Circulating miRNA-1290 as a potential biomarker for response to chemoradiotherapy and prognosis of patients with advanced oral squamous cell carcinoma: A single-center retrospective study

Abstract

MicroRNAs are a class of small, endogenous, noncoding 18- to 24-nucleotide-long RNAs that can regulate multiple processes related to cancer progression. However, their clinical value in patients with oral squamous cell carcinoma has not yet been fully explored. Therefore, the aim of this study was to investigate the clinical significance of circulating microRNAs in oral squamous cell carcinoma patients. The expression levels of circulating miR-1246 and miR-1290 in healthy volunteers and oral squamous cell carcinoma patients were examined by quantitative real-time polymerase chain reaction. The expression levels of both microRNAs in the radioresistant oral squamous cell carcinoma cell line (SAS-R) and the parent cell line (SAS) and in the conditioned medium obtained from these cell lines were also examined by quantitative real-time polymerase chain reaction. In addition, the correlations between circulating microRNA status and various clinicopathological features in 55 oral squamous cell carcinoma patients with locally advanced oral squamous cell carcinoma who underwent surgery following 5-fluorouracil-based chemoradiotherapy were examined. The expression level of miR-1290 was significantly lower in the plasma of oral squamous cell carcinoma patients than in that of healthy volunteers (p < 0.01). The expression levels of microRNAs in the conditioned medium and in the cells varied from cell to cell. In the clinicopathological analyses, the frequency of patients with low miR-1290 levels was significantly higher among cases with lower pathological differentiation and among those with a poor pathological response for preoperative chemoradiotherapy (p = 0.030 each). Furthermore, Cox regression analysis based on the 5-year overall survival and disease-free survival revealed that miR-1290 status was a significant prognostic factor for patients with oral squamous cell carcinoma (hazard ratio = 0.169, p = 0.008, and hazard ratio = 0.186, p = 0.008, respectively). Circulating miR-1290 status could be a valuable biomarker for predicting the clinical response to chemoradiotherapy as well as overall survival in patients with oral squamous cell carcinoma.

Keywords

Oral squamous cell carcinoma circulating microRNA chemoradiotherapy resistance prognosis

Introduction

Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the oral cavity;¹ however, the survival rate has not improved despite advances and advancement in diagnostic modalities and treatments. Thus, the prognosis of advanced OSCC remains poor, with a 5-year survival rate of approximately 50%.² This stagnation in the survival rate is mainly attributed to the existence of high-grade malignant cells that show important hallmarks of cancer,³ such as resistance to chemotherapy and/or radiotherapy,^3,4 abnormal proliferation, and invasion and/or metastasis. Therefore, to further improve patient outcomes, it is essential to investigate potential biomarkers for predicting the patient’s prognosis and for guiding the therapeutic management of OSCC.

MicroRNAs (miRNAs) are a class of small, endogenous, noncoding 18- to 24-nucleotide-long RNAs that can regulate multiple processes essential for cancer progression, including cell death, proliferation, metastasis, and treatment resistance.^5,6 Accordingly, the expression patterns of miRNA are frequently dysregulated in various malignancies, and thus, miRNAs have a clinical value to be exploited as disease biomarkers.⁷ Although considerable previous research on determining the usefulness of miRNA as biomarkers has focused on analysis of tissue specimens, other researchers have focused on circulating miRNA as a potential diagnostic and prognostic biomarker.^8–10

Recently, miR-1246 and miR-1290 were identified as tumor-initiating, cell-specific miRNAs in non-small cell lung cancer.^11,12 Moreover, serum miR-1246 and miR-1290 levels were correlated with the clinical response of patients with lung cancer who were receiving chemotherapies.¹² Furthermore, that study demonstrated using TCGA miRNA-Seq data that across the major types of solid tumors, including head and neck cancer, both miRNAs were strongly upregulated in tumors relative to normal tissues.¹² However, there has been minimal focus on determining the correlations between circulating miRNA expression and clinical characteristics in OSCC; therefore, it remains unclear whether the expression profiles of circulating miRNAs reflect malignant phenotypes of the primary tumor and their clinical significance, especially the treatment response to chemoradiotherapy.

Therefore, we examined the diagnostic value of circulating miR-1246 and miR-1290 in patients with advanced OSCC.

Methods

Clinical characteristics of the patients and sample collection

Plasma samples, more than 500 µL per patient, were obtained from 10 patients with locally advanced OSCC. The collected samples were analyzed for evaluating the expression level of miR-1246 and miR-1290 and compared to those of 10 healthy volunteers. For the clinicopathological analysis, preoperative plasma samples were obtained from 55 patients with locally advanced OSCC who were treated at Kumamoto University Hospital between October 2003 and January 2009. We excluded human papillomavirus (HPV)-positive tumors from the analysis based on the immunostaining result for p16, which is a surrogate marker for HPV infection. All 55 patients underwent curative surgery following preoperative chemoradiotherapy (CRT). The protocol for the preoperative CRT was as previously described.¹³ The staging and determination of the degree of tumor differentiation were performed according to the 2002 TNM Classification of the Union of International Cancer Control¹⁴ and the World Health Organization’s grade classification.¹⁵ The histological response to CRT was graded using specimens obtained during surgery based on the criteria proposed by Shimosato et al.¹⁶ as follows: grade I, tumor structures are not destroyed; grade IIa, destruction of the tumor structure is mild (i.e. “viable tumor cells” are frequently observed); grade IIb, destruction of the tumor structure is severe (i.e. “viable tumor cells” are few in number); grade III, nonviable tumor cells are present; and grade IV, no tumor cells remain.

Cell lines

The human OSCC cell line, SAS, derived from a tongue tumor was obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer (IDAC). Clinically relevant radioresistant (CRR) cell line was established from SAS cells by gradually exposing the cells to increasing doses of X-rays, as previously described.¹⁷ To maintain the CRR phenotype, SAS-R cells were exposed to 2 Gy of X-rays every 24 h, and the cumulated dose was more than 1000 Gy. All the cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and maintained under a humidified 5% CO₂-incubation at 37°C.

RNA isolation and real-time polymerase chain reaction analysis

Total RNA was extracted from plasma samples using miRNeasy Serum/Plasma kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Total RNA was extracted from conditioned medium using exoEasy Maxi kit (QIAGEN) according to the manufacturer’s instructions. Thirty-two milliliters of the conditioned medium was collected from confluent SAS cells or SAS-R cells. Total RNA was extracted from OSCC cells (SAS and SAS-R) using miRNeasy Mini kit (QIAGEN), according to the manufacturer’s instructions. These cells were incubated in 8 mL DMEM with 10% FBS per 10-cm dish and maintained under a humidified 5% CO₂-incubation at 37°C. First-strand cDNA was synthesized using the miScript II RT kit (QIAGEN). Each polymerase chain reaction (PCR) was run using a miScript SYBR Green PCR kit (QIAGEN) on a LightCycler 1.5 system (Roche, Indianapolis, IN, USA). The following primers were used to amplify miR-1246 (HS_miR-1246_2 miScript Primer Assay MS00043491): 5′-AAUGGAUUUUUGGAGCAGG-3′, miR-1290 (HS_miR-1290_1 miScript Primer Assay MS00014518): 5′-UGGAUUUUUGGAUCAGGGA-3′, RNU6B (Hs_RNU6B_2 miScript Primer Assay MS00014000): 5′-TTCCTCCGCAAGGATGACACGC-3′, and miR-16 (HS_miR-16_2_1 miScript Primer Assay MS00008813): 5′-CCAAUAUUACUGUGCUGCUUUA-3′. RNU6B was used as a reference gene to evaluate the intracellular expression of miRNAs; conversely, miR-16 was used as a reference gene to evaluate the extracellular expression of miRNAs because it has been proposed as one of the most stably expressed normalization candidates in previous studies.^18,19 The primer sets were purchased from QIAGEN. The PCR amplification conditions were as follows: pre-denaturation at 95°C for 15 min, followed by 60 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and extension at 70°C for 30 s. The comparative Ct (ΔΔCt) method was used to determine the fold changes in the expression levels using miR-16 or RNU6B as a reference.

Statistical analysis

The expression levels of miR-1246 and miR-1290 were quantified and compared between the plasma samples of healthy volunteers and OSCC patients. The chi-square test was performed to determine the associations between the miR-1246 and miR-1290 expression status and clinical or pathological variables. The overall survival (OS) and disease-free survival (DFS) were defined as the time from treatment initiation (CRT) to the date of death from any cause and the date of recurrence of the cancer or death from any cause, respectively. The Kaplan-Meier method was used to estimate the probability of OS and DFS as a function of time, and the statistical differences in the survival of the subgroups of patients were compared by the log-rank test. Multivariate survival analysis was performed using the Cox proportional hazards regression model to investigate the effects of miR-1290 and miR-1246 expression on OS and DFS. The cT-stage, cN-stage, and pathological response to CRT were used as ordinal variables, and age, sex, primary site, tumor differentiation, and miR-1290 expression status were used as nominal variables. All p values were based on a two-tailed statistical analysis, and p values <0.05 were considered to indicate statistical significance. All statistical analyses were performed using the JMP 9 software program (SAS Institute, Inc., Cary, NC, USA).

Results

Levels of circulating miR-1290 are decreased in patients with OSCC

As shown in Figure 1, the expression levels of miR-1290 were significantly lower in the plasma of OSCC patients than in that of healthy volunteers (Figure 1(b)) (p = 0.00003). Similarly, the expression level of miR-1246 tended to decrease in patients with OSCC, although the difference was not statistically significant (Figure 1(a)) (p = 0.085).

Figure 1.

Circulating miR-1246 and miR-1290 expression in the plasma of OSCC patients and healthy volunteers. (a) Expression of miR-1246 was quantified by qRT-PCR in plasma of OSCC patients (n = 10) and healthy volunteers (n = 10) (p = 0.085). (b) The expression of miR-1290 was quantified by qRT-PCR in plasma of OSCC patients (n = 10) and healthy volunteers (n = 10) (p < 0.01). The comparative Ct (ΔΔCt) method was applied to determine the fold changes in the expression, using miR-16 as a reference. **p < 0.01; NS: not significant.

Levels of miRNAs in conditioned medium and intracellular miRNAs vary from cell to cell

As shown in Figure 2, the expression levels of miR-1290 in the conditioned medium of the radioresistant OSCC cell line (SAS-R) were significantly higher than those in the parent cell line (SAS) (Figure 2(b)) (p = 0.00004). Conversely, the intracellular expression levels of miR-1290 in SAS-R cells were significantly lower than those in SAS cells (Figure 2(d)) (p = 0.0073). Furthermore, the miR-1246 expression in both the SAS-R cell line and the conditioned medium obtained from it was significantly higher than that in the SAS cells (p = 0.0051 and p = 0.0034, respectively) (Figure 2(a) and (c)).

Figure 2.

Expression of miR-1246 and miR-1290 in conditioned medium and cells. Expression of (a) miR-1246, (b) miR-1290, (c) miR-1246, and (d) miR-1290 in the conditioned medium of SAS and SAS-R was quantified by qRT-PCR. The comparative Ct (ΔΔCt) method was applied to determine the fold changes in the expression, using miR-16 or RNU6B as a reference. Data are presented as mean ± SD of three independent experiments. **p < 0.01.

Circulating miR-1246 expression is associated with tumor stage and circulating miR-1290 expression is associated with tumor differentiation and response to CRT

To elucidate the clinical significance of circulating miR-1246 and miR-1290 expression in OSCC patients who underwent preoperative 5-fluorouracil (5-FU)–based CRT, the 55 OSCC patients were dichotomized into two groups according to the median level of miR-1246 or miR-1290 expression. Twenty-nine patients (52.7%) had a high expression level of both miR-1246 and miR-1290 (miR-1246-High and miR-1290-High), whereas 26 patients (47.3%) had lower expression levels of the two miRNAs (miR-1246-Low and miR-1290-Low). The distribution of the clinical background characteristics of the patients in the two groups according to miR-1246 and miR-1290 status is shown in Table 1. The frequency of miR-1246-High patients was significantly higher among cases that showed higher T-stage (p = 0.032); however, no associations were detected for the other clinical factors. The frequency of miR-1290-Low patient was significantly higher among cases that showed lower differentiation and a poor pathological response to preoperative CRT (p = 0.030 each). There was no statistical significance between the miR-1290 expression among other variables such as age, gender, primary tumor site, T-stage, or N-stage. These results indicated that circulating miR-1290 expression is associated with the malignant phenotypes of cancer cells, especially tumor characteristics and resistance to CRT.

Table 1.

Correlation between circulating miR-1246 and miR-1290 expression and clinicopathological factors in 55 OSCC patients.

Characteristics	Total	miR-1246 expression		p value	miR-1290 expression		p value
		High	Low		High	Low
		n (%)	n (%)		n (%)	n (%)
Age (years)
Median	67.18	67.79	66.5		65.51	69.04
Range	39–85	45–85	39–85		39–85	45–85
≤65	20	10 (50.0)	10 (50.0)	0.759	13 (65.0)	7 (35.0)	0.168
>65	35	19 (54.3)	16 (45.7)		16 (45.7)	19 (54.3)
Gender
Male	32	17 (53.1)	15 (46.9)	0.944	15 (46.9)	17 (53.1)	0.305
Female	23	12 (52.2)	11 (47.8)		14 (60.9)	9 (39.1)
Primary site
Tongue	16	8 (50.0)	8 (50.0)	0.065	9 (56.2)	7 (43.8)	0.983
Mandible	20	11 (55.0)	9 (45.0)		11 (55.0)	9 (45.0)
Maxilla	6	6 (100.0)	0 (0.0)		3 (50.0)	3 (50.0)
Oral floor	4	2 (50.0)	2 (50.0)		2 (50.0)	2 (50.0)
Buccal mucosa	9	2 (22.2)	7 (77.8)		4 (44.4)	5 (55.5)
T-stage
T2	20	6 (30.0)	14 (70.0)	0.032*	9 (45.0)	11 (55.0)	0.610
T3	18	11 (61.1)	7 (38.9)		11 (61.1)	7 (38.9)
T4	17	12 (70.6)	5 (29.4)		9 (52.9)	8 (47.1)
N-stage
N0	7	4 (57.1)	3 (42.9)	0.802	2 (28.6)	5 (71.4)	0.171
≥N1	48	25 (52.1)	23 (47.9)		27 (56.2)	21 (43.8)
Differentiation
Well	43	23 (53.5)	20 (46.5)	0.831	26 (60.5)	17 (39.5)	0.030*
Moderate	12	6 (50.0)	6 (50.0)		3 (25.0)	9 (75.0)
Pathological response
Grade ≤IIa (poor ~ partial response)	12	5 (41.7)	7 (58.3)	0.385	3 (25.0)	9 (75.0)	0.030*
Grade ≥IIb (complete response)	43	24 (55.8)	19 (44.2)		26 (60.5)	17 (39.5)

OSCC: oral squamous cell carcinoma.

The chi-square test was used to examine the relationships between circulating miR-1246 and miR-1290 expression and clinicopathologic factors.

p < 0.05.

Circulating miR-1290 expression is associated with patient survival

The median OS was 43.2 months (95% confidence interval (CI) = 52.0–78.4) and the median DFS was 41.3 months (95% CI = 48.5–75.1). Eighteen patients died during follow-up. There was no effect of miR-1246 on OS (p = 0.547) or DFS (p = 0.906), with a median OS of 43.1 months (95% CI = 42.8–79.2) and 42.7 months (95% CI = 52.5–89.2), and a median DFS of 40.9 months (95% CI = 44.1–79.6) and 42.6 months (95% CI = 40.7–81.6) in the miR-1246-High and miR-1246-Low groups, respectively (Figure 3(a) and (b)). However, a lower circulating miR-1290 expression level was associated with poorer survival: the median OS was 47.1 months (95% CI = 63.4–93.9) and 38.8 months (95% CI = 29.4–70.4) (p = 0.030; Figure 3(c)), and the median DFS was 46.1 months (95% CI = 59.4–91.3) and 36.0 months (95% CI = 25.2–66.2) (p = 0.029; Figure 3(d)) in the miR-1290-High and miR-1290-Low groups, respectively.

Figure 3.

Relationships between circulating miR-1246 and miR-1290 expression and cancer-specific survival in patients with oral squamous cell carcinoma (OSCC). In the Kaplan-Meier survival analysis, the patients were divided into two groups based on high or low miR-1246 and miR-1290 expression. (a) Overall survival (OS) of the 55 OSCC patients based on miR-1246 expression. (b) Disease-free survival (DFS) of 55 OSCC patients based on miR-1246 expression. (c) OS of the 55 OSCC patients based on miR-1290 expression. (d) DFS of the 55 OSCC patients based on miR-1290 expression. *p < 0.05.

Multivariate analysis of prognostic factors

The Cox proportional hazards regression model demonstrated that low miR-1290 expression was a significant prognostic factor for OSCC patients with respect to both OS and DFS (Table 2). In addition, T-stage, N-stage, and the pathological response to preoperative CRT emerged as significant prognostic factors.

Table 2.

The results of a multivariate regression analysis for predicting the survival of 55 OSCC patients.

Variables	Assigned score	OS		DFS
		Hazard ratio (95% CI)	p value	Hazard ratio (95% CI)	p value
Age, years
≤65	0	1.244 (0.40–4.52)	0.717	1.014 (0.32–3.69)	0.982
>65	1
Gender
male	0	0.707 (0.21–2.19)	0.553	0.592 (0.17–1.82)	0.364
female	1
T-stage
T2	1	6.410 (1.68–29.9)	0.006**	7.003 (1.84–32.5)	0.004**
T3	2
T4	3
N-stage
N0	0	7.264 (1.31–62.5)	0.022*	5.844 (1.24–36.1)	0.024*
≥N1	1
Primary site
Tongue	1	1.987 (0.33–12.1)	0.446	3.135 (0.61–17.4)	0.169
Mandible	2
Maxilla	3
Oral floor	4
Buccal mucosa	5
Differentiation
Well	0	1.390 (0.37–4.73)	0.611	1.262 (0.36–4.08)	0.707
Moderate	1
Pathological response
Grades 0, I, and IIa	1	0.171 (0.04–0.64)	0.008**	0.101 (0.02–0.38)	< 0.001**
Grade IIb	2
Grades III and IV	3
Circulating miR-1290 expression
High	0	3.351 (1.08–12.0)	0.036*	3.308 (1.08–11.6)	0.035*
Low	1

OSCC: oral squamous cell carcinoma; OS: overall survival; DFS: disease-free survival; CI: confidence interval.

p < 0.05; **p < 0.01.

Discussion

This single-center preliminary analysis demonstrates a potential role of low expression level of circulating miR-1290 in the progression, response to treatment, and survival of OSCC patients, highlighting a novel candidate prognostic biomarker. To the best of our knowledge, only few studies have explored circulating miRNAs to provide a useful information regarding resistance to CRT and disease prognosis.²⁰ Therefore, the present study makes a significant contribution to the literature as it reports the clinical significance of circulating miRNA expression in the setting of OSCC patients receiving CRT.

Recently, several new circulating miRNAs have been discovered and found to be highly stable in various body fluids that can be obtained from patients in a minimally invasive manner.⁷ Recently, the expression profiles of circulating miR-1290 have been reported for various malignancies such as those of the colon, cervix, lung, breast, prostate, and pancreas.^12,21–24 Circulating miR-1246 has also been considered as a potential diagnostic biomarker for several malignances, including those of the liver, cervix, and ovary.^10,25 In contrast to our findings in OSCC (Figure 2), most of these previous studies reported elevated miR-1246 and miR-1290 levels in blood samples as well as the primary tumor compared to those in control samples. However, several researchers have demonstrated that intracellular miRNAs may be preferentially excreted or retained in cells; therefore, only certain types of miRNAs are released into the blood.^26,27 This indicates that the findings of the present study are not that surprising. Especially, the result for the miR-1290 expression in plasma and conditioned medium possibly suggests that miR-1290 is retained in primary and high-grade OSCC in which miR-1290 expression is decreased compared to normal tissues. On the other hand, the variation of sample types (plasma or serum) and experimental protocols (i.e. next-generation miRNA sequencing, real-time polymerase chain reaction (RT-PCR) profiling, or targeted analysis of specific miRNAs) used in the previous studies should be considered when interpreting the present data. Another explanation for the discrepancy between our study and the previous studies is the use of ratios, instead of absolute values, in comparing the levels of miR-16; this may have affected the expression status of miRNAs. Although miR-16 has been reported to be one of the most stably expressed miRNAs in body fluids,^19,20 its expression varied in patients with head and neck cancer.²⁸ Therefore, an appropriate internal control for quantitative real-time polymerase chain reaction (qRT-PCR) analysis or the use of absolute values should be considered in further studies.

Our expression analyses in vitro were in line with the previous studies; however, the expression pattern of intracellular miRNAs varied (Figure 2(a)–(d)). Although an accurate explanation of the discrepancies in these results is difficult, our data suggest that certain miRNAs are selectively excluded from parental cells, while other miRNAs are selectively retained by cells, as some researchers have mentioned.^26,27 Collectively, these data may suggest that a low-miR-1246 expression in plasma may reflect a low-miR-1246 expression in primary OSCC and that low-miR-1290 expression in plasma may reflect a high-miR-1290 expression in primary OSCC. Since there is no available literature or analysis on the expression of miR-1246 and miR-1290 in primary OSCC tissues, further studies are needed for elucidating the expression pattern of miRNAs and their biological significance in OSCC tissues.

We found a positive correlation between high miR-1246 expression and advanced T-stage and between high miR-1290 expression and well-differentiated tumor (Table 1). The correlation between elevated circulating miR-1246 and an advanced malignant phenotype has been reported in several malignancies. Chai et al.²⁹ reported that miR-1246 suppresses the expression of AXIN2 and glycogen synthase kinase 3β in hepatocellular carcinoma and activates the Wnt/β-catenin pathway, thereby promoting a variety of malignant phenotypes, including self-renewal, tumorigenicity, and metastasis. In addition, Zhang et al.¹² reported that miR-1246 promotes tumor growth and metastasis by suppressing MT1G, one of the targets of this miRNA. If a high miR-1246 expression reflects a high endogenous expression of this miRNA, these mechanisms possibly affect the progression of OSCC. Although it was difficult to find a direct evidence for the relationship between a high miR-1290 expression in tissue and keratinocyte differentiation based on literature search, we found a paper that supports the relationship between low miR-1290 expression and well-differentiated tumor. Forkhead box C1 (FOXC1), which is one of the direct targets of miR-1290, has been reported to be upregulated in human normal keratinocytes during terminal differentiation.³⁰ Therefore, if a high miR-1290 expression in the plasma of patients reflects a low endogenous expression of miR-1290 in primary tumors, tumor differentiation status could be mediated via a FOXC1-mediated mechanism.

We also demonstrated that low miR-1290 expression in plasma correlates with poor pathological response to CRT (Table 1). Although the biological significance of miR-1290 in cancer cells has not been fully elucidated, there are several lines of evidence indicating that miR-1290 promotes epithelial–mesenchymal transition and the subsequent acquisition of cancer stem cell (CSC) phenotypes in tumors.¹¹ It has been reported that CSCs in head and neck carcinoma including OSCC exhibit a radioresistant phenotype by regulating reactive oxygen species (ROS);³¹ Glioma pathogenesis–related protein 1 (GLIPR1), a direct target of miR-1290, upregulates the level of ROS and thereby exerts proapoptotic activities on cancer cells.³² Human mutS homolog 2 (hMSH2), another target of miR-1290, has been reported to be a key molecule for sensitivity to 5-FU in colon cancer;³³ Ye et al.³⁴ reported that overexpression of miR-1290 decreases hMSH2 expression and contributes to 5-FU sensitivity. Taken together, if a low miR-1290 expression in the plasma of patient reflects a high endogenous expression of this miRNA in primary tumor, these molecules possibly contribute to the treatment resistance to CRT in OSCC.

On the other hand, our results possibly suggest that the low miR-1290 expression level in plasma could be caused by the decreased expression of miR-1290 in the primary OSCC tissue, explaining the correlation with the treatment resistance phenotype for chemo- and radiotherapy in advanced OSCC, which likely occurs via the upregulation of downstream target genes. Recently, several genes have been identified as new downstream targets of miR-1290, including BCL-2 ³⁵ and N-acetyltransferase 1 (NAT1).³⁶ BCL2 is an antiapoptotic protein that controls proliferation activity by influencing cell cycle entry³⁵ and is associated with resistance to antineoplastic therapy such as anticancer agents^10,37 and irradiation^38,39 NAT1 is a known drug- and carcinogen-metabolizing enzyme, which transfers an acetyl group from acetyl coenzyme A to arylamines.⁴⁰ Overexpression of NAT1 has been shown to confer breast cancer cells with resistance to etoposide.⁴¹ Moreover, Stepp and colleagues⁴² reported that inhibition of NAT1 reduced anchorage-independent growth, one of the most important hallmarks of cancer, in human breast cancer cells. In the present study, we could not examine the differences in miR-1290 expression between blood samples and primary tumor because of limited resources. Therefore, further study is needed for evaluating the relationship between low circulating miR-1290 expression and treatment resistance phenotypes of primary OSCC. Treatment involving the combination of anticancer agents and/or radiotherapy is an important strategy for OSCC;^43–45 thus, continuing to investigate useful predictive markers in patients receiving these treatments is valuable. The results of the present study suggest that miR-1290 expression may be useful for guiding treatment decisions in patients with OSCC receiving chemo- and/or radiotherapy.

Low expression level of circulating miR-1290 in OSCC patients was also associated with shorter OS and DFS. Similarly, Li and colleagues²² reported that patients with elevated circulating miR-1290 expression level had a poorer outcome after resection of their pancreatic cancer. In addition, Mo and colleagues⁴⁶ reported that high miR-1290 levels in non-small cell carcinoma tissue and serum correlated with a poor prognosis. Although the results of these studies are in direct opposition to those of the present work, Endo and colleagues³⁵ reported that miR-1290 expression was downregulated in estrogen receptor α-positive breast cancer tumors and was correlated with the expression of some targets associated with resistance to chemotherapy. Therefore, a possible explanation for the discrepancy among studies is that miRNA expression varies among samples, cancer types, and tumor subtypes. Collectively, these findings demonstrate that further studies are warranted to clarify the prognostic value of circulating miRNAs in OSCC.

Overall, our data demonstrate that whether or not the circulating miRNA status accurately reflects the cumulative effects of the several underlying pathways, determining the expression status of miRNAs in body fluids is an attractive tool for diagnostic and prognostic purposes in OSCC, as has been demonstrated for some other malignancies.^36,47 Thus, prospective validation studies should be pursued based on Reporting Recommendations for Tumor Marker Prognostic Studies guidelines to develop a new prognostic algorithm for improving the treatment and outcome of patients with OSCC. Especially, since Hui et al. reported that miR-16 expression varied in patients with head and neck cancer,²⁸ an appropriate internal control for qRT-PCR analysis or the use of absolute values for expression analysis should be explored to generalize the present data.

Footnotes

Acknowledgements

We thank Editage () for English language editing.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

This study was performed with the approval of the Ethics Committee of Kumamoto University (approval number: 174) and in accordance with the Good Clinical Practice and the Declaration of Helsinki guidelines. This study was performed with the approval of the Ethics Committee of Kumamoto University (approval number: 174).

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a Grant-in-Aid for Young Scientists B (16K20591) from the Japanese Ministry of Education, Culture, Sport, Science and Technology.

ORCID iD

Hikaru Nakashima

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