Upregulation of long non-coding RNA LOC284454 may serve as a new serum biomarker for head and neck cancers

Background: Identication of effective diagnostic and prognostic biomarkers of cancer is necessary for improving precision medicine. Long non-coding RNAs (lncRNAs) play an important regulatory role in tumor initiation and progression. The lncRNA LOC284454 is distinctly expressed in various head and neck cancers (HNCs), as demonstrated by our previous bioinformatics analysis. However, the expression levels and functions of LOC284454 in cancer are still unclear. Methods: We investigated the dysregulation of lncRNAs in HNCs using the GEO database and found that LOC284454 was highly expressed in HNCs. Serum samples from 212 patients with HNCs and 121 normal controls were included in this biomarker study. We measured the expression of LOC284454 in the sera of HNC patients and normal controls using RT-qPCR. Receiver operating characteristics (ROC) analysis is an important statistical method that is widely used in clinical diagnosis and disease screening. ROC was used to analyze the clinical value of LOC284454 in the early diagnosis of HNCs. Results: LOC284454 was signicantly upregulated in the sera of patients with nasopharyngeal carcinoma, oral cancer, and thyroid cancer. LOC284454 upregulation had good clinical diagnostic value in these cancers, as evaluated by area under the ROC curve values of 0.931, 0.698, and 0.834, respectively. Conclusions:LOC284454 may be a valuable serum biomarker for HNCs facilitating the early diagnosis of malignant cancers. Further studies are needed to elucidate the mechanisms underlying the involvement of LOC284454 in HNCs. This study provides the rst evidence that LOC284454 may be a serum biomarker for HNCs.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that do not encode proteins 2, 17-19, 21, 53, 58 . In recent years, many studies have shown that a variety of lncRNAs are frequently expressed in malignant cancers and may participate in the initiation and development of malignant cancers 9, 28, 30-32, 45, 51, 57 . For example, the AFAP1-AS1 lncRNA promotes the proliferation, migration, and invasion of cervical cancer, colon cancer and nasopharyngeal carcinoma (NPC) through different mechanisms 3,4,29 . Additionally, PVT1 lncRNA induces radioresistance by regulating DNA repair and cell apoptosis, while promoting the proliferation of thyroid cancer through polycomb enhancer of zeste homolog 2/thyroid-stimulating hormone receptor 23,25,47 . However, the functional importance of most lncRNAs has not yet been elucidated, including their roles in human tumors. Only a few lncRNAs have been reported to have clinical implications for early screening and prognosis.
Presently, we examined the expression level of LOC284454 in patients' serum with HNCs and evaluated its clinical signi cance as a serum biomarker for early diagnosis.

Serum collection
We used blood collection tubes containing anticoagulants, mixed gently after blood collection. The samples were centrifuged at 1000-3000 rpm for 10 minutes, the supernatant was collected for RNA extraction. Blood samples were transported on ice and stored in -80℃ refrigerator. Discard the hemolysis and hyperlipidemia samples. The patients had not received any radio-chemotherapy or surgery before diagnosis. In total, 333 serum samples were collected from A liated Cancer Hospital of Central South University within 2017. The samples were collected from 121 normal donors randomly and 212 HNC patients. Of the 212 HNC serum, 100 were NPC, 55 were oral cancer, and 57 were thyroid cancer serum samples. Clinical data of patients is available in suppmentary table 1&2, except for 30 cases of NPC patients are not available. This study was approved by the Ethical Committee of Central South University.
Written informed consent was obtained from all patients and healthy donors.

Statistical analysis
GSE61218 is from our group, which aims to identify signi cantly expressed lncRNAs in NPC tissues. GSE68799 is a RNA-Seq data identi ed human transcriptome alterations in NPC. RNA-Seq has been proved a tool with high throughput and coverage, reliable accuracy. GSE53819 is a genome-wide expressing pro ling of NPC included 18 NPC tissue samples versus 18 control samples. They are paired tumor tissues and non-cancerous controls, which we thought can reduce individual heterogeneity. After discovering that LOC284454 is highly expressed in NPC, we also wanted to know whether it is highly expressed in other head and neck cancers, so we randomly selected the GEO dataset of oral cancer (GSE30784) and thyroid cancer (GSE33630). Data were analyzed using SPSS 13.0 (SPSS Inc., USA) and GraphPad 5.0 (GraphPad, USA). Student's t-tests were used to evaluate differences between two groups of samples. P-values< 0.05 were considered statistically signi cant. All the results obtained were from three independent replicates. The area under the curve (AUC), sensitivity, and speci city were obtained by receiver operating characteristic (ROC) curve analysis.

LOC284454 is upregulated in NPC, oral cancer, and thyroid cancer
We explored the dysregulation of lncRNAs in HNCs using the GEO database. LOC284454 was signi cantly upregulated in several cancers, including NPC, oral cancer, and thyroid cancer. In our previous article, we performed gene expression pro ling enrolled six in ammatory normal controls and 10 NPC tissues to identify differentially expressed lncRNAs (accession number GSE61218). 46506 lncRNA probes were included. The data showed that totally 1276 lncRNAs were differentially expressed, including 405 upregulated and 871 downregulated lncRNAs in NPC tissues. We selected top 20 highly expressed lncRNAs to validate. LOC284454 was one of the most signi cant and had not been detected its application as a serum biomarker (heatmap showed in Figure 1A) 11 . In NPC, we integrated three sets of gene expression pro les, including GSE53819, GSE68799, GSE61218, which further demonstrated that LOC284454 is highly expressed in NPC. GSE30784 and GSE33630 were used to analyze oral cancer and thyroid cancer, respectively. The expression levels of LOC284454 were signi cantly higher in NPC ( LOC284454 expression is signi cantly increased in serum of patients with NPC SYBR-quantitative polymerase chain reaction (qPCR) was used to detect the expression of LOC284454 in the serum of 76 NPC patients and 51 healthy donors. LOC284454 expression level was signi cantly higher in the serum of patients with NPC (Figure2a, P<0.001).
Next, to eliminate systematic errors and make the results more reliable, we designed a TaqMan probe for LOC284454 and tested the same serum samples using TaqMan-qPCR. This examination also revealed signi cantly higher expression of LOC284454 in the serum of the NPC patients (Figure 2b, P<0.001). This was consistent with previous conventional RT-qPCR results. Subsequent correlation analysis of the results obtained by SYBR-qPCR and TaqMan-qPCR demonstrated a good positive correlation between the two methods, which veri ed the reliability of this data (Figure 2c, P<0.001).
We also veri ed the expression of LOC284454 in a larger cohort of 121 normal controls and 100 NPC patients (added some new samples to the original cohort). The expression of LOC284454 in the serum of NPC patients was signi cantly higher than that of the normal control group (Figure 2d, P<0.001). Taken together, these results suggested that LOC284454 may be a potential serum marker for NPC.

LOC284454 is highly expressed in serum of patients with oral cancer and thyroid cancer
The results of the GEO database results suggested that LOC284454 may be dysregulated in oral cancer and thyroid cancer. Therefore, we next detected the expression of LOC284454 in the serum of patients with these cancers using TaqMan-qPCR. LOC284454 was signi cantly upregulated in the serum of patients with oral cancer and thyroid cancer compared with those of the normal controls (Figure 3a& 3b, P<0.001). Notably, a signi cant difference was evident in the proportion of men and women with thyroid cancer (43 females and 14 males). To exclude gender effects, we analyzed the expression of LOC284454 in 43 female thyroid cancer patients and 36 normal women. The expression of LOC284454 was higher in the tumor serum than in the normal group (Figure 3c, P=0.024). The collective results demonstrated that the expression level of LOC284454 in the serum of patients with oral and thyroid cancers was signi cantly higher than normal controls.
Diagnostic value of serum LOC284454 for HNC patients ROC is commonly used to assess the diagnostic value of biomarkers. AUC refers to the area enclosed by the curve and the 45-degree diagonal line, which is used to quantify the diagnostic value. An AUC value <0.5 indicates almost no diagnostic value. AUCs of 0.5~0.7, 0.7~0.9, and >0.9 indicate low, moderate, and high diagnostic value, respectively. Values exceeding 0.9 also indicate high speci city and sensitivity.
When the sensitivity (1 speci city) value is the largest, its corresponding row is the best cut-off point.
The AUC values of LOC284454 in NPC (Figure 4a), oral cancer (Figure 4b), and thyroid cancer (Figure 4c) were 0.931, 0.698, and 0.834, respectively, indicating that LOC284454 might be an appropriate diagnostic biomarkers for these cancers (Table 1). However, we analyzed the LOC284454 expression level as well as of patients' clinical characteristics and found that LOC284454 had no signi cant correlation with pathological stages, gender, and age distribution (Supplemental Table1)

Discussion
HNCs rank as the sixth most common type of cancers worldwide. The cancers are often at an advanced stage at the time of diagnosis and display frequent recurrence and metastasis. Thus, prognosis and patient survival are poor. Radiotherapy and chemotherapy have largely improved the treatment of HNCs in recent decades 12,33,34,41,50,56 . However, the 5-year survival rate is still very low. Improving the accuracy of early diagnosis could signi cantly improve the disease-free survival rate of patients.
Compared with other detection methods, liquid biopsy has become the preferred choice for disease screening because of its non-invasiveness, low cost, ease of use, and high stability. Some biomarkers for HNCs, including proteins, miRNAs, and EBV DNA, have been identi ed using liquid biopsies 13, 54 . However, each of these markers has its own disadvantages, including low positive rates, high false positive rate, need for experienced operators, and instrumental limitations. Therefore, nding effective early diagnostic markers in serum is critical for the treatment of HNCs.
LncRNAs have been reported to participate in the pathogenesis of HNCs. LncRNAs circulating in the serum or other bodily uids present promising biomarkers for clinical diagnostic and prognostic applications. For example, serum MALAT1, AFAP1-AS1, and AL359062 can function as diagnostic and prognostic biomarkers for NPC 22 . Notably, the upregulation of the ATB lncRNA can accurately predict papillary thyroid carcinoma and its prognosis 6 . However, few studies have examined novel lncRNAs expression in serum in HNCs.
The LOC284454 lncRNA is located on 19p13.12 and the miR-23-a~27a~24-2 cluster is present upstream of the same transcript. LOC284454 is a nuclear localized and chromatin associated lncRNA. LOC284454 RNA is found only in primates and is highly conserved. In our previous study, we demonstrated that LOC284454 promotes migration and invasion of NPC cells invitro and in vivo, and is associated with skeletal remodeling and adhesion signal pathways 11 . In this study, based on the feasibility of SYBR-qPCR and TaqMan-qPCR tests of serum LOC284454, we found that compared with healthy controls, the expression of LOC284454 was higher in NPC, oral cancer, and thyroid cancer, indicating that LOC284454 might be very important for the diagnosis of HNCs. To con rm this, we used ROC curve analysis to evaluate the diagnostic value of LOC284454. The AUC values of LOC284454 in NPC, oral cancer, and thyroid cancer were 0.931, 0.698, and 0.834, respectively, indicating that LOC284454 might be an appropriate diagnostic biomarker for these cancers. Even though we found that LOC284454 is highly expressed in NPC, oral cancer, and thyroid cancer, that does not mean LOC284454 can be generalized to all cancers. Study have shown that LOC284454 is signi cantly reduced in prostate, uterus, breast, and kidney cancer 7 , suggesting that LOC284454 is speci cly highly expressed in HNC.
Real-time PCR can sensitively detect small changes in nucleic acids based on uorescent dyes and uorescently labeled probes. In TaqMan-PCR, a uorescent reporter group and a uorescence quenching group are labeled on both ends of the probe 1,14,16,55 . When ampli ed, the 5'-3' exonuclease activity of the Taq enzyme degrades the probe. The uorescent reporter group and the uorescence quenching group are separated, so that the uorescence monitoring system can receive the uorescent signal, and the accumulation of uorescent signal is completely synchronized with the formation of the PCR product 20, 24, 37 . Since the qPCR instrument has a multicolor uorescent channel, the experimental group and the control group are allowed to react in the same tube with the same cDNA template, which can reduce systematic errors and improve the speci city and sensitivity of the experiment 40 . This is also one of the highlights of this study and might be very useful for future detection of biomarkers.
We found that LOC284454 is highly expressed in the peripheral blood of HNCs. Why it remains stable in the peripheral blood is still unclear. We suspect that this may be related to exosomes or vesicles. In summary, our results veri ed that LOC284454 is signi cantly upregulated in the serum of patients with NPC, oral cancer, and thyroid cancer based on SYBR-qPCR and TaqMan-qPCR. Moreover, ROC curve data indicate that LOC284454 could be used as a novel diagnostic biomarker for HNCs. Further research should focus on follow-up investigations to study the prognostic value of LOC284454. It is hoped that the development of new technologies, such as digital PCR, will make it easier to detect phenotypic speci c molecular changes, and will increase the sensitivity and speci city of biomarkers.

Conlusions
In this study, we investigated the dysregulation of lncRNAs in HNCs using the GEO database and found that LOC284454 was highly expressed in HNCs (nasopharyngeal carcinoma, oral cancer, and thyroid cancer). We measured the expression of LOC284454 in the serum of HNC patients via Taqman RT-qPCR.
We then used ROC curve to analyze the clinical value of LOC284454 in the early diagnosis of HNCs.

Consent to publish
Each author of this article has read the manuscript and agreed to publish.

Availability of data and material
The authors declared that all the data were available on line or from the corresponding author on reasonable request.

Competing interests
There are no potential con icts of interest. LOC284454 expression is signi cantly higherin sera of patients with NPC. (a). SYBRGreen quantitative polymerase chain reaction(qPCR) assay was used to detect the expression of LOC284454 in the sera of