miRNA-3651: a Diagnostic Marker in Oral Squamous Cell Carcinoma?


 Background and aim: microRNAs are a group of small non-coding single-stranded RNAs that control post-transcriptional gene expression. They can act as oncogenes or tumor suppressors by amplifying or preventing the expression of certain genes. present study was conducted to assess the expression of miRNA-3651 in paraffin blocks of oral squamous cell carcinoma (OSCC) cells using qRT-PCR method in Islamic Azad university, dental branch of Tehran in 1399.Material and methods: This case-control study was conducted on 20 paraffin blocks of irritation fibroma (IF) as control group and 20 paraffin blocks of patients with oral squamous cell carcinoma as case group. After RNA extraction, qRT-PCR was performed. all experiments were repeated 3 times for each sample. Eventually the data were analyzed by SPSS 24 statistics software. Differences in miRNA expression levels between the two groups were compared using the independent samples t-test. In order to evaluate the correlation between mean levels of miRNA-3651 marker and different variables (grade, age and sex of patients), Kruskal-Wallis test, Pearson's correlation coefficient test and independent samples t-test were used, respectively.Results: The results showed that the mean expression of this biomarker method was 10.15± 5.44 rpm in normal tissue and 8.11± 1.57 rpm in cancerous tissue. Despite the lower expression of miRNA-3651 in cancerous tissue samples than normal samples, this decrease was not statistically significant (P> 0.05). There was no significant difference between the mean level of miRNA-3651 marker and different grades, age and sex of patients (p> 0.05).Conclusion: In the end, it seems that the evaluation of changes in the expression of miRNAs such as miRNA-3651, can be a minimally invasive method, in early detection and screening of patients with OSCC. Decreased expression of miRNA-3651 marker in cancerous tissue compared to normal tissue indicates the importance of these biomarkers, including miRNA-3651 in the diagnosis of oral cancer, and the researchers of this project suggest further and broader investigations on the mechanism of action and signaling cascades associated with this marker in oral cancer.


Introduction
Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the head and neck and accounts for more than 90% of oral malignancies (1). Because 5-year survival rate is directly related to disease staging at diagnosis, preventive measures and early intervention treatments reduce or even stop the progression of the disease (2,3). Changes in the occurrence of biomarkers can be helpful in diagnosis, prognosis, and treatment. However, the lack of clinically proven biomarkers still limits treatment decisions. Because most biomarkers lack the necessary speci city and sensitivity and cannot be used as reliable tools (4,5).
MicroRNAs (miRNAs) are a group of small non-coding single-stranded RNAs with a length of about 20 to 22 nucleotides that regulate gene expression after transcription (6). They are responsible for controlling the expression of 50% of all genes (7) and are vital regulators of cellular processes such as proliferation, apoptosis, cell differentiation and movement (8). Depending on the type of tumor, they can act as oncogenes or anti-oncogenes by amplifying or preventing the expression of certain genes and suppressing the host's immune response to cancer cells, which is one of the most important steps in tumorigenesis (9). MiRNAs are known for their role in cell growth and proliferation and regulation of vital pathways for cancer development (10). MiRNAs are thought to be potential biomarkers for various solid tumors, including OSCC (11,12). As a result, they can be used as useful minimally invasive tools for early diagnosis, screening, follow-up of therapeutic responses, and diagnosis of relapse (13,14). MiRNAs are secreted from cells and enter the bloodstream. They are found in various body uids including plasma, serum, blood cells and saliva (15). Therefore, identifying altered patterns of miRNAs in blood and tumor tissue can be considered as biomarkers of cancer (24)(25)(26). Changes in the expression of miRNA-3651 in a number of cancers have been studied (25,26). So far, very few studies (only 3 cases) have been performed on the expression of miRNA-3651 in oral cancers (26-28). Due to the lack of information in this eld and the importance of early diagnosis of OSCC for treatment outcome, the present study aimed to evaluate the expression of miRNA-3651 on para n blocks of oral squamous cell carcinoma in conducting research:

RNA isolation stage
Total-RNA was extracted from tissues using RiboEX (genall south korea) RNA extraction kit based on the manufacturer's protocol. RNA was determined using the A260 / A280 adsorption ratio using a Thermo scienti c-Nanodrop 2000 spectrophotometer.

QRT-PCR
Reverse transcription was performed using the all-in-one miRNA qRT-PCR diagnostic kit. The qRT-PCR step was performed using the qRT-PCR mRNA detection kit. The RT reaction system consisted of 1µl of 2.5 U / µl poly A polymerase, 1ul of RTase mixture and 5µl of PAP / RT × 5 buffer, and 2µg total RNA template and ddH2O without RNase / DNase. The reaction was performed at 37 ° C for 60 minutes and at strand cDNA (1: 5 dilution), 2 µl of universal adapter PCR primer and 2ul of all-in-one miRNA qPCR primer and 4 µl of Was ddH2O. Ampli cation of the reaction was performed under the following conditions: 95 °C for 10 minutes and 40 cycles of 95 ° C for 10 seconds, 60 ° C for 20 seconds and 72 ° C for 10 seconds. A monochrome BT-PCR RT-PCR system was used for ampli cation. MiRNA expression levels were normalized to U6 small nuclear RNA. Alteration of miRNA expression levels was performed by 2-^dCt method in para n blocks. For each sample, all experiments were repeated 3 times (25).

Statistical reviews:
Data were entered into SPSS 24 statistical software. Normality test was performed by Kolmogorov-Smirnov test. Since the distribution of ΔCT values was not signi cantly different from the normal distribution, t-test Independent samples were used to compare the mean miRNA expression in IF and OSCC samples. The obtained P-value indicated the signi cance of the study. The correlation between miRNA and histopathological grade of cancer cells was evaluated by Kruskal-Wallis test. The relationship between patients' age and miRNA-3651 marker was evaluated using Pearson correlation coe cient test. Also, the relationship between patients' sex and miRNA-3651 marker was examined using Independent samples t-test.

Results
The mean age of the patients was 69.05±14.76. The minimum age of the patients is 33 years and the maximum age is 92 years.
In this study, 40% of patients were female and 60% were men.  Total 20 100 Nx= Regional lymph nodes cannot be assessed, N0= No regional lymph node metastasis, N1= Metastasis in a single ipsilateral node ≤3 cm in dimension, N2a= Metastasis in a single ipsilateral node 3 cm & 6 cm in dimension, N2b= Metastasis in multiple ipsilateral nodes, none > 6 cm in dimension  Using The Kolmogorov-Smirnov test, we investigated the signi cance of ΔCT scores in each of the cancerous and normal tissues. This test showed that the distribution of ΔCT values was not signi cantly different from normal distribution (p>0.05) and therefore, normal distribution for these values can be accepted in each group. This result can also be seen from the graph below. This result shows that the use of independent samples parametric t-test to investigate the mean difference of ΔCT between the two groups is acceptable.  To evaluate the predictive accuracy of the miRNA-3651 marker in the diagnosis of oral cancer, the ROC diagram (receiver operating characteristic curve) and the area under the curve (AUC) were used. Youden index is also used to access the best cut-off point. The ROC curve is shown below.

Discussion
According to the human genome, miRNA-3651 is a non-coding RNA gene found in various classes of eukaryotes, including animals. This miRNA is involved in regulating gene expression after transcription. It is located in the nucleus of a human cell on chromosome 9 (9q22.31) and is estimated to be 24 nucleotides in length. Changes in the expression of this miRNA have been studied in various cancers, including esophageal cancer (25), nasopharynx (29), liver cells (32) and colorectal (30,33). Also, according to articles that examined such biomarkers in precancerous lesions compared to cancerous and normal tissue, and observed changes in the incidence of these biomarkers (31), resulting in the possibility of changes in the expression of this miRNA in cancer. The squamous cells of the mouth also made sense. Therefore, the aim of this study was to investigate the expression of miRNA-3651 on para n blocks of squamous cell carcinoma of healthy mouth and mucosa by qRT_PCR method. The results of this study showed that the mean incidence of this biomarker according to the computational method ∆CT in normal tissue was 10.15 ± 5.44 rpm and in cancerous tissue was 8.11 ± 1.57 rpm. Despite the lower expression of miRNA-3651 in cancer tissue samples than normal samples, but this decrease and difference was not statistically signi cant (P = 0.12). Also, the relationship between the expression of this marker and the variables of age, sex and grade was investigated. According to the result of Kruskal-Wallis test, there was no signi cant difference between the mean level of miRNA-3651 marker and different grades (p=0.77). According to the results of Pearson correlation coe cient test, the relationship between patients' age and miRNA-3651 marker was not signi cant (p> 0.05). According to the results of t-test of two independent samples, the difference between the mean level of miRNA-3651 marker between male and female patients was not signi cant (p=0.18).
In a study by Cong Wang et al., The results of measuring the expression levels of miRNA-3651 by qRT-PCR in esophageal squamous cell carcinoma (ESCC) and comparing it with adjacent healthy tissue, as well as examining the relationship between miRNA-3651 expression and clinical signs, showed that The expression of miRNA-3651 in tumor tissues (0.48 ± 0.45) was reduced compared to healthy tissues adjacent to the tumor (1.05 07 0.07) (P <0.001). It was also shown that the expression of this marker decreases with increasing T stage in patients. Despite the similarities between the two studies and the decrease in the incidence of miRNA-3651 biomarker in cancer cells compared to normal tissue, but this decrease was not statistically signi cant in the present study. Despite the same laboratory methods (qRT-PCR) in both studies, it should be noted that the cancers studied in these two studies are different, which can be partially different due to the type of mutations and different mechanisms in tumor production. Justi ed. (25) In a study by Jutta Ries et al., Changes in the incidence of miRNA-3651, miRNA-494, and miRNA-186 in both tissue and blood samples of patients with OSCC were compared with those of healthy individuals.
The study method was similar to the present study, qRT-PCR method, which is one of the common methods in the study of miRNA and has been introduced as the standard Golden Genomic study in many diseases (34). The results of this study did not show a statistically signi cant difference in the incidence of miRNA-494 and miRNA-186 between the case and control groups. While the expression of biomarker was signi cantly reduced in tumor tissue compared to mucosa of normal individuals (p = 0.0001). The result was consistent with the result of the present study. However, unlike tumor tissue, the expression of the miRNA-3651 biomarker was increased in the blood of cancer patients. Differences in the incidence of biomarkers in the tissues and blood of cancer patients may indicate different functions of miRNA-3651 in the bloodstream and tissues. Finally, this study concluded that reducing the expression of miRNA-3651 in tumor tissues could be a diagnostic biomarker (27).
Another study by Jutta Ries et al., Which examined the incidence of miRNA-3651, miRNA-494, and miRNA-186 only in whole blood samples from people with OSCC compared to healthy volunteers, also found an increase in miRNA-3651 levels. And it was repeated. However, this discrepancy between this study and the present study can be explained by testing and marking this marker on tissue and blood. There was also a signi cant correlation between miRNA-3651 levels and lymph node status, clinical stage and tumor grade. Given these cases, it was suggested that altered miRNA-3651 levels in patients' blood samples could be used as a minimally invasive method for screening patients (26).
Another study by Jutta Ries et al. Emphasized the importance of changing the incidence of miRNA-3651, miRNA-494, and miRNA-186, even in the blood of patients previously treated with OSCC, to assess the difference in recurrence in patients. In this study, 4 groups consisting of: 1-with initial diagnosis of OSCC (case group), 2-healthy individuals (control group), 3-individuals with no recurrence of the disease and 4individuals with disease recurrence. An important nding of this study was that the level of miRNA-3651 in the blood of patients with recurrence of the disease was signi cantly increased (Pvalue = 0.001).
Considering the importance of early detection of recurrence in increasing the probability of patient survival, it can be concluded that the study of the expression of this biomarker in the prevention and early detection of recurrence of the disease is necessary and helpful (28).
In a 2018 study by Schneider et al., MiRNAs were examined in the tissues and serum of people with OSCC compared with healthy people. Of the 225 miRNAs examined in tissue, the results were almost identical to those of the present study. The expression level of miRNA3651 in healthy tissue was 7.24 .03 37.03 and in tumor tissue was 12.6 ± 5.58 and a signi cant decrease (p = 0.0068) was observed in the expression of this marker in oral cancer compared to healthy tissue. In the present study, a decrease in the expression of this marker was observed, although this decrease was not statistically signi cant (10).
A 2020 study by Tuncer et al., Which examined changes in the expression pro le of miRNAs in the peripheral blood of people with ovarian carcinoma compared to healthy individuals, found con icting results with the results of the present study. The ndings of this study showed an increase in the expression of some miRNAs, including miRNA3651. Perhaps this difference in the results obtained with respect to the use of peripheral blood in this study compared to the use of tissue in the present study, two different cancers, ovarian cancer and oral squamous cell carcinoma and speci cally the mechanism and activity of different cell cascades in each Attributed to these cancers. But the important point is the change in the expression of this marker that by examining the mechanism of its effect in different cancers, miRNA3651 can be considered a potentially useful marker for early detection of cancers, especially oral cancer (35).
In a study by Li et al. In 2020 entitled The effect of miRNA3651 on promoting the proliferation of colorectal cancer cells through direct inhibition of T box transcription factor 1. In this study, which examined the expression of miRNA-3651 in colorectal cancer tissues compared to healthy tissue, an increase in the expression of this marker was observed in cancer tissues and this increase in expression was related to the TNM stage. The result was inconsistent with the result obtained in the present study. In this study, it was found that reducing the expression of this marker stops the growth of cancer cells and induces apoptosis. Western blot in this study also showed that decreased expression of this marker leads to inactivation of PI3K / AKT and MAPK / ERK signaling pathways in colorectal cancer cells. Finally, this study suggested that this marker has oncogenic potential in colorectal cancer (33).
In a 2019 study by Zhao et al. Entitled The effect of miRNA3651 on promoting the growth and invasion of hepatocellular carcinoma cells targeting PTEN, qRT-PCR was used to evaluate the expression of this marker as well as methods such as transwell invasion Cell invasion) and Western blot were used to evaluate the status of cell apoptosis. In this study, contrary to the present results, an increase in the expression of this marker was observed compared to normal tissue. This increase in expression signi cantly led to the progression of proliferation and invasion of the Huh-7 cancer cell line. Conversely, decreased expression of this marker clearly led to inhibition of growth and invasion of this cell line by stimulation of apoptosis. As a result, this study suggested that miRNA3651 could be considered as a new therapeutic target in hepatocellular carcinoma (32).
Mentioning all these articles indicates that miRNA3651 can be used as a potential diagnostic and therapeutic marker in various cancers, although the type of cancer under study and the different cell cascades that are activated in each cancer lead to various changes in the occurrence of this marker. It turns out that more and more careful study of its effect on any cancer can be a new way to help cancer patients.

Conclusion
Although the expression rate of miRNA-3651 in cancerous tissue samples was lower than normal ones, this decrease and difference were not statistically signi cant (P>0.05). There was no signi cant difference between the mean expression of miRNA-3651 and different histopathological grades, age and sex of the patients (p>0.05). It seems that better identi cation of miRNAs change patterns in tumoral tissue can be considered as cancer biomarkers and as a minimally invasive tool in early diagnosis and screening of cancer patients. The reduction of miRNA-3651 marker expression in cancerous tissue relative to normal tissue indicates the importance of these biomarkers such as miRNA-3651 in the diagnosis of oral cancer, and the researchers of this project suggest further and broader investigations and investigation of the mechanism of action and signaling cascades associated with this marker in oral cancer  Investigation of the normality of data distribution in two groups