LncRNA FOXP4-AS1 serves as a biomarker for nasopharyngeal carcinoma diagnosis and prognosis

Abstract

This study was performed to probe the clinical significance of serum lncRNA FOXP4-AS1 in nasopharyngeal carcinoma (NPC) tumorigenesis. LncRNA FOXP4-AS1 from nasopharyngeal carcinoma patients and healthy volunteers were abstracted and converged. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of FOXP4-AS1. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of FOXP4-AS1. Kaplan–Meier survival analysis and log-rank test were used to assess the patients’ survival prognosis. Independent risk factors for overall survival (OS) and progression-free survival (PFS) were assessed by univariate and multivariate cox proportional hazards regression analysis. In this study, we observed that the levels of FOXP4-AS1 were significantly upregulated in nasopharyngeal carcinoma patients compared to healthy volunteers. Besides, the expression of FOXP4-AS1 was closely associated with T stage, lymph node metastasis, and clinical stage. Meanwhile, ROC analysis found that FOXP4-AS1 had diagnostic values to distinguish tumor patients from healthy volunteers. Furthermore, patients with high FOXP4-AS1 expression level had poorer OS and PFS than those with low FOXP4-AS1 expression. Finally, univariate and multivariate Cox proportional hazards regression analysis found that the T stage, lymph node metastasis, clinical stage, FOXP4-AS1 expression might be independent risk factors for OS and PFS of nasopharyngeal carcinoma patients. This study firstly clarified that FOXP4-AS1 was overexpressed in nasopharyngeal carcinoma. And FOXP4-AS1 may act as a diagnostic and prognostic biomarker, and hopeful therapeutic target for nasopharyngeal carcinoma patients.

Introduction

Nasopharyngeal carcinoma is a type of head and neck cancer that occurs mainly in southern China and Southeast Asia (Bruce et al. 2015). Bad living habits, Epstein-Barr virus (EBV) infection and genetic factors are prone to result in the development of nasopharyngeal cancer (Wu et al. 2018). Early nasopharyngeal carcinoma can be cured by radiotherapy, while advanced nasopharyngeal carcinoma, especially undifferentiated nasopharyngeal carcinoma, usually has a poor prognosis due to the frequent occurrence of distant metastasis and the tendency to relapse after treatment (Wu et al. 2018). Therefore, it is necessary to explore novel molecular mechanism and biomarkers underlying the progression of nasopharyngeal carcinoma.

Long non-coding RNA belongs to a class of RNA with a length more than 200 nucleotides and no protein-coding function. LncRNA has been reported to regulate the formation and progression of nasopharyngeal carcinoma by regulating gene expression (Yang and Deng 2015). For example, novel lncRNA LINC01385 can predict the poor prognosis and facilitate nasopharyngeal carcinoma growth through sponging miR-140-3p and augmenting Twist1 expression (Li and Zhang 2020). LncRNA SNHG5 accelerates nasopharyngeal carcinoma growth by downregulating miR-1179 and upregulating HMGB3 expression (Liu et al. 2020). Meanwhile, it’s been reported that lncRNA can function as biomarkers of tumor diagnosis and prognosis. For instance, lncRNA SNHG12 is an independent poor prognostic factor for nasopharyngeal carcinoma patients and regulates the progression of nasopharyngeal carcinoma (Liu et al. 2018). Three circulating lncRNAs MALAT1, AFAP1-AS1 and AL359062 displayed relative veracity in identifying nasopharyngeal carcinoma from a healthy population (He et al. 2017). In addition, it’s been reported that lncRNA FOXP4-AS1 is overexpressed, promotes cancer progression, and predicts the prognosis for multiple cancers including cervical cancer (Zhao et al. 2020), hepatocellular carcinoma (Wang et al. 2019), gastric cancer (Chen et al. 2019), prostate cancer (Wu et al. 2019), osteosarcoma (Yang et al. 2018), colorectal cancer (Li et al. 2017), esophageal squamous cell carcinoma (Li et al. 2020). Nevertheless, the expression and clinical significance of FOXP4-AS1 in nasopharyngeal carcinoma remain to be explored.

In this study, we reported for the first time that FOXP4-AS1 was highly overexpressed in nasopharyngeal carcinoma and that high level of FOXP4-AS1 was linked with poor survival outcomes of nasopharyngeal carcinoma patients. In addition, ROC analysis proved that FOXP4-AS1 could be used as a novel biomarker of distinguishing nasopharyngeal carcinoma.

Material and methods

Serum samples

A total of 116 NPC and 50 healthy serum samples were collected between February 2012 and February 2015 in Jinan Fourth People's Hospital. These patients’ pathological and clinical data were collected for the associated analysis of FOXP4-AS1 expression. Each participant draw whole blood (20 ml) at the first day of enrollment to this project. Serum samples were acquired by the centrifugation for the obtained whole blood samples at 1200×g for 20 min. Serum samples were conserved using liquid nitrogen until the extraction of FOXP4-AS1. All participants signed the informed consents. The Ethics Committee of Jinan Fourth People's Hospital approved this project. The clinical baseline of all patients was shown in Table1.

Table 1.

Correlation between the expression levels of FOXP4-AS1 and clinicopathological features in nasopharyngeal carcinoma

Characteristics	No.	FOXP4-AS1 expression
		High	Low	p-value
Gender
Male	120	62	58	0.6032
Female	46	21	25
Age
< 60	109	57	52	0.5134
≥ 60	57	26	31
T stage
T1–T2	105	42	63	0.0012**
T3–T4	61	41	20
N stage
N0–N1	89	43	46	0.7557
N2–N3	77	40	37
M stage
M0	144	75	69	0.2521
M1	22	8	14
Lymph node metastasis
Positvie	106	63	43	0.0020**
Negative	60	20	40
Clinical stage
I–II	86	51	35	0.0195*
III–IV	80	32	48
Histological grade
I	90	44	46	0.8763
II–III	76	39	37
Total	166	83	83

Chi-square test. *p < 0.05, **p < 0.01

RNA extraction and quantitative real-time PCR analyses (qRT-PCR)

Total RNA was extracted using TRIzol reagent RNA extraction kit (Invitrogen, Carlsbad, CA, USA). The First Strand cDNA Synthesis Kit was used to produce the first cDNA strand (Merck Millipore, Darmstadt, Germany). PrimeScript RT reagent Kit (Takara, Dalian, P.R. China) was used for reverse transcription of cDNAs. Real-time PCR analysis was carried out to measure the relative expression of FOXP4-AS1 on a Stratagene Mx3005P qRT-PCR system (Agilent, CA, USA) using SYBR Green Master Mix (Bio-Rad, Hercules, CA, USA). GAPDH was used to control. Primers: GAPDH, forward: 5′-CACCCACTCCTCCACCTTTG-3′, reverse: 5′-CCACCACCCTGTTGCTGTAG-3′; FOXP4-AS1, forward: 5′-GTGAGCTTCTGGGTTCGACA-3′, reverse: 5′-ATTGAGGGTTAGGGCAGCAC-3′. Data were analyzed according to the comparative CT method (2^−ΔΔCt). All procedures were carried out in triplicate.

Statistical analysis

The chi-squared test was used to analyze the association between clinical characteristics and FOXP4-AS1 expression. The Kaplan–Meier method test and log-rank test was performed to calculate the progression-free survival (PFS) and overall survival (OS). Univariate and multivariate cox proportional hazards regression analysis was carried out to analyze the relationship between the clinical features and prognosis. The diagnostic value of FOXP4-AS1 was determined by the receiver operating characteristic (ROC) curve. All analysis was processed by GraphPad Prism 7 (La Jolla, CA, USA) software. Difference with p < 0.05 was considered to be statistically significant.

Results

Serum FOXP4-AS1 was highly expressed and had diagnostic value in nasopharyngeal carcinoma

As shown in Fig. 1a, qRT-PCR assay indicated that serum lncRNA FOXP4-AS1 was dramatically increased in nasopharyngeal carcinoma patients compared to that from healthy volunteers (p < 0.01). To explore the diagnostic value of serum lncRNA FOXP4-AS1 in the nasopharyngeal carcinoma patients, the receiver operating characteristic (ROC) curve was plotted and the areas under the ROC curve (AUC) are 0.7974 (95% CI, 0.7226–0.8721, Fig. 1b, p < 0.001), which clearly clarified that serum FOXP4-AS1 could be a potential and effective biomarker to differentiate cancer patients from healthy population.

Fig. 1.

a The expression of serum lncRNA FOXP4-AS1 was meaningfully upregulated in nasopharyngeal carcinoma. **p < 0.01. b The receiver operating characteristic (ROC) curve (AUC = 0.7974, P < 0.001) was used to assess the diagnostic value of lncRNA FOXP4-AS1 in the nasopharyngeal carcinoma patients, which clearly clarified that serum FOXP4-AS1 could be a potential and effective biomarker to differentiate cancer patients from a healthy population

The association between the lncRNA FOXP4-AS1 and clinical features of nasopharyngeal carcinoma

To further explore the association between lncRNA FOXP4-AS1 expression and clinic pathological features of nasopharyngeal carcinoma, the patients with nasopharyngeal carcinoma were categorized into low FOXP4-AS1 group (≤ median expression of FOXP4-AS1) and high FOXP4-AS1 group(≥ median expression of FOXP4-AS1). As shown in Table 1, chi-square test exhibited that FOXP4-AS1 expression was expressively related with T stage, lymph node metastasis, and clinical stage. Nevertheless, the FOXP4-AS1 expression displayed no connection with other clinical features, such as gender, age, N stage, M stage, histological grade.

FOXP4-AS1 was significantly implicated with poor prognosis of nasopharyngeal carcinoma

Next, we further explored the interaction between the FOXP4-AS1 expression and prognosis of nasopharyngeal carcinoma. Kaplan–Meier analysis and log-rank test proved that nasopharyngeal carcinoma patients with high FOXP4-AS1 expression were prominently associated with shorter progression-free survival (PFS) (Fig. 2, p < 0.01) and overall survival (OS) (Fig. 3, p < 0.01) than patients with low FOXP4-AS1 expression. In addition, univariate and multivariate Cox proportional hazards regression analysis found that the T stage, lymph node metastasis, clinical stage, FOXP4-AS1 expression was also correlated with deficient PFS and OS (Table 2 and Table 3), which also indicated that FOXP4-AS1 might be an independent prognostic factor for survival outcome of nasopharyngeal carcinoma.

Fig. 2.

Kaplan–Meier analysis and log-rank test revealed that higher expression of FOXP4-AS1 was meaningfully interrelated with the reduced progression-free survival (PFS) of nasopharyngeal carcinoma patients compared with patients with low FOXP4-AS1 expression. p < 0.01

Fig. 3.

Kaplan–Meier analysis and log-rank test revealed that higher expression of FOXP4-AS1 was meaningfully interrelated with the reduced overall survival (OS) of nasopharyngeal carcinoma patients compared with patients with low FOXP4-AS1 expression. p < 0.01

Table 2.

Univariate and multivariate analysis of PFS in nasopharyngeal carcinoma patients

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	p value	HR	95% CI	p value
Age	1.357	0.655–1.446	0.321	–	–	–
Gender	1.465	0.792–1.622	0.213	–	–	–
T stage	3.324	1.983–6.436	0.001	3.126	1.873–5.462	0.006
N stage	1.236	0.758–2.424	0.176	–	–	–
M stage	1.542	0.675–2.328	0.202	–	–	–
Lymph node metastasis	2.892	1.876–5.621	0.004	2.596	1.373–4. 683	0.009
Clinical stage	3.567	1.421–4.898	0.004	3.076	1.528–4.382	0.001
Histological grade	1.569	0.806–2.768	0.304	–	–	–
FOXP4-AS1 expression	4.042	1.531–6.764	0.007	3.762	1.408–5.465	0.008

Table 3.

Univariate and multivariate analysis of OS in nasopharyngeal carcinoma patients

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	p value	HR	95% CI	p value
Age	1.262	0.576–1.748	0.654	–	–	–
Gender	1.359	0.686–1.519	0.536	–	–	–
T stage	2.988	1.741–5.687	0.007	2.651	1.623–4.786	0.002
N stage	1.369	0.478–2.589	0.164	–	–	–
M stage	1.764	0.566–2.781	0.243	–	–	–
Lymph node metastasis	3.553	1.627–4.652	0.002	3.249	1.189–3.763	0.004
Clinical stage	3.342	1.561–5.372	0.001	2.766	1.422–4.108	0.006
Histological grade	1.268	0.674–2.335	0.128	–	–	–
FOXP4-AS1 expression	3.798	1.467–5.689	0.005	3.213	1.326–4.678	0.007

Discussion

In this study, we first identified FOXP4-AS1 was remarkably upregulated in nasopharyngeal carcinoma patient serum samples and the expression of FOXP4-AS1 was correlated with T stage lymph node metastasis, tumor histological grade, and clinical stage. The FOXP4-AS1 was one potential diagnostic biomarker for nasopharyngeal carcinoma patients. Patients with high FOXP4-AS1 expression showed lower PFS and OS compared with those with low FOXP4-AS1 expression. The FOXP4-AS1 was established as an independent prognostic factor. These data elucidated that FOXP4-AS1 served on a prognostic and diagnostic biomarker in the development and progression of nasopharyngeal carcinoma.

Recently, increasing evidence has shown that lncRNAs act as diagnostic and prognostic markers in nasopharyngeal carcinoma. Zou et al. found that lncRNA ANRIL was markedly upregulated in advanced-stage nasopharyngeal carcinoma and multivariate analyses suggested that ANRIL expression could play a part as an independent foreteller of OS and PFS (Zou et al. 2016). Zhang et al. observed that LINC00312 expression could tell apart non-cancerous patients from nasopharyngeal carcinoma patients by ROC analysis and low expression of LINC00312 was an independent predictor for OS by multivariate analyses (Zhang et al. 2013). Additionally, Bo et al. disclosed that AFAP1-AS1 expression was positively connected with distant metastasis in nasopharyngeal carcinoma, and the significant upregulation of AFAP1-AS1 was compactly relevant with inferior prognosis for nasopharyngeal carcinoma (Bo et al. 2015). LncRNA NEAT1 had also been reported to be considerably interrelated with the shorter OS but definitely related with more advanced clinical stages in nasopharyngeal carcinoma patients (Lu et al. 2016). Consistently, the data from this study showed that FOXP4-AS1 might be a useful biomarker for nasopharyngeal carcinoma diagnosis and prognosis prediction.

In addition, Zhao et al. reported that lncRNA FOXP4-AS1 participated in cervical cancer evolvement through modulating miR-136-5p and CBX4 network, which indicated that FOXP4-AS1 played an oncogenic role and provided novel therapeutic biomarker against cervical cancer (Zhao et al. 2020). Li et al. found that lncRNA FOXP4-AS1 was an adverse prognostic element and regulated growth in colorectal cancer (Li et al. 2017). Yang et al. reported that FOXP4-AS1 participated in the osteosarcoma tumorigenesis through combining with lysine-specific demethylase 1 (LSD1) and enhancer of zeste homolog 2 (EZH2) to decrease LATS1 expression, which predicted that FOXP4-AS1 might be a diagnostic and prognostic biomarker for osteosarcoma (Yang et al. 2018). Coincidentally, this study also provided the evidence for FOXP4-AS1 acting as a diagnostic and prognostic biomarker for nasopharyngeal carcinoma.

In short, our results firstly disclosed that FOXP4-AS1 was upregulated and relevant with unfavorable prognosis of nasopharyngeal carcinoma patients, which entirely stated that FOXP4-AS1 might be an underlying therapeutic target for nasopharyngeal carcinoma treatment. This study focused on the clinical significance and prognostic value of FOXP4-AS1. But the biological function and underlying mechanism for nasopharyngeal carcinoma development and progression should be explored in our future study.

Abbreviations

qRT-PCR

Quantitative real-time PCR

ROC

Receiver operating characteristic

OS

Overall survival

PFS

Progression-free survival

NPC

Nasopharyngeal carcinoma

EBV

Epstein-Barr virus

LSD1

Lysine-specific demethylase 1

EZH2

Enhancer of zeste homolog 2

Author contributions

LY designed the experiments. TTW performed all experiments. XYW helped complete the animal experiments. TTW analyzed the data. XYW wrote the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors of this work declare that they have no conflict of interest.