1. Introduction

Nasopharyngeal carcinoma (NPC) is a common head and neck malignant tumor [1, 2]. In 2018, there were about 72,987 patients who died from NPC and 129,079 new cases of NPC worldwide, most of which were distributed in Southeast Asia [3]. The preferred treatment for early-stage NPC is intensity-modulated radiotherapy [4–6]. However, more than 80% of patients are locally advanced during initial diagnosis [7, 8]. For these locally advanced patients, the treatment of choice is platinum-based concurrent chemoradiotherapy. Nonetheless, 5%–15% NPC patients still developed nasopharyngeal or local lymph node recurrence after synchronous radiotherapy, and 15%–30% NPC patients developed distant metastasis [9–12]. Therefore, exploring the pathogenesis and searching for novel molecules are imperative for the diagnosis and individualized treatment of NPC.

MicroRNA (miRNA) is a class type of noncoding RNA consisting of 17–23 nucleotides [13]. miRNA has an important effect on the differentiation, migration, proliferation, and apoptosis of cancer cell by binding to 3′-UTR and regulating translation of target genes [14]. MiR-150-5p is abnormally expressed in the non-small cell lung carcinoma, rectal carcinoma, cervical carcinoma, and other carcinomas, and is closely related to their tumorigenesis and development [15–17]. However, the mechanism of miR-150-5p in NPC remains unclear.

In order to explore the role of miR-150-5p in NPC, miRNA microarrays and sequencing data of NPC were collected from online databases, and the miR-150-5p expression in NPC was determined. Subsequently, the potential target genes were obtained by combining the differentially expressed genes (DEGs) of NPC with the genes predicted using miRWalk. In addition, the DAVID database was used to identify the potential pathways and mechanisms from two aspects: Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Finally, Cytoscape software and the STRING database were used to screen the hub genes.

2. Materials and Methods

3. Results

3.1. The Expression of miR-150-5p Was Down-Regulated in NPC

This study finally retrieved fourteen mRNA datasets and seven miRNA datasets from the GEO public database (Table 1). The selection process is visualized with Figures 1 and 2. The expression of miR-150-5p in individual datasets was represented using violin plots and ROC curves (Figures 3 and 4), and four datasets, including GSE22587, GSE32960, GSE36682, and GSE43039, showing that P < 0.05 in the violin plots and ROC curves. Since the individual studies were too small to draw stable conclusions, we integrated all theincluded datasets. Due to the large heterogeneity (I² = 91.8%, P < 0.05) Figure 5(a), the random effect model was adopted. The SMD of miR-150-5p was =−1.20 (95% CI: −2.23, −0.17; Figure 5(a)). The sensitivity analysis revealed that GSE32960 largely affected the stability of the SMD (Figure 5(b)). The heterogeneity was significantly reduced after excluding GSE32960, and the fixed effect model was adopted (I² = 25.7%, P = 0.241, Figure 5(c)). Therefore, the SMD was −0.66 (95% CI: −0.98, −0.33; Figure 5(c)). The above results revealed that the expression of miR-150-5p was distinctly down-regulated in NPC. Furthermore, Begg’s test did not indicate obvious publication bias (Figure 5(d)).

Table 1. Characteristics of GEO datasets included in this study.

Publication year	ID of datasets	Platform	Cancer group	Normal controls
2013	GSE22587	GPL8933	8	4
2012	GSE32906	GPL11350	12	6
2012	GSE32960	GPL14722	312	18
2012	GSE36682	GPL15311	62	6
2015	GSE43039	GPL16414	20	20
2014	GSE46172	GPL16770	4	4
2015	GSE70970	GPL20699	121	17
2009	GSE13597	GPL96	25	3
2016	GSE40290	GPL8380	25	8
2014	GSE53819	GPL6480	18	18
2020	GSE61218	GPL19061	10	6
2019	GSE95166	GPL15314	4	4
2019	GSE126683	GPL16956	3	3
2019	GSE118719	GPL20301	7	4
2013	GSE39826	GPL6244	3	3
2016	GSE68799	GPL11154	42	4
2014	GSE63381	GPL11154	4	0
2017	GSE102349	GPL11154	113	0
2017	GSE64634	GPL570	12	4
2012	GSE34573	GPL570	15	3
2008	GSE12452	GPL570	31	10

To further determine the ability of miR-150-5p to distinguish NPC and non-NPC nasopharyngeal tissues, we generated a sROC curve with the positive and negative diagnostic likelihood ratios, pooled sensitivity, and pooled specificity. The pooled AUC was 0.89 (95% CI; 0.86, 0.92; Figure 6(a)). The pooled sensitivity and pooled specificity were 0.70 (95% CI; 0.38, 0.90) and 0.89 (95% CI; 0.68, 0.97; Figures 6(b) and 6(c)), respectively. The negative and positive diagnostic likelihood ratios were 0.34 (95% CI; 0.14, 0.81) and 6.22 (95% CI; 2.26, 17.11; Figures 6(d) and 6(e)).

3.2. The Target Genes of miR-150-5p Were Obtained

Overall, 12,599 predicted target genes were obtained from miRwalk. The DEGs of each datasets were visualized using volcano plots (Figure 7(a)). In addition, 1904 upregulated DEGs were screened out. The SMDs of 1904 up-regulated DEGs were displayed in Table S1. The genes that intersected two times were regarded as the target genes, and finally, 1316 target genes of miR-150-5p were obtained (Figure 7(b)).

3.3. The Results of Enrichment Analysis

In this study, KEGG and GO analysis of 1316 target genes were performed using DAVID to further explore the molecular mechanisms of miR-150-5p in NPC. Regarding BP, the target genes mainly focused on G1/S transition of mitotic cell cycle, cell division, and DNA replication (Figure 8(a)). For the CC, the target genes focused on the nucleoplasm, cytoplasm, and spindle midzone (Figure 8(b)). Regarding MF, the target genes focused on the protein binding and protein-kinase binding (Figure 8(c)). As for KEGG, a total of seven important pathways were identified, including “pathways in cancer” (Figure 8(d)).

3.4. The Hub Genes of the Network

The PPI network was constructed using the 72 target genes of “pathways in cancer” (Figure 9). The Centiscape were adopted to screen hub genes. Six hub genes were screened, including EGFR, TP53, HRAS, CCND1, CDH1, and FGF2.

3.5. The mRNA Expression of the Six Hub Genes

The mRNA expression of the six hub genes, including EGFR, TP53, HRAS, CCND1, CDH1, and FGF2, was assessed using SMD. As shown in Figure 10, SMDs of all six hub genes exceeded 0, and 95% CI was over 0. The above results indicated that the six genes were highly expressed in NPC.

4. Discussion

This study comprehensively explored the role of miR-150-5p in NPC and confirmed that the down-regulation of miR-150-5p promoted the tumorigenesis of NPC by regulating the target genes.

NPC is a malignant tumor prevalent in Southeast Asia and Southern China [18]. Due to the lack of biomarkers for early diagnosis and a high degree of malignancy, most patients have local metastases at initial diagnosis [19, 20]. Therefore, exploring the pathogenesis and discovering new biomarkers are imperative. miRNA is a type of endogenous RNA with 17–23 nucleotides in length [13]. It regulates the transcription of target genes by combining with the 3′-UTR region, thereby modulating cell growth, differentiation, and apoptosis [21]. Many miRNAs are aberrantly expressed in NPC and regulate relevant target genes, thus affecting proliferation, invasion, and metastasis of tumor cells. For example, miR-142 is highly expressed in NPC and enhances the invasiveness and proliferation of NPC by inhibiting PTEN expression [22]. MiR-129-5p inhibits NPC lymph node metastasis and lymphangiogenesis by reducing ZIC2 expression [23]. MiR-18a plays its carcinogenic role by inhibiting SMG1 expression and activating the mTOR pathway in NPC [24]. LINC01551 promotes the metastatic ability of NPC by regulating miR-132-5p [25]. These indicate that miRNAs are critical in NPC tumorigenesis and development. Exploring the mechanism of miRNAs contributes to the treatment and diagnosis of NPC.

The miR-150-5p has been demonstrated to have an important effect on proliferation, invasion, and metastasis of multiple tumors. For example, miR-150-5p contributes to the epithelial–mesenchymal transition and cell growth of cervical carcinoma cells by regulating SRCIN1 [17]. In addition, miR-150-5p enhances the metastasis and proliferation of gastric cancer by interacting with circLMTK2 [26]. However, the mechanisms of miR-150-5p in NPC remain unclear. This study is helpful in revealing the mechanism of miR-150-5p in NPC tumorigenesis and progression.

In this study, a pooled analysis based on 539 NPC samples and 75 non-NPC nasopharyngeal samples demonstrated that the expression of miR-150-5p was down-regulated in NPC. The sROC curve indicated that miR-150-5p had high specificity for differentiating NPC from non-NPC nasopharyngeal samples. Subsequently, 1316 target genes were screened to perform GO and KEGG enrichment analysis. Finally, 72 target genes of “pathways in cancer” were selected to construct the PPI network, and six hub genes were screened, including EGFR, TP53, HRAS, CCND1, CDH1, and FGF2.

EGFR was demonstrated to have an important effect on the proliferation, invasion, and metastasis of multiple tumors [27]. After being activated by ligands, EGFR dimerizes and triggers the production of tyrosine kinase activity to activate downstream signal pathways and participate in cell proliferation, differentiation, division, survival, and cancer development [28, 29]. Leong et al. found that EGFR was overexpressed in most NPC patients [30]. Nimotuzumab is a monoclonal antibody against EGFR. Combined treatment with nimotuzumab and celecoxib can enhance the radiosensitivity of CNE2 cells by inhibiting the EGFR pathway [31]. A retrospective analysis showed that nimotuzumab combined with concurrent chemoradiotherapy achieved exciting efficacy in the treatment of locally advanced NPC [32]. TP53 is a tumor suppressor gene that encodes a protein with a molecular weight of 53 kDa. It participates in the processes of cell differentiation, proliferation, apoptosis, senescence, autophagy, inflammation, and metabolism by regulating the transcription of various genes [33]. TP53 is abnormally expressed in thyroid carcinoma, gastric cancer, and esophageal carcinoma and is associated with tumorigenesis and progression [34–36]. CCND1 is an important regulator of cell cycle [37, 38]. MiR-584 targets CCND1 to inhibit invasion and proliferation of pancreatic carcinoma [39]. In colorectal carcinoma, miR-628-5p can downregulate CCND1 expression to induce apoptosis and restrain cell proliferation [40]. In ovarian cancer, the inhibition of CCND1 expression by cisplatin can reduce cell proliferation and increase cell apoptosis [41]. LncRNA HCG18 can act as a ceRNA of miR-140 to upregulate the expression of CCND1 and promote the progression of NPC [42]. HRAS is an important member of the RAS oncogene family. As an essential part of the signal network that controls cell proliferation, differentiation, and survival, the RAS protein plays a vital role in tumorigenesis and progression [43]. In NPC, circZNF609 affects cell proliferation, invasion, migration, and glycolysis by regulating the miR-338-3p/HRAS axis [44]. CDH1 encodes E-cadherin. As a calcium-dependent transmembrane protein, E-cadherin is involved in cell–cell adhesion and has an important effect in maintaining epithelial structure and function [45]. As a vital angiogenic factor, FGF2 induces the migration and growth of endothelial cells [46]. FGF2 is produced by tumor cells and surrounding stromal cells, and promotes angiogenesis, migration, proliferation, and invasion of tumor cells [47].

All in all, the expression of miR-150-5p was down-regulated in NPC and affected tumorigenesis and progression of NPC by regulating target genes. We will further validate the effects of miR-150-5p on the six hub genes by experiments and further explore the key pathways.

5. Conclusions

This study confirmed the low expression of miR-150-5p in NPC, and that miR-150-5p regulated the tumorigenesis and progression of NPC by targeting EGFR, TP53, HRAS, CCND1, CDH1 and FGF2.

Conflicts of Interest

The authors declare no conflicts of interest.

Authors’ Contributions

Gang Chen and Ren-Sheng Wang directed this project. Jia-Ying Wen, Jian-Di Li, and Li-Ting Qin obtained and further analyzed the data. Jia-Yuan Luo and Juan-He wrote the manuscript. The manuscript was revised by all authors.