The expression tendency and prognostic value of PDGFRβ in oral cancer

Background Oral cancer is a common malignant tumor in head and neck with poor prognosis. This study aimed to determine the expression tendency and prognostic value of PDGFRβ in oral cancer. Methods The mRNA expression level of PDGFRβ in the oral cancer tissues and adjacent normal tissues of oral cancer patients were detected by quantitative real-time polymerase chain reaction (qRT-PCR). And the association of PDGFRβ expression with clinicopathological characteristic was analyzed via chi-square test. Then we used Kaplan-Meier analysis to analyze the effects of PDGFRβ expression on the overall survival of oral cancer patients. The multivariate cox analysis was used to evaluate its prognostic value. Results The results indicated that the mRNA expression level of PDGFRβ was signicantly increased in oral cancer tissues compared with that in the adjacent normal tissue ( P < 0.001). And its expression is positively associated with clinical stage, T stage, lymph node metastasis and histological grade. Kaplan-Meier analysis revealed that patients with high expression of PDGFRβ had markedly worse overall survival than those with low expression of PDGFRβ (log rank test, P < 0.05). Additionally, cox regression analysis revealed that the high expression of PDGFRβ was an independent prognostic maker in oral cancer patients. Conclusion PDGFRβ is up-regulated and involved in the development of oral cancer. Moreover, it could be an independent prognostic bio-marker for oral cancer.


Background
Oral cancer is the most common malignant tumor in head and neck, most of which are characterized by invasive growth, invasion of surrounding tissue and easy to occur cervical lymph node metastasis [1]. Approximately 90% of oral neoplasms are oral squamous cell carcinoma (OSCC) [2]. Although surgical resection with chemotherapy and radiotherapy act as the most effective therapeutic method for treatment oral cancer, the prognosis for patients suffering OSCC remain poor [3]. Therefore, it is of important to identify the molecular markers that are associated with oral malignancy; which may further improve the clinical management and therapeutic development.
Platelet-derived growth factor receptors (PDGFRs) are part of the class III receptor tyrosine kinase family, including PDGFRα and PDGFRβ two subtypes [4,5]. The receptor for PDGF-BB, PDGFRβ, located on chromosome 5q33.1, has an important role in regulating mesenchymal cells development, including pericytes, broblasts, and vascular smooth muscle cells [6,7]. PDGFRα and PDGFRβ are important biological markers of cancer-associated broblast (CAFs) that can promote the occurrence, growth, invasion and metastasis of tumor, which are involved in the process of tumor cell proliferation and migration [8][9][10]. PDGFRβ has been found aberrantly expressed in many diseases and in tumor progression. However, the clinical signi cance of PDGFRβ in the prognosis of oral cancer was few reported.
In this study, we investigated the expression level of PDGFRβ in clinical oral cancer tissues and adjacent normal tissues. And we also investigated the relationship between PDGFRβ expression and clinicopathological characteristics of patients. Besides, the prognostic value of PDGFRβ was estimated.

Patients and specimens
A total of 156 patients with oral cancer, who underwent surgery at Chinese PLA General Hospital were included in this study. Among them, there were 74 females and 82 males. They were all histopathologically con rmed and without a prior history of cancer or previous chemo-or radiotherapy.
And all the cancer tissues and adjacent normal tissues were extracted from patients and immediately put into liquid nitrogen after surgical resection and then stored at -80 ℃until RNA extraction, respectively. The clinicopathology of the cases were summarized in Table 1. A 5-years' follow-up was conducted and patients who were died from unexpected events or other diseases were excluded from our study. Rna Extraction And Quantitative Real-time Rt-pcr (qrt-pcr) The PDGFRβ gene was ampli ed via qRT-PCR. Total RNA was extracted using Trizol reagent (Invitrogen, Carisbad, CA, USA) according to the manufacturer's instructions. Then the rst-strand complementary DNA (cDNA) was synthesized using the PrimeScript RT reagent kit (Takara, Dalian, China). QRT-PCR reaction was performed using a SYBR Green Premix Ex Taq (Takara, Dalian China) on the Applied Biosystems 7900 Fast Real-Time PCR system (Applied Biosystems, Foster City, California, USA). β-actin was used as internal control for normalization of data and the expression of PDGFRβ at mRNA level was calculated via the comparative cycle threshold (CT) method. QRT-PCR primer sequences for PDGFRβ were as follows: forward, 5'-GTGGTGAGCACACTGCGTCTG-3' and reverse, 5'-GTAACGT GGCTTCTTCTGCCA-3' [11]. Each sample was examined in triplicate.

Statistical analysis
All statistical analyses were performed using SPSS 22.0 statistical software (SPSS, Inc., Chicago, IL, USA). All data were presented as mean ± standard deviation (SD). The associations between protein expression and clinicopathological factors were assessed using the χ 2 test. The mRNA level of PDGFRβ between tumor and normal groups was compared by Student's t test. The association between PDGFRβ and overall survival was examined using the Kaplan-Meier method and log-rank test. Multivariate analysis was performed to identify the independent risk factor for oral cancer. P values less than 0.05 were regarded as statistically signi cant.

Results
The expression of PDGFRβ was increased in oral cancer To test role of PDGFRβ in the progression of oral cancer, we measured the expression levels of PDGFRβ in a total 156 oral cancer tissues and adjacent normal tissues specimens. As shown in Fig. 1, the mRNA expression level of PDGFRβ was signi cantly higher in oral cancer tissues compared with adjacent normal tissues (P < 0.001).

Relationship between PDGFRβ and clinicopathological characteristics of oral cancer
The correlation between PDGFRβ and clinicopathological features was analyzed via chi-square test. To explore whether PDGFRβ was involved in the development of oral cancer, oral cancer samples were classi ed into the low expression group (n = 75) and high expression group (n = 81) according to the median expression level of all the samples. As shown in Table 1, the expression of PDGFRβ was positively associated with clinical stage (P = 0.036), T stage (P = 0.010), lymph node metastasis (P = 0.013) and histological grade (P = 0.037). However, there was no signi cant association was observed between PDGFRβ expression and other parameters including age, gender and differentiation (P > 0.05).
Association of PDGFRβ expression with prognosis in oral cancer patients To investigate the prognostic role of PDGFRβ in oral cancer, we performed a 5 years' follow-up. As shown in Fig. 2, our results suggested that patients with high expression of PDGFRβ had a shorter overall survival than those with low expression (log rank test, P = 0.000). The multivariate analysis using the cox regression analysis adjusted for all variables was performed which manifested that the high expression of PDGFRβ (Table 2, HR = 2.467, 95% CI = 1.340-4.544, P = 0.004) was an important factor for predicting poor outcome and it might be an independent prognostic bio-marker.

Discussion
The occurrence of oral cancer is related to many factors, such as smoking, alcohol use, smokeless tobacco products and HPV infections [12,13]. The 5-year survival rate of patients with early stage oral cancer ranges from 69-82%, but the 5-year overall survival rate of patients with nodal metastases remain unsatisfactory when compared to the early stage patients [14][15][16]. The accurate prognostic bio-markers are meaningful for the prediction of the prognosis for oral cancer. Therefore, nding effective prognostic factors is crucial to improve the prognosis and therapy of oral cancer patients.
Recently, many researchers have been devoted to examine speci c biological markers with prognosis in head and neck cancer, including oral cancer [17][18][19]. RUMI YOSHIHAMA et al. indicated that SOX2, KLF4 and brachyury serve important roles in tumor progression, and these transcription factors may thus represent clinically useful prognostic markers for OSCC [20]. Acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) was found commonly increased in OSCC and ANP32A protein could act as a potential biomarker for prognosis assessment of oral cancer patients with lymph node metastasis [21].
In this study, we detected the expression of PDGFRβ at mRNA level using qRT-PCR analyses. And PDGFRβ was found to be up-regulated in oral cancer tissues compared to that in adjacent non-cancerous tissues which indicated that it might be a tumor oncogene in oral cancer. Then we further explored the role of PDGFRβ in the development of oral cancer via investigating the relationship between its expression and clinical factors. The high expression of PDGFRβ was involved in the progression of oral cancer due to its tightly correlation with clinical stage, T stage, lymph node metastasis and histological grade. Kaplan-Meier analysis with the data from follow-up showed that patients with a high PDGFRβ expression had worse overall survival compared to patients with low expression, which suggested that PDGFRβ expression was related to the prognosis of patients with oral cancer. According to cox regression analysis, the result showed high PDGFRβ expression was an independent and signi cant prognostic factor for oral cancer.

Conclusion
In conclusion, PDGFRβ expression is increased in oral cancer patients and its expression is in uenced by some clinical factors. The present evidence provides support for PDGFRβ to be a potential prognostic marker. However, it still remains to be further warrant its prognostic utility.  The mRNA expression of PDGFRβ in oral cancer tissues and normal tissue. The expression level of PDGFRβ in oral cancer tissues was higher than in the adjacent normal specimens ( P < 0.001).