Relationship of Common Variants in miR-27a-3p Gene with Susceptibility and Prognosis of Oral Cancer

Background: miR-27a-3p has been found dysexpressed in various cancers. The aim of the present study was to clarify the prognostic value of miR-27a-3p in patients with oral cancer. Methods: We used quantitative real-time polymerase chain reaction (qRT-PCR) assay to detect the expression of miR-27a-3p in the tissue of oral cancer and adjacent normal specimens. The association of miR-27a-3p with clinicopathological characteristics was analyzed via the Chi-square test. Kaplan-Meier survival and Cox regression analysis were performed to evaluate the prognostic value of miR-27a-3p in oral cancer patients. Results: The down-regulated expression of miR-27a-3p was found in oral cancer tissues compared with the matched noncancerous samples (P<0.05). And its expression was inuenced by TNM stage (P=0.032), T stage (P=0.014) and lymph node metastasis (P=0.025). Kaplan–Meier analysis result showed that the decreased level of miR-27a-3p expression was associated with a poor overall survival of oral cancer patients. Additionally, multivariate cox regression analysis revealed that the low expression of miR-27a-3p was an independent prognostic maker in oral cancer patients (HR=0.462, 95% CI=0.223-0.957, P=0.038). Conclusions: Taken together, the expression pattern of miR-27a-3p was decreased in oral cancer tissues. The decreased expression of miR-27a-3p was a potential prognostic biomarker in patients with oral cancer.

(P=0.032), T stage (P=0.014) and lymph node metastasis (P=0.025). Kaplan-Meier analysis result showed that the decreased level of miR-27a-3p expression was associated with a poor overall survival of oral cancer patients. Additionally, multivariate cox regression analysis revealed that the low expression of miR-27a-3p was an independent prognostic maker in oral cancer patients (HR=0.462, 95% CI=0.223-0.957, P=0.038).
Conclusions: Taken together, the expression pattern of miR-27a-3p was decreased in oral cancer tissues. The decreased expression of miR-27a-3p was a potential prognostic biomarker in patients with oral cancer.

Background
Oral cancer is one of the most common malignancies in head and neck that de ned as any cancerous tissue growth located in the oral cavity [1]. According to the American Cancer Society, approximately 45,780 new cases and 8,650 cancer deaths attributed to this condition occur in 2015 in United States [2] Approximately 90% of oral neoplasms are oral squamous cell carcinoma (OSCC), most of which are invasive growth, invasion of surrounding tissue and easy to occur lymph node metastasis [3,4]. Surgical resection with chemotherapy and radiotherapy are the the most effective therapeutic methods for treatment oral cancer, but the 5-year survival rate of patients with in advanced clinical stages remained around 50-55% over the past several decades [5,6]. Therefore, it is of great importance to identify reliable molecular markers that may be bene cial to improve the poor prognosis of oral cancer.
Recent studies suggest that aberrant microRNAs (miRNAs) expressions have been found in various human cancers [7][8][9]. miRNAs are a group of small, noncoding, single-stranded RNAs of [19][20][21][22][23][24][25] nucleotides in length that play important roles in modulating cell differentiation, growth, apoptosis and proliferation [10][11][12]. miRNAs are found highly stable and abundant in serum, urine and tissues, which play crucial roles in tumorigenesis and cancer diagnosis or prognosis in various cancers [13][14][15]. miR-27a-3p found by Zeng et al. could inhibit the YAP1 directly by post-transcriptionally silencing and potentially suppress EMT process, suggesting it plays pivotal role in effectively manipulating the invasion and metastasis in oral squamous cell carcinoma cells [16,17]. However, the clinical signi cance of miR-27a-3p in the prognosis of oral cancer was still unclear.
In this study, we mainly focused on the serum expression levels of miR-27a-3p and investigated the relationship between its expression and clinicopathological characteristics in patients with oral cancer. Thereby further evaluating its value as a prognostic marker in oral cancer.

Methods And Materials
The subjects under study included 136 patients with oral cancer who underwent surgery in Chinese PLA General Hospital. Patients with oral cancer who had received prior radiotherapy to the head and neck area, were excluded from the study. All the fresh oral cancer tissues and adjacent normal tissues specimens were surgically removed and put immediately into liquid nitrogen, then stored at -80℃ until use, respectively. Clinicopathological characteristics of the patients are summarized in Table 1, including age, gender, TNM stage, T stage, lymph node metastasis, differentiation and smoking status. A 5-years' follow-up was conducted.

Statistical analysis
All statistical analyses were performed using the SPSS 21.0 software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Data are presented as mean normalized gene expression ± standard deviation (SD) from independent experiments. Student's test was used to evaluate the difference between tumor and normal groups. The associations between mRNA expression and clinicopathological factors were assessed using the χ 2 test. The survival curves were constructed by the Ksplan-Meier method and compared using the log-rank test. A multivariate analysis with cox regression analysis was conducted to evaluate the prognostic value of miR-27a-3p in oral cancer. The levels of signi cance was set at P < 0.05.

Results
The expression of miR-27a-3p was decreased in oral cancer We used qRT-PCR analysis to measure miR-27a-3p expression levels in oral cancer tissues and adjacent normal tissues. As shown in Fig. 1, the miR-27a-3p expression levels were signi cantly lower in oral cancer tissues compared with adjacent healthy tissues (P < 0.01).
Relationship between miR-27a-3p and clinicopathological characteristics of oral cancer To explore whether miR-27a-3p was involved in the development of oral cancer, we further explored the association between the expression of miR-27a-3p and clinicopathological characteristics of patients. We divided the patients into two classed (high-and low-expression group) using the average level of patients as the cutoff value. As indicated in Table 1, miR-27a-3p expression was positively associated with TNM stage (P = 0.032), T stage (P = 0.014) and lymph node metastasis (P = 0.025). However, there was no signi cant relationship between miR-27a-3p expression and other parameters, including age, gender, differentiation and smoking status (all P > 0.05).

Association of miR-27a-3p expression with prognosis in oral cancer patients
Kaplan-Meier survival analysis was done to evaluate the association of miR-27a-3p expression with overall survival. Patients with negative expression of miR-27a-3p had signi cantly poorer overall survival as compared to patients with high expression of miR-27a-3p (Fig. 2, log rank test, P < 0.05). We further evaluate the correlation between clinicopathological parameters and patients outcomes using multivariate Cox regression analysis. The cox analysis results suggested that the expression of miR-27a-3p was an independent prognostic factor in patients with oral cancer (  High miR-27a-3p expression ---HR: hazard radio, 95% CI: 95% con dence interval. P < 0.05 was considered to be statistically signi cant.

Discussion
As oral cancer is generally asymptomatic at an early stage, most of patients are diagnosed at advanced stage, resulting the poor prognosis. Currently, the advances in surgery operation, chemotherapy and radiotherapy have been performed, and the prognosis of patients with oral cancer has slowly but steadily improved, but the overall 5-year survival rate is still showing poor [18]. Thus, it is important to identify new biomarkers that, using surgical samples, predict cancer recurrence in oral cancer.
Recent studies have demonstrated that miRNAs can play important roles in cancer development and progression for several types of cancer including oral squamous cell carcinoma (OSCC) [19][20][21], and as such they may be useful as biomarkers and treatment targets..
It has been reported that miR-27a-3p dysexpressed in many cancers [22][23][24][25][26][27]. Wataru Nakata et al. identi ed miRNAs that are up-regulated in clear cell renal cell carcinoma (ccRCC) and revealed that high levels of miR-27a-3p correlated with a worse progression-free survival rate [23]. XU et al. showed that overexpression of miR-24-3p and miR-27a-3p could promote cell proliferation and observed that miR-27a-3p is upregulated in glioma tissues [24]. So far, dysregulation of miR-27a-3p has not been reported in oral cancer. Therefore, we were interested to validate its roles in in oral cancer after the aberrant overexpression of miR-27a-3p was further con rmed in paired oral carcinomas and normal tissues.
In the present study, we investigated the expression pattern of miR-27a-3p in oral cancer. The results indicated that the level of miR-27a-3p was decreased in oral cancer tissues than in adjacent normal tissues, which suggests that miR-27a-3p may be a tumor suppressor gene in oral cancer. The downregulated expression of miR-27a-3p was positively associated with TNM stage (P = 0.032), T stage (P = 0.014) and lymph node metastasis (P = 0.025), whereas no signi cantly associations was found between miR-27a-3p and other characteristics. These data above suggested that miR-27a-3p plays crucial role in patients with oral cancer and is associated with the development of oral cancer.
Furthermore, the prognostic value of miR-27a-3p was also been investigated in our study. Kaplan-Meier analysis result showed that patients with a low miR-27a-3p expression had poor overall survival (log rank test, P < 0.05), which suggested that miR-27a-3p expression was related to the prognostic value for patients with oral cancer. Then we used Cox regression analysis to further examine the prognostic value of miR-27a-3p. The results showed that the low miR-27a-3p expression was an independent and signi cant prognostic factor for oral cancer.

Conclusions
In conclusion, the down-regulated expression of miR-27a-3p was observed in oral cancer and its expression is in uenced by some clinical factors in this study. The present evidence provides support for miR-27a-3p to be a potential prognostic marker. Although further studies are needed to con rm the results of our study, our ndings may serve as the basis for future researches about the clinical value of miR-27a-3p in monitoring treatment e cacy and forecasting prognosis of oral cancer. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.
Availability of data and materialsT he datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.
Authors' contributions L.W. design of the work; S.Y. the acquisition, analysis, H.S. interpretation of data; L.W. the creation of new software used in the work; S.Y. have drafted the work or substantively revised it. All authors read and approved the nal manuscript.

Figure 1
The relative miR-27a-3p expression in oral cancer tissues and adjacent normal tissue. The expression of miR-27a-3p in oral cancer patients tissues was signi cantly lower than that in adjacent normal tissues; All the data were presented as mean ± SD; ** compared with controls p< 0.01.