Overexpression of NLRPs as Independent Indicator for Prognosis of Patients with Head and Neck Squamous Cell Carcinoma

NOD-like receptor proteins (NLRPs) are an important part of gene regulation and play a vital role in many cancers. Recently, some studies have shown that members of the NLRP family are a crucial component in the progression of head and neck squamous cell carcinoma (HNSCC) and can be used as the main therapeutic targets. To explore this hypothesis, bioinformatics analyses using Gene Expression Proling Interactive Analysis (GEPIA), Oncomine, UALCAN, the Human Protein Atlas, and the cBioPortal database were performed in this study to determine the intracellular roles and functions of NLRPs. String and Cytoscape were also employed to investigate and visualize the proteins in intracellular pathways related to the regulation of HNSCC carcinogenesis. Further analysis revealed that the overexpression of NLRPs was signicantly related to the HNSCC stage and the survival of patients. We also explored the miRNAs downstream of NLRPs involved in nasopharyngeal carcinoma using GEO2R, TargetScan, and miRDB, and found that NLRPs can serve as prognostic biomarkers for the survival of HNSCC patients. Venn used analyze the NLRPs targeted by The target miRNAs related to NPC through the above rigorous research process. We eventually determined that the NLRPs are regulated by three miRNAs: miR-199a-3p regulates the expression NLRP1, miR-125a-5p regulates and miR-22 regulates signaling, regulation of the production of type I interferons, interleukin-1, interleukin-18, pyroptosis, NLRP3 inammasome complex assembly, female gamete generation, response to interferon-gamma, positive regulation of organelle organization, protein processing, and positive regulation of cellular component biogenesis. KEGG analysis conrmed the functions of the NLRP1 inammasome in the regulation of the innate immune responses to cytosolic DNA and interleukin-1 processing.


Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world.
According to data from the Global Cancer Observatory (GLOBOCAN), a 30% increase in the incidence of HNSCC is predicted to occur by 2030 (1.08 million new cases per year) [1] [2]. More remarkably, the 5-year survival rate for HNSCC ranges from 40-50% [3] and is only 34.9% for patients with advanced HNSCC [4] .
Thus, obtaining an accurate diagnosis of HNSCC at the early stage and predicting the prognosis of HNSCC have become the research focus for head and neck cancer treatment under the current circumstances. In recent years, there have been many reports on the mechanisms, development, and metastasis of HNSCC, but the biomarkers and potential therapeutic targets for HNSCC remain unde ned.
Members of the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domaincontaining (NLRP) family have been suggested to play critical roles in the carcinogenesis of HNSCC.  [5]. In recent years, miRNA-based post-transcriptional control of NLRP3 has received substantial research attention, especially as a potential therapeutic strategy.
A comprehensive study of NLRPs can uncover the molecular mechanisms related to the oncogenesis and metastasis of HNSCC and may reveal potential targets for the treatment of HNSCC. In the present study, we analyzed the relevant evidence using bioinformatics methods to investigate the expression of NLRPs in HNSCC patients and their relationships with clinical indicators. In addition, we predicted and analyzed the neighboring genes for the NLRP family. Finally, we examined the miRNAs downstream of NLRPs involved in nasopharyngeal carcinoma (NPC), providing a research foundation for better treatment of NPC.

Microarray data
The Gene Expression Pro ling Interactive Analysis (GEPIA) server [6][7] is a web-based tool that facilitates data mining on the RNA sequencing expression data repository. The resource was constructed using samples from the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases and covers 33 different cancers. We used GEPIA to obtain the expression levels of 12 NLRPs in different cancer tissues and the prognostic value of NLRP expression.

Data processing
The Oncomine database (www.oncomine.org) [7] integrates RNA and DNA-seq data from the Gene Expression Omnibus (GEO) and TCGA with published literature sources to provide cancer genetic information. Oncomine is usually used to identify new biomarkers. We performed differential expression analysis and co-expression analysis with Oncomine to nd genes that are differentially expressed in certain cancers with the goal of identifying target genes and guiding future research. UALCAN (http://ualcan.path.uab.edu/index.html) [8] uses TCGA level 3 RNA-seq and clinical data from 31 cancer types to analyze the relative expression of a queried gene(s) across tumor and normal samples.
The tool can also analyze various tumor sub-groups based on individual cancer stages, tumor grades, races, body weights, and other clinicopathological features; estimate the effect of gene expression levels and clinicopathological features on patient survival; and identify the top over-or under-expressed (up and down-regulated) genes in individual cancer types. This resource provides a platform for in silico validation of target genes and for identifying tumor sub-group-speci c candidate biomarkers. We studied the expression levels of 12 NPRLs and the associated clinicopathologic parameters using UALCAN. p < 0.05 was considered statically signi cant (student's t-test).
The cBio Cancer Genomics Portal (http://cbioportal.org) is an open-access resource for interactive exploration of multidimensional cancer genomics datasets. It currently provides access to data from more than 5,000 tumor samples from 20 cancer studies, allowing users to query datasets across genes, samples, and data types to examine different biologically and/or clinically relevant hypotheses [9]. This online tool simpli es the molecular expression pro le data for cancer tissues and cell lines into easy-tounderstand genetic (gene), epigenetic, gene expression, and protein events. The website also provides graphical gene-level abstracts, network visualization analysis, survival analysis, patient-centric queries, and software programming entries. In this study, we used this online platform to analyze the genome pro les of 12 NLRPs that contained mutations based on putative copy number changes obtained from the GISTIC program and an mRNA z-score (RNASeq V2 RSEM) threshold of ±1.8. For the statistical analysis, Kaplan-Meier diagrams were used to visualize the mutations in NLRP genes and their association with overall survival (OS) in HNSCC patients. The log-rank test was performed to identify the signi cance of the differences between the survival curves. When the p-value was <0.05, the difference was considered statistically signi cant.

Functional and pathway enrichment analyses
Metascape is a powerful gene function annotation analysis tool that not only performs biological pathway enrichment analysis and protein interaction network structure analysis but also presents the results in a high-quality graphical language. The data in Metascape are updated quickly (updated faster than DAVID) and cover a wide range of genes and diseases. Inside the cell, a vast number of macromolecules interact dynamically with each other at all times and it is necessary to understand these interactions. String and Cytoscape are tools that can be used to create molecular maps, that is, protein interaction networks. In this study, we effectively combined these tools to determine the protein network for NLRPs. Annotation and integration using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were carried out with Metascape and visualization was performed with Cytoscape software. GO enrichment analysis can predict the functional roles of NLRP mutations and genes signi cantly associated with NLRPs, while KEGG analysis can de ne the pathways related to the NLRPs.
The Human Protein Atlas (https://www.proteinatlas.org) integrates various omics technologies, including antibody-based imaging, mass spectrometry-based proteomics, transcriptomics, and systems biology to map the organization and cell distribution information for 26,000 human proteins in 64 cell lines and 48 human normal tissues, as well as protein expression in 20 kinds of tumor tissues. Using this open resource, we identi ed tumor-type speci c proteins that were differentially expressed and performed a direct comparison of protein expression in different NLRPs between normal and HNSCC tissues in humans.
The GEO database is a gene expression database created and maintained by the National Center for Biotechnology Information (NCBI) and is used for differential analysis of expression pro le chips. In the present study, we used this tool to identify differentially expressed miRNAs (DEM) in NPC. The GSE32960 dataset includes the miRNA pro le of 312 NPC specimens and 18 normal nasopharyngeal tissues. Statistically signi cant DEMs between NPC samples and normal samples were obtained based on the false discovery rate (FDR) with an adjusted p-value less than 0.05 and a fold change (FC) less than 2. miRNAs in mammals can play a post-transcriptional regulatory role by binding to the 3'untranslated region (UTR) region of the transcript sequence. TargetScan [10] is a tool that speci cally organizes mammalian miRNA target genes into a database based on existing analysis results. Similar to the other web-based resources, the miRNA Target Prediction Database (miRDB) provides the expression pro les for over 1000 cell lines. It also provides target data tailored for speci c cell models, thereby enabling the prediction of miRNA target gene information for humans, mice, and other species through MirTarget software. In this study, we combined these two software tools to investigate the miRNAs downstream of NLRPs involved in NPC.

Expression of NLRPs in patients with carcinoma
Both the transcription and translation of NLRPs were analyzed using GEPIA (http://gepia.cancer-pku.cn/), UALCAN (htttp://ualcan.path.uab.edu) and the Human Protein Atlas (http://www.proteinatlas.org) to determine the prognostic and potential therapeutic values of the NLRPs in patients with carcinoma. The expression levels and mRNA expression patterns of 14 NLRPs in 31 types of cancers were obtained from the GEPIA database and 20 types of cancers were obtained from the Oncomine, respectively ( Figure 1; Table 1). Compared to normal samples, 12 NLRPs (A-K: NLRP1/2/3/4/6/7/9/10) were signi cantly overexpressed in primary NPC tissues (p < 0.05) but there was no statistical difference in the expression of NLRP11, NLRP12 , NLRP13 and NLRP14 (p > 0.05), while there was no data of NLRP 5 and 8 ( Figure 2; Table 2).
The relationship between the expression of NLRPs and the clinical manifestations of HNSCC We employed UALCAN (http://ualcan.path.uab.edu) and The Human Protein Atlas (https://www.proteinatlas.org) to examine the relationship between the expression of NLRPs and the clinical manifestations of HNSCC, including the cancer stage and tumor grades for each patient, and found that the mRNA expression of 12 NLRPs (NLRP1/2/3/4/6/7/9/10/11/12/14) had an obvious association with the HNSCC cancer stages. This nding demonstrated that patients with advanced cancer tend to exhibit higher NLRP expression (Figure 3, Figure 4; Table 3).

Genetic mutations in NLRPs and their prognostic value
We investigated the genetic alteration in NLRPs using cBioPortal. As shown in Figure 5,

Prediction of functions and pathways of NLRPs
After analyzing the genetic changes in members of the NLRP family and their prognostic value for HNSCC patients, we explored the functions and pathways of the NLRPs with Metascape by constructing an integrated network of neighboring genes (Figure 7) that GO enrichment and KEGG pathway analyses in Metascape were performed to understand the intracellular biological pathways of the integrated functions of NLRPs (Figure 8 9 10). The results showed the involvement of NLRPs in the pathways pertaining to the regulation of innate immune responses to cytosolic DNA, interleukin-1 processing, and the NLRP1 in ammasome. evidently connect to the NLRPs using String and Cytoscape ( Figure 11).

Prediction of target miRNAs in nasopharyngeal carcinoma patients
After a series of studies on the role of the NLRPs in HNSCC patients, we obtained data from the NCBI-GEO dataset GSE32960 to establish the value of the NLRP family in the treatment of NPC. GSE32960 contains data for 312 NPC tissue samples and 18 normal tissue samples. Applying the cut-off criteria of < 0.05 for the adjusted p-value (FDR) and FC > 1 resulted in a total of 82 DEMs that were considered statistically signi cant. The results are shown in a volcano map (Figure 12).
To explore the target miRNAs of NLRPs in NPC, we employed two online analysis tools, miRDB (http://ww.mirdb.org/miRDB/) and TargetScanHuman 7.2 (http://ww.targetscan.org/) for miRNA prediction. The resulting Venn diagram was used to analyze the NLRPs targeted by miRNAs. The target miRNAs related to NPC survival were identi ed through the above rigorous research process. We eventually determined that the NLRPs are regulated by three miRNAs: miR-199a-3p regulates the expression of NLRP1, miR-125a-5p regulates the expression of NLRP2, and miR-22 regulates the expression of NLRP3.

Discussion
In recent years, the use of traditional clinicopathological indicators for the early HNSCC diagnosis and prediction of the prognosis has been insu cient [11; 12]. Therefore, new prognostic biomarkers that fully re ect the biological characteristics of tumors and the treatment e cacy of patients with HNSCC have been under investigation. To date, many studies have been conducted to identify predictive biomarkers in HNSCC patients. These potential biomarkers are divided into single-molecule protein markers and gene markers that can be identi ed through high-throughput analysis and gene expression pro ling [13; 14; 15].
NLRP are involved in the progression of a variety of cancers [16]; however, their functions in HNSCC remain unknown. Our results showed that the mRNAs and proteins of 12 NLRP family members were overexpressed in HNSCC. The overexpression was signi cantly associated with the cancer stage in HNSCC and was also remarkably associated with shorter OS. Additionally, the mutation and genetic alteration rates of NLRPs were higher in HNSCC patients. Analysis of the functions and pathways of NLRPs and their altered neighbor genes suggested the involvement of NLRPs in the following biological processes: defense response to other organisms, I-kappaB kinase/NF-kappaB signaling, regulation of the production of type I interferons, interleukin-1, interleukin-18, pyroptosis, NLRP3 in ammasome complex assembly, female gamete generation, response to interferon-gamma, positive regulation of organelle organization, protein processing, and positive regulation of cellular component biogenesis. KEGG analysis con rmed the functions of the NLRP1 in ammasome in the regulation of the innate immune responses to cytosolic DNA and interleukin-1 processing.
Previous studies have suggested that NLRPs play a role in various cancers. Polymorphism or mutation of NLRP1A is closely related to the progression of cancer [17]. NLRP3 plays a vital role in the metastasis and prognosis of NPC and other cancers [18; 19; 20]. NLRP4 (a cytosolic receptor in several tissues [21]) has recently been con rmed to not only inhibit autophagy [22; 23] but also to function in the immune response to viral infections (eg Epstein-Barr virus, human papilloma virus) [24], thereby indirectly contributing to the development of NPC. NLRP7 had been reported in previous studies as the main pathogenic gene for recurrent hydatidiform moles (RHMs) [25]. NLRP12 de ciency leads to colitis and in ammation-induced tumorigenesis in mice [26; 27].
The NLRP family is signi cantly related to the progression of NPC in studies. Therefore, we further explored the miRNAs downstream of NLRPs involved in NPC. The results indicated that miR-199a-3p regulates the expression of NLRP1, miR-125a-5p regulates the expression of NLRP2, and miR-22 regulates the expression of NLRP3. It has previously been demonstrated in some cancers that miR-22 targets NLRP3 during carcinogenesis [28]. miR-125a-5p reportedly targets ERBB3 and inhibits cell proliferation, cell cycle progression, and cell migration in head and neck cancers [29]. In another study, upregulated miR-125a-5p was found to enhance the metastatic ability of HNSCC cell lines [30; 31]. In addition, miR-125a-5p has been identi ed as an important cancer treatment target in a series of cancers, including gastric cancer [32], cervical cancer [33], and lung cancer [34]. miR-199a-3p is not only expressed abnormally in a variety of cancers but also regulates ITGA3 to inhibit metastasis in head and neck cancer [35]. In a recently published study, bioinformatics analysis established miR-199a-3p as an important target for the treatment of head and neck cancer [36].
Our research has certain limitations. First, due to the small sample size in the online databases used, the breadth of our research ndings is limited. A larger sample size is needed for further research to con rm our ndings and explore the role of NLRPs in HNSCC. Second, our study lacks information on the diagnostic value of NLRPs in HNSCC; therefore, further research is needed to investigate whether NLRPs can be used as diagnostic biomarkers. Finally, because we did not uncover the underlying mechanisms for NLRP in HNSCC, our research lacks depth.

Conclusion
In brief, our research results con rm that the over-expression of 12 NLRPs is associated with the cancer stage in HNSCC patients. Multivariate analysis showed that higher expression of NLRPs was signi cantly associated with shorter OS in HNSCC patients. This study demonstrates that the NLRP family can be prognostic biomarkers for HNSCC patients. We also explored the miRNA targets downstream of NLRPs in patients with NPC, laying the foundation for further research.

Declarations
Ethics approval and consent to participate   Table 3 The relationship between the expression of NLRPs and tumor grade  Visualization of NLRPs and their altered neighbor genes in HNSCC patients (String and Cytoscape).
NLRPs marked in red.

Figure 12
Volcano plot of differentially expressed miRNAs (GEO2R). The red dots represent upregulated miRNAs and the blue dots represent downregulated miRNAs.