The expression and biological function of DKK1 in oral squamous cell carcinomas by bioinformatics analysis

Background To investigate the expression of transcription factor Dickkopf-1 (DKK1) in oral squamous cell carcinomas (OSCC) by bioinformatics analysis and to clarify the connection between expression of DKK1 and clinicopathological features of OSCC, so as to elucidate the early diagnosis and prognostic significance of OSCC by DKK1. Methods This study used the GEPIA database in conjunction with the TCGA database to analyze the expression level of DKK1 in OSCC tissues and then verify it by QRT-PCR and Western-blot analysis in vitro . The LinkedOmics database was used to describe the correlation between DKK1 expression and clinical pathological parameters of OSCC and its impact on prognosis. DKK1 was knocked down by RNA interference approach in SCC-4 and SCC-25 OSCC cell lines. In addition, the proliferation ability was assessed by MTT assay. Results DKK1 was highly expressed in OSCC and positively correlated with OSCC pathological grade and T stage. The results of TCGA showed that high DKK1 mRNA expression was associated with overall survival in OSCC. Besides, both DKK1 mRNA and protein expression was confirmed increased significantly in oral squamous cell carcinomas SCC-25 and SCC-4. Furthermore, MTT analysis investigated that knockdown of DKK1 caused reduced proliferation ability of OSCC cells. Conclusions The TCGA database analysis found that DKK1 is highly expressed in OSCC and is associated with multiple pathological indicators of OSCC, which will provide important theoretical guidance for subsequent oral squamous cell carcinoma research.


Background
Oral squamous cell carcinomas (OSCC) is a common oral malignant tumor, accounting for 90% of the incidence and ranking the 6th place among systemic tumors [1]. The etiology of OSCC is complex. At present, many scholars believe that the disrupted balance between oncogene activation and tumor suppressor gene suppression may be one of the important causes of OSCC [2,3]. It has brought difficulties to the clinical treatment of OSCC because of the insidious onset, high degree of malignancy, rapid disease progression, high rate of relapse, and hardly to diagnose in the early stage [4,5].
Therefore, the exploration of oncogenes closely related to OSCC is expected to provide a new direction for tumor gene-targeted therapy.
Dickkopf-1 (DKK1) is part of the DKK family of proteins that includes DKK2, DKK3 and DKK4. This family of secreted proteins share similar conserved cysteine domains and inhibits the Wnt/β-catenin pathway [6,7]. DKK1 is involved in cell apoptosis through the Wnt signaling pathway [8]. DKK1 dysregulation has been implicated in the pathogenesis of a variety of cancers. Many lines of evidence show that upregulation of DKK1 contributes to the development of cancer in prostate tissue and in non-small cell lung carcinoma [9][10][11][12][13]. On the other hand, in gastric and colorectal cancer, DKK1 has been shown to be under-expressed [14]. In chronic lymphocytic leukemia (CLL) it is expressed at normal levels, but unable to affect Wnt/β-catenin pathway, while in multiple myeloma, DKK1's role is shown to change from tumor suppressor to a stress responsive gene involved in the JNK pathway [15,16]. As all these studies have shown, the activity and expression levels of DKK1 varies in different cancers. But the role of DKK1 in OSCC remains unclear and needs to be further investigated.
In this study, the role of DKK1 in oral squamous cell carcinoma was analyzed through the database website and verified by Quantitative real-time PCR and Western-blot analysis, so as to provide a theoretical basis for determining whether its mechanism and possible use as a predictive marker for the prognosis of OSCC patients.

GEPIA database
GEPIA (Gene Expression Profiling Interactive Analysis) database is dynamic Analysis of Gene Expression data, developed by Beijing university online database (http://gepia.cancer-pku.cn) combined with TCGA GTEx and analyze Gene Expression in different tumors in the database. In this study, the expression of DKK1 and its correlation with pathological analysis were analyzed in OSCC tissues and normal tissues.

Linkedomics database
Linkedomics database is third-party online tools for analyzing TCGA database (http://linkedomics.org/login.php). In this study, the website was used to analyze the RNAseq data in TCGA to understand the relationship between the mRNA level of DKK1 and the clinicopathological characteristics of OSCC. Using this site to analyze data requires only 5 steps: (1) select the type of tumor to be analyzed, oral squamous cell carcinoma was selected in this study; (2) RNAseq data of oral squamous cell carcinoma were selected; (3) Input the name of the gene to be analyzed, and fill in DKK1 here;(4) Select the data content of joint analysis, and "Clinical data" is selected in this step;(5) Select statistical method and non-parametric test. After submitting, wait for the analysis result, and click the corresponding option to view.

String-DB database
String database (https://string-db.org/) is a database for analyzing the interaction between genes or proteins, including direct physical interaction between proteins and indirect functional correlation between proteins. In addition to containing experimental data, PubMed abstracts, and other database data, it also contains results predicted using bioinformatics methods.In this study, "DKK1" was input, "human" was selected for species, "Medium0.4" for confidence, and 20 for maximum number interaction.

Discussion
OSCC is the most common tumor in the world, and its incidence has been younger in the past 30 years, which has seriously endangered human survival and health [17,18].
Although the current treatment plan for OSCC is constantly improving, unfortunately the 5-year survival rate of OSCC patients is still less than 50%. Therefore, it is urgent to understand the occurrence and development of OSCC and discover effective genetargeted treatment methods [19].
The DKK1 is frequently overexpressed in prostate cancer, non-small cell lung carcinoma, breast cancer, gastric and colorectal cancer [20][21][22][23]. It is suggesting that aberrant DKK1 expression contribute to progression of malignancies [24]. However, few reports have evaluated DKK1 expression in OSCC [25]. Here, we studied expression and prognosis of DKK1 in OSCC samples by analyzing the TCGA database.
Through GEPIA data analysis, we found that compared with normal oral epithelial tissues, the expression of DKK1 in OSCC was significantly higher, and it was positively correlated with clinical staging. Besides, the relationship between DKK1 and OSCC clinicopathological characteristics through the LinkedOmics database showed that DKK1 expression was related to pathological staging and T staging. At the same time, the effect of DKK1 expression level on the overall survival rate of OSCC through GEPIA found that patients with high DKK1 expression have a poorer prognosis trend.
Therefore, combining the DKK1 expression status with the tumor stage is useful to predict the prognosis of OSCC.
Hence, to confirm that DKK1 served as an oncogene in OSCC, we conducted cell line verification. PCR and Western blot analysis in Figure 5 revealed that DKK1 was upregulated in OSCC cell lines. Knockdown of DKK1 inhibited the proliferation of OSCC cells. These findings indicated that DKK1 promoted the proliferation of OSCC cells.
However, the mechanism of DKK1 becomes an oncogene in OSCC remains unknown, but accumulating evidence reveals that up-regulation of DKK1 is related to the accumulation of β-catenin. For example, Jing et al. reported that DKK1 promotes migration and invasion of non-small cell lung cancer via β-catenin signaling pathway [26].

Conclusions
In conclusion, we demonstrate that DKK1 is overexpressed in OSCC. Moreover, knockdown of DKK1 suppresses the cell growth of OSCC. DKK1 may play a potential therapeutic strategy for predicting the prognosis of patients in early disease stage.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This current work was not supported by any sources of findings.

Authors' contributions
Huijie Yu and Tianhua Li are responsible for experimental design, experiments, data analysis and interpretation, and writing of the manuscript. Xuemei Mao is the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors have read and given final approval of this version of manuscript to be published. The expression levels of DKK1 in different tumors, and the type of tumor with red at the top indicates that DKK1 is highly expressed in this tumor compared with normal tissues.

Fig. 2 Expression of DKK1 mRNA in OSCC and normal oral epithelial tissues
Quantitative PCR analysis showed that DKK1 mRNA expression significantly increased in OSCC (n=519) as compared with in normal oral epithelial tissues (n=44) (*P < 0.05).    and 72 h post-transfection with si-DKK1 or its control. The data corresponds to the results of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.0001.