is mainly related to the regulation of cell cycle, the involvement of DNA damage and repair, and the conduction of some signaling pathways. Table 2 reveals some representative pathways and related functions of enrichment.  Gene sets with NOM p-val and FDR q-value<0.05 are considered as signi cant.

Immunohistochemical results
Immunohistochemical results showed that PRMT6 protein was positively expressed in the nucleus and was brownish yellow or brown in color. The positive rate was 87.5% in glioma and 25% in normal tissue, with statistical difference, and a P value of 0.0055.
Additionally, the PRMT6 expression in normal tissues, LGG and GBM was signi cantly different, with a signi cant upward trend. Based on the obtained clinical information, we drew a survival analysis curve, which further veri ed the signi cant correlation between high and low PRMT6 expression and prognosis. Discussion PRMT6 is a key epigenetic enzyme in the PRMT's family [26], which is a histone modi cation associated with transcriptional activation with unique self-methylation activity [27,28]. PRMT6 involves in various regulatory processes, including signal transduction and transcriptional activation [29]. At present, there have been more and more studies found that PRMT6 plays an important role in many tumors, but there is still a lack of relevant research in glioma. Hence, considering these conditions, we investigated the PRMT6 expression in pan-cancer and analyzed the correlation of PRMT6 expression from multiple perspectives such as immunity and mutation. At the same time, we found the close relationship and potential value of PRMT6 and gliomas. Moreover, the potential clinical value of PRMT6 was veri ed from the perspective of clinical indicators., so the potential value of PRMT6 as a tumor marker of glioma and its role in immunity were rstly discussed in this study..
The results of our study suggest that the expression level of PRMT6 plays an important role in various tumors, which is consistent with some known studies, for example, PRMT6 could be used as a target for colon cancer in the intestinal tract [30]; the PRMT6 expression was up-regulated in endometrial cancer samples and promoted the growth and metastasis of tumor cells by activating related pathways. Additionally, it affected aerobic glycolysis through signalling pathways in hepatocellular carcinoma [31], and also showed special value in gliomas. The gene expression analysis showed that PRMT6 expression was low in normal brain tissue, but increased signi cantly in glioma. And both gene expression pro le and immunohistochemical results have been veri ed.
At present, people pay more and more attention to the role of immune cells in tumor, and the type of immune cell in ltration and the subtype of immune cells are closely related to the occurrence and development of tumor [32,33]. This study found that the PRMT6 expression in various tumor was mainly related to T cell and macrophage in ltration. In glioma, the primary immunoin ltrating cell are T cells, CD4 memory resting cells and dendritic cells. People also pay more and more attention to the immunotherapy for tumors, which is an emerging direction for the immunotherapy that stimulates the immune system to enhance the anticancer ability [34]. A number of studies have shown that immunotherapy on autologous dendritic cells can improve patient survival [35]. Referring to our results, we speculated that there is an intrinsic relationship between PRMT6 expression and immunotherapy targeting dendritic cells.
Recent studies have also shown that regulatory T cells (Treg) function in immune regulation is associated with PRMT. And there was an autoimmune response in mice with PRMT5 de ciency when Treg cells were in ltrated [36]. The results of this study showed that in glioma, the up-regulated PRMT6 expression was positively correlated with the in ltration of CD4 T cells, suggesting that PRMT6 is a valuable therapeutic target in immunotherapy for gliomas.
At the gene level, we also compared the correlation between the expression level of PRMT6 and the expression of some immune checkpoint genes in tumors. In the immune checkpoint genes analogous to LGG, the genes signi cantly connected with the activation of T and B lymphocytes and macrophages, such as CD40, CD44, CD80 and CD86. Besides, NRP-1 gene was associated with immune regulation and cell migration and interacted with Tregs [37]. The HAVCR2 gene encoded TH1-speci c cell surface proteins, which was associated with regulating macrophage activation [38]. All these evidence was further expressed that PRMT6 is strongly associated with in ltrating immune cells in LGG. Moreover, this study linked arginine methylation with immunity building a bridge between epigenetics and immunology.
TMB is a promising biomarker, and there have been studies shown that high TMB is positively correlated with immunotherapy [39]. Furthermore, TMB is closely related to the overall survival rate and tumor proliferative activity of patients with glioma [40], which is an important reference factor in the treatment and prognosis of glioma. This association may be closely related to PRMT's involvement in cell cycle regulation and DNA damage, such as catalyzed cyclin expression, leading to spontaneous DNA damage, checkpoint deletion, and chromosome instability. These factors will lead to gene coding errors, base substitutions, and gene insertion or deletion errors in somatic cells, further increasing TMB and thereby promoting glioma proliferation.
In the GSEA results, we found that PRMT6 expression is related to the regulation of cell cycle, DNA damage and repair, and signal transduction in gliomas, acting as an important role in DNA repair and regulation of DNA polymerase β (Pol-β), and an enzyme involved in basic repair [41]. PRMT6 is co-activated in staining as a nuclear factor to facilitate the transcription process [42]. From this evidence, we can infer that PRMT6 can promote the transcription of tumor cells in gliomas and thus increases the proliferation of tumor cells.
To verify the clinical value of PRMT6, we analyzed its association with clinically common risk factors and studied it as an independent prognostic risk factor. One of the most noteworthy was the correlation with chemotherapy. Studies have shown that PRMT6 can lead to dysfunctions of P21 CDKN1A (P21) in cancers, causing it to be methylated and promoting phosphorylation of threonine 145 on P21, making cancer cells more resistant to anti-tumor drugs [43,44]. At present, the imbalance of arginine or lysine methyltransferase in cancer has been gradually noticed [45], and new anticancer drugs targeting these sites are in clinical trials [46]. The development prospects of PRMT6 inhibitor chemotherapeutic drugs in the treatment of glioma are considerable.

Conclusions
According to the above evidence, we can conclude that high PRMT6 expression is a potential tumor marker for gliomas with important predictive value for the prognosis of patients. Additionally, PRMT6 may be involved in the regulation of glioma cell cycle through signal transduction, promote the RNA transcription process, and further improve the proliferation and invasion of glioma cells. In clinical treatment, immunotherapy and chemotherapy are both areas to be developed.