Relationship of Common Variants in Krüppel-like Factor 7 Gene With Susceptibility and Prognosis of Oral Squamous Cell Carcinoma

Background: The aim of this study was to assess the prognostic value of Krüppel-like factor 7 (KLF7) for patients with oral squamous cell carcinoma(OSCC). Methods: The expression of KLF7 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) in pairs of tumor tissues and adjacent non-tumor tissues of OSCC. Chi-square (χ 2 ) test was applied to evaluate the association of KLF7 expression with clinicopathological characteristics of OSCC patients. Overall survival was estimated using the Kaplan-Meier method with log rank test. The cox proportional hazards model was used for univariate and multivariate analyses. Results: The expression of KLF7 was remarkably increased in OSCC tissues compared with adjacent non-tumor tissues (P<0.001). KLF7 expression was related to TNM stage (P=0.006), tumor size (P=0.010), smoking (P=0.006) and drinking (P=0.000). Kaplan-Meier analysis showed that OSCC patients with high KLF7 expression had a poorer overall survival than those with low expression (log rank test, P=0.018). Moreover, multivariate analyses showed that KLF7 was an independent prognostic factor for OSCC (P=0.002 HR=2.645 95%CI: 1.426-4.906). Conclusion: Decreased expression of KLF7 may be a potential unfavorable prognostic factor for patients with OSCC.


Background
Oral squamous cell carcinoma (OSCC) is one of the most common forms of oral cancer worldwide, and its morbidity has been rapidly increasing in Asian countries in the past decade [1]. There are several risk factors con rmed for patients with OSCC, such as tobacco smoking, alcohol consumption, human papillomavirus infection, etc [2][3][4]. The conventional treatments for OSCC include surgical treatment, chemotherapy, radiotherapy. In the past few years, the therapeutic strategies for OSCC have been signi cantly improved, but the ve-year survival of the patients still remains unsatisfactory, especially those diagnosed with advanced stages [5][6][7]. The aggressive tumor progression and high recurrence rate may be responsible for the poor clinical outcomes [8]. Until now, OSCC progression evaluation is mainly dependent on TNM staging. However, the TNM system is based on clinical parameters of the cases. The histopathological features as well as the interaction between the tumor and host are not considered, which may cause bias to the nal results [9]. Therefore, novel biomarkers are in urgent need to evaluate tumor progression and guide treatments in patients with OSCC.
Krüppel-like factors (KLFs) are a group of DNA-binding transcriptional regulators which contain zinc ngers. The family plays important roles in a variety of biological processes, such as cell stemness, proliferation, differentiation, apoptosis, and energy metabolism [10][11][12]. Krüppel-like factor 7 (KLF7) is a member of the KLF family, and three C2H2 type zinc ngers are located at its C-terminus that bind to the promoters of target genes [13]. KLF7 could regulate the function of the nervous system and adipose tissue [14]. Knockdown the expression of KLF7 may cause damages to neuronal and cardiomyocytic differentiation of embryonic stem cells [15]. Besides, the abnormal expression of KLF7 may be also involved in regulation of blood disease, type 2 diabetes, obesity, etc [16][17][18] [19]. However, to our knowledge the prognostic signi cance of KLF7 in OSCC remained unclear.
In the present study, we analyzed the relative expression level of KLF7 in OSCC carcinoma tissues, as well as its association with clinicopathological factors. In addition, a ve-year follow up investigation was performed for the patients, and the clinical signi cance of KLF7 for prognosis prediction in OSCC patients was also investigated in the current study.

Materials And Methods
Patients and tissue samples The study was approved by the Ethic Committee of Chinese PLA General Hospital. All patients provided written informed consents in advance.
In the present study, a total of 111 OSCC cases were recruited from Chinese PLA General Hospital. All patients were pathologically diagnosed with OSCC and did not receive any treatment prior. The tumor tissues and adjacent non-cancerous tissue specimens were collected from each patient, then the specimens were immediately frozen in liquid nitrogen and stored at -80 ℃ until use.
All the patients were enrolled in a ve-year follow up investigations. The survival information during the follow-up was recorded for survival analysis. The patients whose death was not related to OSCC would be excluded from the study.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from OSCC tumor specimens and matched adjacent non-tumor tissues using

Statistical analysis
Statistical analysis were performed using SPSS statistics 18.0 (SPSS, Chicago, IL, USA) and the graph was plotted using GraphPad Prism 5.0 (GraphPad Software Inc., USA). The expression value of KLF7 in tissue specimens were shown as mean ± standard deviation (SD), and its comparison between tumor and non-cancerous specimens were carried out using student's t test. The relationship between KLF7 expression levels and clinicopathologic characteristics was estimated by chi-square test. Kaplan-Meier curve was applied to analyze the effects of KLF7 expression on overall survival of OSCC cases. The prognostic value of KLF7 in OSCC was evaluated using Cox regression model. The P values less than 0.05 were considered statistically signi cant.

Results
The expression level of KLF7 in OSCC A total of 111 OSCC cases including 53 females and 58 males were collected in our study. QRT-PCR were used to investigate the expression pattern of KLF7 in the OSCC tissues and adjacent non-tumor tissues. The results showed that the relative expression of KLF7 was signi cantly increased in OSCC tissues compared with adjacent non-tumor tissues (P < 0.001) (Fig. 1).

The relationships between KLF7 expression and clinicopathological features
To assess the correlation of KLF7 expression with clinicopathological parameters of patients, the patients were divided into high KLF7 expression group (n = 56) and low KLF7 expression group (n = 55) based on their mean expression value. The expression levels of KLF7 were not correlated with age and gender (all P > 0.05 for both) (Table 1). However, the patients with high expression of KLF7 were more likely to exhibit advanced TNM stage (P = 0.006), large tumor size (P = 0.010), smoking (P = 0.006) and drinking (P = 0.000) ( Table 1). The prognostic value of KLF7 expression in OSCC In order to investigate the prognostic value of KLF7 expression for OSCC, we assessed the association between KLF7 expression levels and patient survival using Kaplan-Meier analysis with log-rank test. Survival analysis indicated that OSCC patients with high KLF7 expression had worse overall survival than those with low KLF7 expression (log rank test, P = 0.018) (Fig. 2). Univariate analysis revealed that KLF7 expression (P = 0.024), TNM stage (P = 0.029), smoking status (P = 0.026) and drinking status (P = 0.026) were potential prognostic indicators for OSCC patient. As shown in Table 2

Discussion
Oral squamous cell carcinoma (OSCC) is an aggressive human malignancy worldwide. Despite advances in prevention and treatment, the ve-year survival rate of OSCC patients remains low due to metastasis and local recurrence [8,20]. Tumor progression and prognosis evaluations are a great challenge for OSCC patients in clinic, due to the lack of effective and reliable biomarkers. Growing evidences have demonstrated that genetic factors play a pivotal role in progression of OSCC. Therefore, to investigate the OSCC-related genes may explore novel and effective biomarkers for patients with OSCC. In the present study, we investigated the prognostic value of KLF7 in OSCC patients.
KLFs are a family of zinc-nger transcription factors, which are widely expressed in multiple human organs or tissues [21]. KLF family is involved in diverse biological processes, and their abnormal expression may lead to human diseases, like cancer [22]. In oral cancer, several members of the KLFs family had been proved to be implicated in the pathogenesis of the cancers. For examples, Uchida D et al. had demonstrated that knockdown the expression of KLF2 would achieve the anti-tumor effects in oral cancer cells [23]. KLF4 and KLF5 might regulate the dedifferentiation and differentiation of oral carcinoma cells, thus leading to oral cancer carcinogenesis [24][25][26]. KLF8 was up-regulated in oral cancer cells, and its down-regulation might suppress the proliferation and clone formation of the cells [27]. In addition, the cellular levels of KLF13 was signi cantly correlated with proliferation ability and therapy sensitivity of oral cancer, which might be a potential diagnostic biomarker and therapeutic target for oral cancer [28]. However, to our knowledge, the function of KLF7 as well as its clinical signi cance had been rarely reported in OSCC.
In the present study, we found that KLF7 expression levels were up-regulated in OSCC tissues compared with adjacent non-tumor tissues. Additionally, the expression of KLF7 was positively correlated with tumor size, TNM stage, smoking status and drinking status. This data indicated that KLF7 might be involved in the development of OSCC. High expression of KLF7 predicted malignant clinical characteristics for the patients with OSCC. The study scheduled by Ding et al. have demonstrated that over-expression of KLF7 via mediating the expression of snail enhanced the migratory potential of OSCC cells, and induced epithelial-mesenchymal transition (EMT) and lymph node metastasis [19]. However, the carcinogenic mechanisms of KLF7 in OSCC was not investigated in the current study. Further relevant researches were still needed to address the issues.
Given its functional roles in OSCC development, we investigated the clinical performance of KLF7 in prognosis evaluation in OSCC population. Kaplan-Meier survival analysis with log-rank test suggested that high-expression group had obviously shorter overall survival than the low-expression group. Multivariate analysis with a Cox proportional hazards model indicated that high KLF7 expression was independently linked to poor survival of patients with OSCC. KLF7 might serve as an independent prognostic factor for patients with OSCC. Although we con rmed the prognostic value of KLF7 in OSCC, there were several limitations in our study. First, the sample size was relatively small. Second, only one single population was enrolled in our researches. Due to the diverse genetic background, the expression pro le of KLF7 in other races might be different. In addition, despite of the diverse biomarkers con rmed for OSCC, few of then were applied in clinic. Thus, subsequent investigations still required to analyze the clinical application of KLF7 for OSCC cases.

Conclusions
In conclusion, the expression of KLF7 is increased in OSCC, and shows positive association with aggressive clinical parameters of OSCC cases. KLF7 may be an independent biomarker for prognosis prediction in OSCC. This study was supported by the Ethics Committee of Chinese PLA General Hospital and also has been carried out in accordance with the World Medical Association Declaration of Helsinki.

Abbreviations
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 materialsAll data generated or analysed during this study are included in this published article.
Competing interestsThe authors declare that they have no competing interests.
Authors' contributions L.W. design of the work; L.W. the acquisition, analysis, H.S. interpretation of data; H.S. 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 expression of KLF7 in OSCC patients was detected using qRT-PCR. Results showed that KLF7 expression was signi cantly increased in OSCC tissues compared with adjacent non-tumor tissues (***: indicated P<0.001).

Figure 2
Kaplan-Keier survival curves of OSCC patients based on KLF7 expression levels. Patients with high KLF7 expression had signi cantly poorer prognosis than those with low expression (log rank test, P=0.018).