The Expression and Biological Function of DKK1 in Oral Squamous Cell Carcinomas by Bioinformatics Analysis

Research Article

Austin Dent Sci. 2021; 6(1): 1034.

The Expression and Biological Function of DKK1 in Oral Squamous Cell Carcinomas by Bioinformatics Analysis

Huijie Yu#, Tianhua Li#, Xuemei Mao*

Department of Stomatology, The People’s Hospital of Dongying, Shandong, China

#Contribute Equally to this Work

*Corresponding author: Xuemei Mao, Department of Stomatology, The People’s Hospital of Dongying, Shandong, China

Received: March 13, 2021; Accepted: April 21, 2021; Published: April 28, 2021

Abstract

Objective: To clear the expression of transcription factor Dickkopf-1 (DKK1) in Oral Squamous Cell Carcinoma (OSCC) using the method of bioinformatics analysis. And to clarify the relationship between the expression of DKK1 and the clinicopathological characteristics of OSCC using the method of molecular biology and cytobiology, in order to determine the early diagnosis and significance of OSCC according to the marker of DKK1.

Methods: In this study, the expression level of DKK1 in OSCC tissues was analyzed using GEPIA and TCGA databases, and then verified in vitro by qRTPCR and Western-blot analysis. The correlation between DKK1 gene expression and the clinical pathological parameters of OSCC, and also the impact of DKK1 on prognosis were determined using the LinkedOmics database. In addition, DKK1 was knocked down by RNA interference in SCC-4 and SCC-25 OSCC cell lines and the proliferation ability of OSCC cells was assessed by MTT assay.

Results: High expression of DKK1 in OSCC is positively correlated with the pathological grade and T stage of OSCC. According to the TCGA results, DKK1 mRNA was highly expressed and it is related to the overall survival rate of OSCC. In addition, the expression level of both DKK1mRNA and protein were significantly raised in the cell line SCC-25 and SCC-4. Furthermore, MTT analysis showed that DKK1 knockdown resulted in reduced proliferation of OSCC cells.

Conclusions: TCGA database analysis showed that DKK1 was highly expressed in OSCC, and it is closely correlated to the pathological parameters of OSCC, which will provide important theoretical guidance for the subsequent study of oral squamous cell carcinoma.

Keywords: DKK1; Oral Squamous Cell Carcinomas (OSCC); Bioinformatics analysis; Biomarker

Introduction

Oral Squamous Cell Carcinomas (OSCC) is one of the most common oral malignant tumors, 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, highly malignancy, rapid 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 proteins family. The secreted proteins family shares a similar conserved cysteine domain and inhibits the Wnt/ß-catenin pathway [6,7]. DKK1 participates in apoptosis through the Wnt/ß-catenin signaling pathway [8]. DKK1 disorder is associated with the pathogenesis of a great many cancers. There is much evidence showed that DKK1 upregulation contributes to the development of many cancers such as prostatic cancer and nonsmall cell lung carcinoma [9-13]. On the other hand, DKK1 has been shown to be under-expressed in colorectal cancer and gastric cancer [14]. In Chronic Lymphoblastic Leukemia (CLL), DKK1 is expressed at normal level, but does not affect the Wnt/ß-catenin pathway. In multiple myeloma, DKK1 has been proved to be a stress response gene involved in the JNK pathway [15,16]. As mentioned above, these studies have shown that the activity and expression level of DKK1 are different in different cancers. However, the role of DKK1 in OSCC is still 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 its regulatory mechanism and whether it can be used as a predictor of prognosis in patients with OSCC.

Materials and Methods

GEPIA database

GEPIA (Gene Expression Profiling Interactive Analysis) is a database for 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 present study; (2) Select the specific RNAseq data of oral squamous cell carcinoma; (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 experimental data, PubMed abstracts, and other database data, it also contains predicted results using the bioinformatics methods. In this study, “DKK1” was input, “human” was selected for species, “Medium 0.4” for confidence, and 20 for maximum number interaction.

Quantitative real-time PCR

Quantitative Real-Time PCR (QRT-PCR) was used to inoculate HOK cells from normal oral epithelial cells, SCC-25 cells from oral squamous cell lines and SCC-4 cells into 6-well plates at a density of 1.5×105 cells per well (grown in RPMI 1640 medium at 37oC under 5% CO2). After 24h, the mRNA of cells was extracted by Trizol (Invitrogen Carlsbad, USA) according to the manufacturer’s instructions. RNA was quantitated with a NanoDrop spectrophotometer (Thermo, USA). According to the manufacturer’s instructions, the mRNA samples were reverse transcribed into cDNA using a commercial reverse transcription system (Thermo scientific, USA). The relative PCR quantification was performed using a commercial RT-PCR Kit (TaKaRa, Japan). Using the –ΔΔCT method, the gene expression data were normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Primer sequences of DKK1: forward: AACGCTATCAAGAACCTGC, reverse: GATGACCGGAGACAAACA, target fragment of 460bp. Primer sequences of GAPDH: forward: GGGAGCCAAAAGGGTCATCATCTC, reverse: CCATGCCAGTGAGCTTCCCGTTC, target fragment of 353bp. All the primers used in this study were synthesized by AUGCT (Beijing, China).

Western-blot analysis

Total proteins of the cells were dissolved in lysis buffer and extracted following the manufacturer’s protocol (Beyotime Institute of Biotechnology, Haimen, China). The concentration of the target proteins was determined using the Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific, Wilmington, DE, USA). Equal amounts of protein were separated by SDS-PAGE using 10% horizontal gels. And then the proteins were transferred onto a polyvinylidene difluoride membrane (EMD Millipore Corp., Billerica, MA) in a wet blotting system. Membranes were blocked for 1h at room temperature and then incubated with the specific primary antibodies overnight at 4oC. After being washed, membranes were incubated with a secondary Horseradish Peroxidase (HRP)-coupled antibody and processed for enhanced chemiluminescence detection using Immobilon HRP substrate (EMD Millipore Corp., Billerica, MA). Signals were visualized and analyzed on a VisionWorks LS (UVP, BioSpectrum Imaging System, USA). The integrated density of the bands came from the different proteins was quantified using ImageJ Software (National Institute of Health, Bethesda, MD, USA). The ratio of the integrated density of the target protein to that of GAPDH as the loading control was calculated to represent the expression level of protein. Antibodies were used as follows: anti- DKK1-1 diluted 1:1,000, and anti-GAPDH diluted 1:1,000, which applied from Santa Cruz (Santa Cruz Biotechnology Inc., CA).

MTT assay

SCC-4 and SCC-25 cells were both inoculated into 96-well plates at 3,000 cells per 100μl culture media per well, and transfected the next day. A total of 10μl MTT reagent (5mg/ml) was added to each well at each time point after the transfection (24, 48 and 72 h respectively), and 150μl dimethyl sulfoxide was added after 4h. The FLUOstar OPTIMA microplate reader (BMG) was used to measure the cell viability of the sample at 490nm. Following transfection with si-DKK1 or its control, the cells were further cultivated for an additional 1-3 days. Each experiment contained three replicates and was repeated at least twice.

Statistical analysis

All the experiments were performed at least three times in the same environment, and data were all expressed as means ± S.E.M. Significance was established with the SPSS 22.0 software (IBM, USA). Student’s t-test and ANOVA analysis were used if the quantitative data between groups show normal distribution. If not consistent with the normal distribution, using the Wilcoxon-Mann-Whitney test, and a P value <0.05 was considered as significant.

Results

DKK1 mRNA was highly expressed in OSCC and normal oral epithelial tissues

The GEPIA database was used to analyze the expression of DKK1 in different tumors. The results showed that the expression level of DKK1 was significantly increased in most tumors, such as esophageal cancer, squamous cell carcinoma, pancreatic cancer, including head and neck squamous cell carcinoma (Figure 1). The expression of DKK1 mRNA in OSCC (n=519) and normal head and neck epithelial tissue (n=44) was further analyzed. And the results showed that compared with normal head and neck epithelial tissue, DKK1 mRNA was expressed extremely high in OSCC. The difference was statistically significant (P <0.05) and was shown in Figure 2. These results suggest that DKK1 may play an important role in the occurrence and development of OSCC.