Summary

METHODS
SRF was identified as a potential target gene of miR7111-5p and miR6870-5p using the TargetScan, Mirwalk, and Mirdb miRNA-target prediction tools. SRF, miR7111-5p, and miR6870-5p expression levels were examined by RT-qPCR in OSCC compared with normal tissues.

RESULTS
Our results showed that miR7111-5p and miR6870-5p were significantly downregulated in 30 pairs of OSCC tissue samples (p<0.05) relative to adjacent normal oral tissues. In the present study, we found that the high expression level of SRF was negatively correlated with the down regulation of miR7111-5p and miR6870-5p and was statistically correlated with the tumor stage.

CONCLUSION
miR7111-5p, miR6870-5p, and SRF may be potential prognostic biomarkers for OSCC diagnosis and monitoring.

Introduction

Considering the asymptomatic nature of oral cancer at its early stage, early detection of OSCC plays a critical role in prognosis and reduces treatment-related morbidity and mortality.[4] Recent attention has focused on the potential application of biomarkers for patient assessment in multiple clinical stages. Cancer biomarkers can be used to estimate the risk of cancer, screen for occult primary cancers, distinguish benign from malignant tumors, diagnose cancer, develop a prognosis, detect recurrence, and assess response to therapy.[5]

MicroRNAs (miRNAs) have a molecular regulatory role and have emerged as diagnostic biomarkers of various human cancers, including OSCC. Several studies have recently investigated the association between the expression levels of some of these miRNAs and the initiation, progression, survival rate, and metastatic potential of cancer.[6,7] miRNAs are small non-coding RNAs (18-25 nu-cleotides long) that negatively regulate gene function at the posttranscriptional level of target genes by suppressing translation or degradation of the target mRNA.[6,8] A number of studies have identified that increased expression of miRs in OSCC and OSCC-derived cells compared to normal tissues have known as the most prevalent risk factors in the OSCC development and tumorigenesis.[9-13]

Several miRs are characterized to act as tumor suppressors and/or oncogenes in OSCC. They regulate gene expression through interaction with components in the signaling pathways influencing cell proliferation, differentiation, migration, apoptosis, and other important involved mechanisms at cancer development.

Identification of miRNA expression patterns in OSCC compared to normal tissues can lead to earlystage diagnosis and prognosis.[14]

Serum response factor (SRF) is a MAD-box containing transcription factor which has an important role in the regulation of many involved-specific genes in cell growth, proliferation, differentiation, and apoptosis.[15] It has been firmly established that SRF is a direct target for regulation by miRNAs and also executes its regulatory role in microRNA expression and biogenesis where miRNAs are direct transcriptional targets of SRF.[16,17] A previous study has reported that miR7111-5p and miR6870-5p were upregulated in OSCC[8] but the role of two miRs in the regulation of SRF expression in OSCC remains elusive.

This study was designed to investigate the expression of miR7111-5p, miR6870-5p, and SRF as their target gene as well as to identify their possible expression association in OSCC and normal tissue.

Methods

Table 1: Association between SRF expression and the clinicopathological features of patients with OSCC

Predicting microRNA Target Genes
The TargetScan (http://www.targetscan.org), Mirwalk (http://mirwalk.uni-hd.de), and miRDB (http://mirdb. org) databases were used to predict potential miRNA target genes. The selected gene for both miRNAs was subjected to analysis.

RNA Extraction and Quantitative RT-PCR (qRTPCR)
The total RNA was extracted from frozen tissue samples using RiboEx? reagent (GeneAll Biotechnology, Korea), according to the manufacturer"s instructions. In addition, the extracted RNA was treated with RNAse-free DNAase to eliminate any genomic DNA contamination. The quality and quantity of extracted RNAs were evaluated using 1.5% agarose gel electrophoresis and Nanodrop (Thermo scientific- Nanodrop 2000) spectrophotometer analyses, respectively. miRNA was reverse transcribed with 1 µg total RNA using BioFACT cDNA Synthesis kit (Daejeon, South Korea), according to the manufacturer"s protocol. PCR was performed on an Exicycler? 96 thermocycler (Bioneer, Korea) using a SYBR-Green kit (Ampliqon). Quantitative real-time PCR on miRNAs and SRF was conducted with the following thermal cycling profile at 95°C for 2 min, followed by 40 cycles comprising 95°C for 15 s, 60°C for 30 s, and 72°C for 20 s. U6 served as an endogenous control for miR7111-5p and miR6870-5p and GAPDH used as a reference control for SRF target gene. The primer sequences are summarized in Table 2. The formula 2-^ΔCq was utilized to determine the expression level of miR7111-5p, miR6870-5p, and SRF.[18]

Table 2: Sequence of the primers used in this study

Statistical Analysis
All experiments were repeated three times, and numerical data are expressed as the means±standard deviation. Statistical analysis was performed using GraphPad Prism software 5.0 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS (GraphPad Prism 5 software (version 18.0; SPSS, Inc., Chicago, IL, USA). Expression data were controlled for normal distribution by one-sample Kolmogorov-Smirnov (K-S test). A one-way ANOVA was used to determine statistically differences in SRF gene expression levels in different tumor sizes. The associations between miR7111-5p, miR6870-5p, and SRF levels and clinic- pathological parameters of OSCC patients were assessed using independent sample test and independent sample Kruskal-Wallis test. The correlation between miR7111-5p, miR6870-5p, and SRF expression was performed by Pearson correlation analysis. p<0.05 (≤0.05) was considered to indicate a statistically significant.

Results

Fig 1: Downregulated expressions of miR-7111-5p (a) and miR-6870-5p (b) in oral squamous cell carcinoma. Data are shown as means±SD of three separate experiments. *p<0.05.

SRF Expression Analysis by RT-qPCR
In the present study, the potential targets of miR7111- 5p and miR6870-5p were identified with promising miRNA-target prediction tools (The TargetScan, Mirwalk, and Mirdb). According to conserved seed matching sequence principles, the predicted miRs and potential target gene were selected by identifying the intersection of databases.[20] SRF was identified as a potential target for miR7111-5p and miR6870-5p based on predicted databases. We used qRT-PCR to investigate SRF expression in 30 pairs of OSCC tissues and adjacent normal oral epithelial tissues. Statistical analysis revealed that SRF expression level was significantly upregulated (~5-folds) in OSCC tissues compared with that in adjacent normal tissues (p=0.025) (Fig. 2). Furthermore, the expression level of SRF in OSCC tissues was negatively correlated with corresponding tumor-adjacent normal tissues (p=0.241).

Fig 2: Expression level of serum response factor showed upregulation in oral squamous cell carcinoma (p=0.025). Data are shown as means±SD of three separate experiments. *p<0.05.
SRF: Serum response factor.

The Association between SRF Expression and Clinicopathological Characteristics
To examine the potential diagnostic value of SRF expression rate in OSCC, we analyzed the SRF expression levels at OSCC patients with different clinical feature categories (Table 1). The results revealed that SRF expression was significantly associated with clinical tumor stages (p=0.020) and family history (p=0.045). The patients were assessed for the history of cancer in the family in which 20% (n=6) of the patients had a family history of cancer, and 80% (n=24) with no familial history. The expression of SRF was increased in all patients and was higher in patients with a family history of cancer. A significant association was found between SRF upregulation and family history of cancer (p<0.05). We found high expression of SRF was not statistically associated with clinical features including lymph node status (p=0.88), tumor grade and size (p=0.33, p=0.69), vascular invasion (p=0.32), necrosis (p=0.77), metastasis (p=0.88), perineural invasion (p=0.40), sex (p=0.31), and smoking (p=0.55).

The Correlation between the Expression of miR7111- 5p, miR6870-5p and SRF
To study miR7111-5p, miR6870-5p levels positively and negatively correlated to SRF expression in OSCC, a correlation analysis by Pearson method performed in 30 pairs OSCC tissues and adjacent normal tissues. We did not find a significant correlation between downregulation of miR7111-5p, miR6870-5p, and increased expression of SRF gene in OSCC tissues and adjacent normal tissues (Fig. 3a, b).

Fig 3: No significant correlation was found between the expression of serum response factor, miR-7111- 5p (a) and miR-7111-5p (b) in oral squamous cell carcinoma patients" tissues. *p<0.05.
SRF: Serum response factor.

Discussion

Many miRNAs, including miR-10b,[23] miR- 21,[24] miR-31,[25] miR-34b/c,[26] miR-146a-5p,[15] miR186,[21] miR-196a/b,[16] miR-223, miR-320, miR-494,[21] miR-543,[27] miR-1179[14], and miR- 4513,[28] have been reported to be dysregulated in OSCC. miR7111-5p and miR6870-5p have been confirmed to be upregulated in hepatocellular carcinoma through targeting the KSR2 gene.

To date, no study has attempted to evaluate the expression of MiR7111-5p and miR6870-5p in OSCC, except the research by Chamorro-Petronacci et al.,[8] in which only microarray-based miRNA expression profiling was taken into account and found to be upregulated. In the present study, qRT-PCR was employed to evaluate dysregulated expression levels of miR7111-5p, miR6870-5p in OSCC.

Our results showed that miR7111-5p, miR6870-5p downregulated in OSCC patients which were not in a manner consistent with the upregulated level obtained from the previous report. miRNAs perform their regulatory roles through the translation repression or the degradation of their target mRNAs. Therefore, recognition of regulation relations of miRNAs on target genes for a specific type of cancer can be a crucial step in early detection and to improve patient survival rates.

Regardless of the regulatory function of SRF at the expression of various miRNAs involved in the MAPKinase Signaling Pathway, it has been demonstrated to be a direct target for regulation by many miRNAs in several types of cancer.[15] SRF was selected as a potential target gene for miR7111-5p and miR6870- 5p based on analysis using TargetScan, Mirwalk and, miRDB online bioinformat-ics software.

Although research of the deregulated functions of miR7111-5p and miR6870-5p in different cancers is in progress, their regulatory function by targeting SRF in OSCC still remains largely unclear. To the best of our knowledge, this is the first evaluation of the correlation between deregulated expression levels of miR7111-5p, miR6870-5p, and SRF in OSCC. We also investigated the correlation between clinico-pathological stages of OSCC and SRF expression.

Our results revealed that the miR7111-5p and miR6870-5p expression levels were significantly decreased in OSCC compared with adjacent normal tissues. Moreover, SRF expression was significantly upregulated in OSCC and was not correlated with downregulated expression levels of miR7111-5p and miR6870-5p.

Family history has been reported as an important risk factor for oral cavity cancer and many of cancers. Therefore, understanding the relationship between family history and oral cavity cancer provides great potential in screening and preventive strategies resulting reduce the risk of cancer.[29,30] In our study among investigated clinicopathological characteristics, the upregulation of SRF only was significantly correlated with tumor stage (p=0.020) and family history (p=0.045) probably due to the small sample size and needs to further research in the future.

According to the results of the present study, the expression of SRF gene has increased in higher stages (IV) which may be a potential marker for early detection of OSCC in the future. OSCC is among the most common causes of cancer death in the world, accounting for more than 90% of all oral cancers and has a 5-year survival rate of <50%.

Ghafari et al.[31] revealed that a changing trend of histopathologic pattern with an increasing number of moderately differentiated OSCC and a higher increasing rate in tumor size and vascular, perineural invasion during years of 2006-2015 in Iran. Altered expression miRNAs have been implicated in various forms of cancer. Therefore, identification of miRNAs as a biomarker may provide new insights into clinical diagnosis at earlier stages and treatment of OSCC.[32,33] The identification of OSCC patients in the advanced clinical stage provides the main obstacle to correct treatment and save lives in Iran. Therefore, our findings can be an effective step in creating specific miRNAs signatures in predicting and diagnosing of OSCC.

Our study suggested that downregulation of miR- 7111-5p and miR-6870-5p alone and or with other altered expression miRNAs profiles may serve as a prognostic marker to predict and early detection of OSCC. Given that, the relatively small sample size was a major limitation in this study, therefore whether miRs down expression impacted the expression of SRF and or SRF acts as oncogene contributes to OSCC requires further investigation.

Conclusion

Peer-review: Externally peer-reviewed.

Conflict of Interest: All authors declared no conflict of interest.

Ethics Committee Approval: The study was approved by the Research Medical Ethics Committee of Imam Khomeini Hospital (No: IR.IAU.SRB.REC.1399.099, Date: 19/11/2020).

Financial Support: None declared.

Authorship contributions: Concept - F.J.; Design - F.J.; Supervision - F.J.; Funding - M.S.; Materials - M.S.; Data collection and/or processing - M.S.; Data analysis and/or interpretation - M.S.; Literature search - A.G.; Writing - A.G.; Critical review - A.G.

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