A panel of three serum microRNA can be used as potential diagnostic biomarkers for nasopharyngeal carcinoma

Abstract Background Nasopharyngeal carcinoma is cancer with unique epidemiological characteristics, showing obvious ethnicity, gender, and geographical prevalence. More and more evidence shows that microRNAs are stable in serum and are specific to different tumor types. Therefore, miRNA is a new non‐invasive biomarker for cancer detection. Methods The experiment is divided into three stages, namely, the screening stage, the training stage, and the verification stage. We took 54 patients with nasopharyngeal carcinoma and 108 healthy controls as the research objects. We use the receiver‐operating characteristic (ROC) curve and area under the ROC curve (AUC) to evaluate the diagnostic value of miRNA. Finally, a three‐miRNA panel with high diagnostic efficiency was constructed. In addition, we conducted biological information analysis of these miRNAs to explore their functions. Results In NPC patients, the expression of five serum miRNAs (miR‐29c‐3p, miR‐143‐5p, miR‐150‐5p, miR‐145‐3p, and miR‐205‐5p) is significantly dysregulated. Among them, the diagnostic value of these three miRNAs (miR‐29c‐3p, AUC = 0.702; miR‐143‐5p, AUC = 0.733; and miR‐205‐5p, AUC = 787) is more prominent. The diagnostic panel constructed by them has a higher diagnostic value (AUC = 0.902). Through the analysis of the TCGA data set, the target gene of the three‐miRNA panel may be KLF7, NRG1, SH3BGRL2, and SYNPO2. Conclusion The three‐miRNA panel (miR‐29c‐3p, miR‐143‐5p, and miR‐205‐5p) may become a novel non‐invasive biological marker for nasopharyngeal cancer screening.


| INTRODUC TI ON
The distribution of nasopharyngeal carcinoma (NPC) shows obvious ethnicity, gender, and geographical prevalence. Nasopharyngeal cancer has the highest incidence rate in Southeast Asia, such as Singapore, Maldives, and Indonesia with an incidence rate of 7 per 100,000. 1 Generally, the prognosis of women is better than that of men. 2,3 So, it is a kind of cancer with unique epidemiological characteristics. 1 According to research, during 2000-2007, the 5-year survival rate of European adult NPC was only 49%. 4 The current diagnosis method is a primary nasopharyngeal tumor biopsy guided by an endoscope. This is an invasive test with poor patient compliance, so it is not suitable for screening. Currently, the commonly used screening methods are plasma EBV DNA and primer/probe analysis for the BamHI-W region of the EBV genome. However, this examination needs to be performed in duplicate and at least 4 weeks apart. 5 In addition, the role of EBV in keratinizing cancer is not obvious. In summary, we urgently need a new, non-invasive, and more reliable screening method.
MicroRNAs (miRNAs) are short (19)(20)(21)(22) nucleotides) non-coding and highly conserved single-stranded RNAs that can regulate gene expression after transcription. 6 More and more evidence shows that the expression profile of microRNA can distinguish normal and tumor tissues and is specific to different tumor types. 7 Studies have found that miRNAs are stably present in serum, which makes it possible to screen for cancer through circulating miRNA analysis. 8 In summary, the expression pattern of human plasma miRNAs may have the potential to diagnose certain types of cancer.
Our purpose of this study is to find a novel biomarker for the screening of NPC. In this study, we used real-time quantitative polymerase chain reaction (qRT-PCR) to find a set of plasma miRNAs, which can be used as a novel biomarker for the screening of NPC. In this experiment, a three-phase study was used to identify miRNAs with diagnostic value and construct an efficient diagnostic panel.
In addition, we also analyzed the biological information of these miRNAs.

| Participants and ethics statement
All patients were diagnosed as NPC based on histopathological evaluation and did not receive any treatment before sample col-

| Research design
We conducted a three-phase study, as shown in Figure S1. Select differentially expressed miRNAs as candidate biomarkers from GSE32960 published on the Gene Expression Omnibus. 9 In the next step, to determine these candidate biomarkers, we conducted a testing phase and verification phase study. First, in the screening phase, we selected 10 differentially expressed miRNAs, which were related to NPC (Table S1 lists the corresponding reference abstracts of the 10 candidate miRNAs). The primer sequences of these miRNAs are listed in Table S3. Then, in the testing phase, we randomly select 15 serum samples from NPC patients and 30 serum samples from HCs, analyze the expression profiles and diagnostic capabilities of these miRNAs. Finally, focus on verification in the verification phase, and backward stepwise logistic regression analysis is used to construct the panel with the highest diagnostic ability. And there was no significant difference in each group.

| Collect serum samples and extract RNA
All participants did not receive any treatment before taking serum samples; first, 2-ul miR-54 (cel-miR-54-5p) (10 nm/L, RiboBio) was added to each sample. The purpose of this is to allow it to be used as an internal reference for the RT-qPCR process and to normalize the variability in the extraction process. Then, total RNA was extracted from serum samples using TRIzol LS isolation kit (Thermo Fisher Scientific) and measured RNA concentration and purity with NanoDrop 2000c spectrophotometer (Thermo Scientific).

| Quantitative reverse transcriptionpolymerase chain reaction (RT-qPCR)
To detect the expression level of these miRNAs, the reverse transcription-specific primers of the Bulge-LoopmiRNA qRT-PCR primer set (RiboBio) were used to amplify miRNAs. Then, Taqman probe was used to perform RT-qPCR on the LightCycler480 Real-Time PCR system (Roche Diagnostics). The qPCR reaction was performed at 95°C for 20 s, 95°C for 10 s, 60°C for 20 s, and 70°C for 10 s, with 40 cycles. Finally, the 2 −ΔΔCq method was used to analyze the relative expression level of target miRNA. 10 is considered statistically significant, only when it is less than 0.05.

| Bioinformatic analysis
To identify the function of candidate miRNAs in NPC, a series of miRNA targets were predicted in miRWalk2.0 (http://zmf.umm.uniheide lberg.de/mirwalk2), and miRWalk is used to predict and experimentally validate miRNA-target interactions. 11 Enrichr database (http://amp.pharm.mssm.edu/Enric hr/) was used to perform enrichment analysis and to perform functional annotation on the predicted target gene. 12 This study used gene ontology annotation (GO) and Kyoto Encyclopedia of Genome (KEGG) approach analysis. Besides, we used OncoLnc (http://www.oncol nc.org/) to generate Kaplan-Meier survival curves and correlate TCGA survival data with the expression levels of miR-29c-3p and miR-143-5p and miR-205-5p. 13 Table 1. Among the two stages, there was no significant difference between NPC and HC both in age and in gender. Parameters were shown as numbers (percentage). Statistical contrast was exerted through the Wilcoxon-Mann-Whitney test.

| Confirm candidate miRNAs during the testing phase
For the ten candidate miRNAs screened out, we verify them during the testing phase. There are randomly selected 15 cases of NPC patients and 30 cases of HCs. Through qRT-PCR analysis, the results are shown in Figure 2. We can see that out of ten candidate miRNAs, there are still significant differences in expression between NPC patients and HCs in five of the ten candidate miRNAs (miR-29c-3p, miR-143-5p, miR-150-5p, miR-145-3p, and miR-205-5p). Therefore, we conducted further studies on these five miRNAs.

| Detect the diagnostic value and expression level of these five miRNAs in the verification set
We further studied these five miRNAs to verify whether their expression may be used as serum biomarkers in the screening of NPC. We selected 39 NPC patients and 78 HCs for further study.
The results are shown in Figure 3. From the figure, we can know that the relative expression level of miR-205-5p is significantly increased in NPC patients, while the relative expression level of miR-29c-3p, miR-143-5p, miR-150-5p, miR-145-3p is decreased in NPC patients.

F I G U R E 2
The expression levels of 10 candidate miRNAs in the testing phase. Screening conditions: p-value < 0.05, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001. The serum of 15 NPC patients and 30 HCs patients was used for testing phase  Figure 4, by drawing the ROC curve of the three miRNAs joint diagnostic panels, we found that its AUC is 0.902 (95% CI: 0.833-0.949; sensitivity = 87.18%, specificity = 88.46%; Table 2).

| Bioinformatics analysis of candidate miRNAs
MiRWalk2.0 is used to predict the possible target genes of miR-29c-3p, miR-143-5p, and miR-205-5p. Select two or more miRNApredicted genes as target genes, and in total 283 genes were have also shown that miR-29c-3p has been shown to be associated with gene expression in the development of nasopharyngeal carcinoma (NPC). 18 Combining these studies with ours, miR-29c-3p may

F I G U R E 4
The receiver-operating characteristic (ROC) curve analyses for the three-miRNA panels. The AUC of the group consisting of three microribonucleic acids (miR-29c-3p, miR-143-5p, and miR-205-5p) is 0.903 (95% confidence interval: 0.833-0.949; sensitivity = 87.18%, specificity = 88.46%). The red curve represents the receiver-operating characteristic (ROC) curve. The green curve represents 95% ROC confidence interval. The blue line represents diagonal play an important role in intercellular molecular signaling pathways in the occurrence and development of NPC.
A large number of studies have shown that miR-143-5p is downregulated in a variety of tumors. It participates in cancer metastasis by targeting HIF-1α/EMT-related signaling pathways and is a potential therapeutic target. 19 The study also found that HAGLR, as a competitive endogenous RNA of miR-143-5p, increases the expression of LAMP3, thereby promoting the proliferation, invasion, and migration of cancer cells. 20 In this study, we first revealed the role of miR-143-5p in NPC screening (AUC = 0.733; 95% CI: 0.644-0.811; Figure 3D).
Studies have shown that the up-regulation of miR-205-5p expression is closely related to the cell proliferation, cell migration, and clonogenic activity of HNSCC cells. 21 Another study showed that miR-205-5p and its direct interaction with VENTXP1 regulate HNSCC cell proliferation and tumorigenicity. ANKRD2 has been identified as a miR-205-5p target and plays an important role in regulating NF-kB signaling. 22 Our research also shows that miR-205-5p is a good serum biomarker for NPC screening (AUC=0.787; 95% CI: 0.701-0.857; Figure 3J). cancers. According to reports, SYNPO2 can inhibit the activity of YAP/TAZ. 27 In summary, these four genes play a role in the occurrence and development of a variety of cancers, so these genes may also have a close relationship with nasopharyngeal carcinoma. We will further explore the role of these genes in the occurrence and progression of nasopharyngeal carcinoma in future research.
There is no doubt that our research results are meaningful, but there are still some limitations. First of all, the number of patients included in this study is relatively small and the serum is taken from the patients before treatment, so we cannot know whether the levels of these miRNAs change after the patients receive treatment.
In addition, although There are many miRNAs in the serum of nasopharyngeal cancer patients that are significantly different from HCs, in order to avoid possible negligence in data processing, we only included 10 miRNAs in this study. Therefore, in future research, we will continue to explore the value of other miRNAs in nasopharyngeal cancer screening and explore their biological effects.
In summary, this three-miRNA panel has high diagnostic efficiency (AUC = 0.902;95% CI: 0.833-0.949; sensitivity = 87.18%, specificity = 88.46%; Table 2), and we believe that the three-miRNA panel is likely to become a non-invasive and novel biomarker for early screening and diagnosis of NPC. More studies are needed to confirm our findings.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.