Introduction

Determining serum biomarkers is considered a valuable method for diagnosis, setting treatment goals, and making prognosis assessments for various types of tumors. The concentration of serum biomarkers changes during tumor formation and development. These biomarkers can be proteins, nucleic acid, or metabolites. These markers can inform the prognosis, recurrence, or metastasis of a tumor because the growth of malignant tumors changes their concentration [1, 2]. Using a serum is easier, cheaper, and more acceptable to patients than a biopsy. However, the use of serum biomarkers for various types of cancers still requires further studies [3, 4]. One of these biomarkers is miRNA, which is a single-stranded non-coding RNA and plays a role in various physiological processes such as cell growth and division, differentiation, cell aging, apoptosis, stress and immune response, and carcinogenic processes [3,4,5,6,7]. MiRNAs are secreted in different body fluids such as saliva, serum, and urine [3]. The expression of miRNA in some tumors changes significantly compared to healthy tissue, and in addition, it has been determined that there is a specific type of miRNA for each type of cancer [5, 8]. Dysregulation of some miRNAs such as miR-138, miR-99a, miR-21, miR-375, miR-181a, miR-24, miR-222, and miR-7 has been implicated in the progression and metastasis of head and neck cancer and oral squamous cell carcinoma [3, 6, 9]. One of these miRNAs is miR-31-5p, whose gene is located in the p21.39 regions. Its function includes increasing cell migration and increasing the power of tumor invasion. New evidence has shown that miR-31-5p is upregulated in various neoplasms including OSCC and acts as an oncogenic miRNA, and high expression of miR-31-5p is associated with a poor overall survival rate [10] and that miR-31-5p can act as a useful biomarker for examining clinical prognosis [11, 12]. Regarding oral lichen planus (OLP), several miRNAs, including miR125, miR137, miR147a, miR155, miR181, and miR223, have been investigated and have been associated with the pathogenesis and malignant changes of OLP through their tissue expression and function [13]. Therefore, considering that OSCC is a malignant lesion and OLP is a premalignant lesion, a quick and timely diagnosis of OSCC and OLP is very useful for the effective treatment of this disease. In this study, we decided to evaluate the expression level of miR-31-5p in the serum of patients with OSCC, OLP, and a healthy control group to achieve a primary diagnostic biomarker.

Materials and methods

Study subjects and serum sample collection

The study was approved by the Department of Research Council of Mashhad University of Medical Sciences, Faculty of Dentistry, Mashhad, Iran (No. 991557), and by the Ethics Committee of Mashhad University of Medical Sciences [IR.MUMS.DENTISTRY.REC. 1399.165]. All participating subjects provided signed informed consent. The total sample size for the study was 99 participants in three groups: OLP (n = 32), OSCC (n = 35), and a healthy control group (n = 32).

Inclusion and exclusion criteria

Healthy control subjects had no history of malignant, inflammatory, or systemic disease. Also, they were similar to the patient groups in age and sex. Eligible patients had not received anti-tumor therapy whose disease was confirmed by biopsy; their samples had the quality to perform real-time PCR tests; and they had no other history of malignancy. Participants with a history of other malignancies and an unwillingness to cooperate to continue the project, as well as samples that were not of the required quality, were excluded from the study.

Serum processing and RNA isolation

In this study, miRNAs were manually extracted from serum samples with high quality and purity, which was one of the most important achievements of this study. The procedure was as follows: 400–450 μl of each sample was collected in a 1.5-ml microtube and 800 μl of RNXplus (SinaClon, Iran) was added to it, and the samples vortexed for 15 s to homogenize and then were placed at room temperature for 3–4 min. 250 μl of chloroform was added to each microtube and shaken by hand for 15 s, incubated for 3–5 min at room temperature, and then centrifuged for 20 min (12,000 RPM, 4 °C). About 500 μl of supernatant was carefully transferred to new 1.5-ml DNase and RNase-free microtubes. Then, for RNA precipitation, 500 μl of isopropanol (Merck Co.) was added to each tube. The microtubes were gently covered and incubated overnight at − 20 °C. After the incubation period, the samples were centrifuged for 45 min (12,000 RPM, 4 °C) and supernatant was removed, and the RNA pellet was washed with 1 ml of cold 80% ethanol and centrifuged for 20 s (12,000 RPM, 4 °C). This step was done twice. Then, the pellet was dissolved in 20 μl of DEPC water and incubated for 5 min at room temperature. To determine the quantity and purity of the extracted miRNAs, NanoDrop 2000c (Thermo Scientific 2000, USA) was used based on absorbance ratio of 260/280 nm. RNAs with sufficient quantity (50 mg/μl) and purity (ratio 2–1.5) were used for the synthesis of complementary DNA (cDNA).

Reverse transcription

An Adscriptc DNA synthesis kit (REF: 22,701, Bio-Tech, Addbio, Korea) was used to synthesize cDNA using an ABI thermocycler (One Step, USA) in a final volume of 20 µl containing 10 µl of 2X reaction buffer, 2 µl of 10 mM dNTP mixture, 6 µl of RNA, 1 µl of RT primer (1 pM) for U6 or miR 31 5p, and 1 µl of 20X enzyme. Reverse transcription (RT) was performed using a temperature cycle procedure that included preincubation at 25 °C for 10 min, RT at 50 °C for 60 min, RT inactivation at 80 °C for 5 min, and holding at 12 °C.

Real-time PCR (qPCR)

The qPCR was performed to examine relative expression of miR-31-5p compared with U6 as a reference gene using SYBR Green method. The following primers were used: miR-31-5p forward, 5′-GAGGCAAGATGCTGGC-3′ and reverse, 5′-GTCGTATGCAGAGCAGGGTCCGAGGTATTCGCACTGCATACGACAAAATATGG-3′; U6 forward, 5′-AAGGATGACACGCAAATTC-3' and reverse, 5′-GTCGTATGCAGAGCAGGGTCCGAGGTATTCGCACTGCATACGACAAAATATGG-3′. All reactions were carried out in duplicate in separate wells of 20 µl total volume. 0.5 µl of each primer (10 pM), 10 µl of SYBR Green master mix (XX) (SMOBIO, Taiwan), 7 µl of distilled sterile water, and 2 µl of cDNA were used in each reaction. Following a preincubation phase at 95 °C for 30 s, 50 cycles at 95 °C for 5 s for denaturation, and 60 °C for 30 s for annealing were performed using the Light Cycler 96 (Roche, Germany). The ΔΔCT technique was utilized to examine relative expression of qPCR data. Additionally, we examined the melting curve to verify precise target amplification (Fig. 1).

Fig. 1
figure 1

The melting curve (right) and amplification plot (left) of miR-31-5p and U6 expressions in real-time PCR

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Analytical statistics

SPSS software was used for all data analysis (version 25). The relative miRNA expression level was computed as 2−ΔΔCT, which is a frequently used measure of miRNA expression after the data had been normalized by the threshold cycle number ΔCt. A P value less than 0.05 was considered significant.

Results

Characteristics of study subjects

In this study, miR-31-5p expression was evaluated in the serum of 35 OSCC, 32 OLP patients, and 32 healthy controls. A total of 99 individuals were studied, including 51 women (51.5%) and 48 men (48.5%). Age and sex values of the study groups and the control group were compared, and differences were not statistically significant (P > 0.05). The results showed that the expression level of miRNA-32-5p in the study groups was higher than that in the control group, and the differences were statistically significant (Table 1 and Fig. 2). The increase in the expression of miRNA-31-5p qualitatively and quantitatively in the OSCC group was significant compared to the other two groups. However, the expression of miRNA-31-5P in OSCC and OLP groups was not significantly different from each other.

Table 1 Comparison of the expression levels of miR-31-5p between the study groups
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Fig. 2
figure 2

Expression level of miRNA-32-5p in the study groups

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Multiple comparisons of miR-31-5p expression in study groups

The Kruskal–Wallis test was performed to determine which specific group rejected the null hypothesis. According to this test, the OSCC group showed statistically significant differences from other groups (P < 0.05) (Table 2).

Table 2 Multiple comparisons of miR-31-5p expression in study groups
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Comparison of miR-31-5p expression in the OSCC group based on grades and stages

The statistical analysis by Fisher’s exact test showed differences between grades I, II, and III in OSCC patients (P < 0.05). The quantitative expression of miR-31-5p in grade III was higher than in grades II and I (Table 3). The average qualitative expression of miRNA-31-5P in grades I, II, and III was 3.08 ± 1.49, 5.20 ± 4.80, and 5.52 ± 2.70, respectively (Table 4). Our findings revealed a significant difference between OSCC stages (P < 0.05). The mean quantitative expression of miR-31-5p in the early stage was 2.49 ± 1.02, and in the advanced stage, it was 6.67 ± 4.27. The mean expression of miR-31-5p in early-stage patients was higher than in advanced-stage patients (Fig. 3).

Table 3 Comparison of miR-31-5p quantitative expression in OSCC group (by grades) and control
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Table 4 Qualitative expression of miR-31-5p in OSCC group (by grades)
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Fig. 3
figure 3

Quantitative expression of miR-31-5p in OSCC patients based on stages

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Comparison of miRNA-31-5P quantitative expression in the OSCC group (by tumor size)

In Table 5, it can be seen that the average quantitative expression of miRNA-31-5P in CM2 tumor size was 3.11 ± 1.7, and in CM2 tumor size it was 5.01 ± 7.23. The average expression of miRNA-31-5P in patients with tumor sizes greater than 2 cm was significantly higher than the average expression of the gene in patients with tumor sizes less than 2 cm (P 0.05).

Table 5 Comparison of miRNA-31-5p quantitative expression in OSCC group at the levels of lymph node involvement and tumor size
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Comparison of miRNA-31-5P quantitative expression in the OSCC group at the levels of lymph node involvement

In Table 5, it can be seen that the quantitative expression ranges of the miRNA-31-5P gene related to level two are greater than level one and zero and this difference was significant (P  < 0.05).

Discussion

Human body tissues and fluids are a rich source of biomarkers that are used in clinical diagnosis. Many researchers have shown that microRNAs can be used as non-invasive markers for diagnosis and prognosis. Studies conducted on cancer cells and laboratory animals show that the dysregulation of miRNAs in their primary form or by suppressing a miRNA can effectively play a role in tumorigenesis or tumor progression [14, 15]. Therefore, today, early diagnosis based on serum biomarker analysis, such as miRNAs, is one of the most important perspectives of modern medicine, which will be effective for screening, early diagnosis, treatment, and prognosis of these patients with oral malignancies. Oral cancer is one of the most common cancers in the world [16, 17]. Epidemiological studies show that the prevalence of oral cancer is different in different parts of the world, and its frequency varies from below 0.1% to above 40%. OSCC is the most common head and neck malignancy and has a poor survival rate. Its 5-year survival rate is 80% when it is detected in the early stages (T1), and it is remarkably low and reaches 20–40% if it is detected in the late stages (T3–T4) [18]. Therefore, the use of miRNA as a biomarker in serum is a fast, non-invasive, and accessible method for the early identification and diagnosis of patients [19]. We investigated the expression of miR-31-5p in three groups: OSCC and OLP patients, as well as healthy individuals. For the first time, we compared serum miR-31-5p expression in patients with pre-malignant and malignant lesions with healthy controls. This is also the first study of miR-31-5p expression in OLP patients. We report significant up-regulation of miR-31-5p in the serum samples of OSCC and OLP patients. The results of our study showed that 80% of OSCC patients and 62.5% of OLP patients demonstrated high expression of miR-31-5p, while the control group exhibited 90.6% lower expressions. So, the levels of miR-31-5p expression in serum revealed significant sensitivity and specificity to differentiate between OSCC, OLP patients, and healthy controls. It was so significant in OSCC patients compared to the OLP and health control groups. The average quantitative expression of miRNA-31-5P in the early stage was significantly higher than in the advanced stage in OSCC patients (P < 0.05). The average expression of miRNA-31-5P in tumor size, lymph node involvement, and TNM levels in OSCC patients had a significant difference (P < 0.05). Scholtz et al. [20] showed that the expression of miR-31-5p can be a suitable biomarker for the prognosis and diagnosis of OSCC. Lu et al. [3] evaluated the expression of miR-31-5p in serum, in healthy individuals and patients with oral cancer. Their results also showed that miR-31-5p expression is a biomarker and therapeutic target for the development of diagnosis in patients with OSCC. Lai et al. [21] demonstrated that increasing miR-31-5p levels was effective in the treatment and migration of cancer cells in OSCC patients. Previous studies suggested that miR-31-5p can be used as a novel biomarker for predicting the development and prognosis of colorectal cancer [22,23,24] and that high miR-31-5p expression promotes colon adenocarcinoma progression [25]. Furthermore, our experiment was consistent with previous findings. In this study, we also examined the expression of miR-31-5p in OLP patients, which was higher than in healthy controls. Based on our searches in different databases, there are no studies for the evaluation of miR-let-7a-5p as a biomarker during precancerous changes of OLP. Therefore, we are reporting novel biomarkers (miR-31-5p) in serum that we have identified to find lesions with malignant transformation potential.

Conclusion

Due to the novelty of the subject and the technical problems and difficulties regarding the study of microRNAs in patients' serum, very few studies have been conducted in this field. According to the searches conducted in reliable databases, the present study is the first study that investigated the expression of miRNA-31-5p in three groups: OSCC, OLP, and a healthy control group. According to the results of the present study, it is recommended that this microRNA be considered a target for future therapeutic strategies and an effective biomarker.