Expression and function of dinkkopf 4 in oral squamous cell carcinoma and in vitro


 Background Oral squamous cell carcinoma accounts for about 90% of malignant tumors of the head and neck, and its 5-year survival is less than 50%. The number of new cases of oral cancer worldwide is expected to increase by 62% in 2035. Wnt/β-catenin signal pathway plays a role in the tumorigenesis and progression of cancer. dinkkopf 4 is involved in a variety of cancers as a molecule in Wnt/β-catenin signal. To our knowledge, the study of DKK4 in OSCC has not been reported. Methods The well differentiated oral squamous cell carcinoma and normal oral mucosa samples were collected from Department of Pathology of Xiangya Stomatological Hospital of Central South University. Human oral epithelial cell HOEC and human oral squamous cell carcinoma cell TSCC1 were cultured for experiments. The plasmid vector was constructed to inhibit the expression of DKK4 to form TSCC1-shDKK4 cells and TSCC1-NC cells were transfected with blank plasmid. Cell proliferation was detected by CCK-8 test. Apoptosis was detected by Annexin V-FITC/PI assay. Cell migration was detected by Transwell assay. IBM SPSS Statistics 24.0 and GraphPad Prism 8 were performed for statistical analysis and graphing. Results We found that the expression of DKK4 and β-catenin increased in OSCC and in vitro (P<0.05). dinkkopf 4 may promote the expression of β-catenin in OSCC. The proliferation and migration of TSCC1-shDKK4 cells decreased and the apoptosis of TSCC1-shDKK4 cells increased (P<0.01). Conclusions dinkkopf 4 may promote the proliferation and migration of OSCC cells and inhibit the apoptosis of OSCC cells in vitro by regulating Wnt/β-catenin signal pathway.

cancer was mainly prevalent in developing countries [6] . European cancer analysis showed that the 5-year survival rate of oral cancer was less than 50% [7] . The prognosis of cancer is related to early diagnosis, so it's very important to prevent and detect OSCC early [2] .
The Wnt ligand binds to the cysteine-rich domain (CRD) of the Frizzled (FZD) receptor, which binds to the low density lipoprotein receptor related protein (LRP5/6), recruiting Disheveled (Dsh) to the cell membrane,and initiating Wnt signal [9,11] . GSK3β and CK1α phosphorylate the cytoplasmic domain of LRP5/6, and recruit Axin to the phosphorylated tail of LRP. The interaction between FZD and cytoplasmic protein Dsh results in the phosphorylation of Dsh by CK1α and binding to GSK3β. These interactions cause the inactivation of APC/Axin/GSK3β/CK1α complex and stabilization of cytoplasmic β-catenin, inhibiting the ubiquitination of β-catenin in the destructive complex [8,11] . The destruction complex is saturated with phosphorylated β-catenin, resulting in the accumulation and transport of newly synthesized β-catenin into the nucleus [10] . β-catenin is translocated to the nucleus and interacts with T-cell factor/lymphocyte enhancer factor (TCF/LEF) to form β-catenin-TCF/LEF complex, activating the transcription of Wnt target genes [9] . β-catenin binds to DNA-binding TCF transcription factors in the nucleus. When Wnt is "off", TCFs interacts with Groucho transcriptional suppressors to prevent gene transcription. When Wnt is "on", binding to β-catenin transformed TCF into a transcriptional activator of target gene [10] . β-catenin, called Armadillo (ARM) in Drosophila, consists of 781 amino acid residues and a central region (residue 141-664). This region consists of 12 imperfect ARM repeats (R1-12), flanked by Nterminal domain (NTD) and C-terminal domain (CTD). NTD and CTD may be structurally flexible, while the central region forms a relatively rigid scaffold. The scaffold acts as a platform for the interaction of β-catenin binding protein in the cell membrane, cytoplasm and nucleus. The competition for βcatenin among different intracellular β-catenin binding protein is important for the regulation of classical Wnt signals. The conformational change of β-catenin helps to regulate its binding properties.
NTD and CTD fold in the central region, affecting the binding to TCF/LEF [12] . The accumulation of cytoplasmic β-catenin increased the invasion and migration of OSCC cells [13,14] . The expression of β-catenin is up-regulated in the cytoplasm/nucleus of oral potentially malignant disorders and OSCC, and gradually increased in the development of normal oral mucosa to OSCC, might being a sign of malignant transformation of OSCC [15,16] . β-catenin is also related to tumor size, stage and invasive growth of OSCC [17,18] .
Dickkopf (DKK) family consists of four secretory proteins DKK1, DKK2, DKK3 and DKK4, containing 255-350 amino acids and 2 conserved sequences of CRD. The CRD sequence of DKK4 is located in the regions of C24-73 and C128-201 [19] . The DKK family induces endocytosis of the Wnt/LRP5/6 complex by interacting with LRP5/6 receptors, inhibiting the activity of Wnt/β-catenin signal pathway [8,9] . It was found that the expression of DKK3 in OSCC was higher than that in NOM, and DKK3 might promote the proliferation, invasion and metastasis of squamous cell carcinoma. However, the study of DKK4, another member of DKK family, in OSCC has not been reported [20,21] .The expression of DKK4 is up-regulated in colorectal cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, ovarian cancer, esophageal cancer and endometrial carcinoma [19] . DKK4 enhances the migration, invasion and angiogenic potential of colon cancer cells [22] . There is a positive correlation between the expression of DKK4 and nuclear β-catenin in colorectal cancer. The increased expression of DKK4 may be a side effect of the activation of Wnt/β-catenin signal pathway [23] . DKK4 may be the target gene of Wnt/β-catenin pathway. Its overexpression in colorectal cancer may reflect the activation of Wnt/βcatenin signal as a downstream target of TCF/β-catenin, and affect the tumorigenesis and progression of colorectal cancer in other signal pathways [22,23] .
Wnt/β-catenin signal pathway is involved in the tumorigenesis and progression of a variety of cancer, and it is abberant activated in OSCC without a clear mechanism [8,9] . β-catenin plays a role in promoting tumorigenesis of OSCC [13][14][15][16] . The expression and mechanism of DKK4 varies in different cancers, and there was no relevant research reported in OSCC. This study aimed to investigate the expression and function of DKK4 in OSCC and in vitro. replaced the antibody as negative control group. The ImageJ 1.51K software (Wayne Rasband, National Institutes of Health, USA) and IHC Profile were conducted to detect the results of immunohistochemical staining [24] .

Cell culture
Human normal oral epithelial cell (HOEC, Wuhan University, China) and human oral squamous cell carcinoma cell (TSCC1, Fudan University, China) were cultured in DMEM (SH30243.01, Hyclone, USA) containing 10% fetal bovine serum (16000-044, GIBCO, USA) in a incubator at 37 ℃ and 5% CO 2 Real-time PCR RNA was extracted according to the Real-time PCR kit (AQ131-01, TransGen, China). The mRNA sequences of DKK4, β-catenin and GAPDH were found from NCBI and primers were designed by clonemanager software (Table 1). The reverse transcription and Real-time PCR amplification were performed by the reverse transcription kit (# K1622, Fermentas, USA). The data were analyzed by ABI Prism 7300 SDS Software. Table 1 Primers and sequences of GAPDH, β-catenin and DKK4

Primers
Sequences The pLkO.1-AcGFP-C1 (Addgen, USA) was used to construct DKK4 interfere plasmid vector. The specific RNAi sequence of DKK4 was designed by WI siRNA genes Selection Program ( Table 2). The synthesized shRNA double strands were annealed, and the linearized vectors were recovered and transformed. The transformants grown on the plate were re-suspended in 10 µl LB culture medium, which was taken as a template for colony PCR identification. The cells were divided into two groups: TSCC1-shDKK4 (DKK4 RNAi plasmid) and TSCC1-NC (negative conntrol). The cells in logarithmic phase were digested by trypsin-EDTA (T1300-100, Solarbio, USA). Preparing the transfection solution, slowly added complex to the corresponding culture medium, shake well, put it in the incubator at 37 ℃ for 6 hours, and change it into the complete culture medium. 48 hours after transfection, cell precipitation was collected for follow-up experiment.  The results of Real-time PCR showed that the expression of DKK4 mRNA in TSCC1 cells was significantly higher than that in HOEC cells (P < 0.001), and the expression of β-catenin mRNA in TSCC1 cells was significantly higher than that in HOEC cells (P < 0.001) (Fig. 1).There was no correlation between the expression of DKK4 and β-catenin and the age and sex of OSCC patients (P > 0.05). No correlation was found between the expression of DKK4 and β-catenin in OSCC and NOM (P > 0.05).

DKK4 increased expression of β-catenin
The results of immunohistochemistry showed that the expression of DKK4 in OSCC was higher than that in NOM, so a plasmid vector was constructed to inhibit the expression of DKK4 to form TSCC1-shDKK4 cells as the experimental group. TSCC1-NC cells were transfected with blank plasmid as control group. The results of Rearl-time PCR showed that the expression of DKK4 mRNA in TSCC1-shDKK4 cells was significantly lower than that in TSCC1-NC cells (P < 0.001), and the expression of βcatenin mRNA in TSCC1-shDKK4 cells was significantly lower than that in TSCC1-NC cells (P < 0.001) (Fig. 2). The results of Western Blot demonstrated that the expression of DKK4 protein in TSCC1-shDKK4 cells was significantly lower than that in TSCC1-NC cells (P < 0.001), and the expression of βcatenin protein in TSCC1-shDKK4 cells was significantly lower than that in TSCC1-NC cells (P < 0.001) (Fig. 3).

DKK4 promotes proliferation and migration of OSCC cells and inhibits apoptosis
The results of CCK-8 assay showed that the proliferation of TSCC1-shDKK4 cells in 72 h was significantly lower than that of TSCC1-NC cells (P < 0.001) (Fig. 4). The results of apoptosis assay showed that the number of apoptosis in TSCC1-shDKK4 cells was significantly higher than that in TSCC1-NC cells (P < 0.001) (Fig. 5).The results of transwell migration assay demonstrated that the number of migration in TSCC1-shDKK4 cell was significantly lower than that of TSCC1-NC cell migration (P < 0.01).

Discussion
It was demonstrated that DKK4 inhibits the growth, migration and invasion of hepatoma cells in vitro, and reduces the tumorigenicity in mouse [25,26] . Pendas-Franco et al. found that DKK4 enhances the migration, invasion and angiogenic potential of colorectal cancer cells in vitro [22] . Hirata et al.
identified that DKK4 promotes the proliferation, migration and invasion of renal cancer cells in vitro, and promotes tumor growth in nude mice [27] . Wang et al. reported that DKK4 promoted the invasion of ovarian cancer cells in vitro [28] . We found that the positive expression rate of DKK4 in OSCC tissues was significantly higher than that in NOM tissues (P < 0.01) and the expression of DKK4 mRNA in TSCC1 cells was higher than that in HOEC cells (P < 0.01). The expression of DKK4 in OSCC was similar to that in colorectal cancer, renal cell carcinoma and ovarian cancer, but opposite to that in hepatocellular carcinoma [22,25,27,28] . We found that the decreased expression of DKK4 inhibits the The β-catenin was found to accumulate in the cytoplasm of OSCC cells [14] . It was found that βcatenin was significantly correlated with tumor size, stage, invasive growth, overall survival and disease-free survival of OSCC [17,18,29] . Duan et al. showed that β-catenin promoted the proliferation, colony formation and tumorigenesis of tongue cancer [30] . Chaw et al. reported that the cytoplasmic/nuclear expression of β-catenin increased in moderate and severe dysplasia of oral mucosa and OSCC [15] . Fujii et al. demonstrated that the expression of cytoplasmic β-catenin protein increased with the progression of OSCC [16] . We found that the positive expression rate of β-catenin in OSCC tissues was significantly higher than that in NOM tissues (P < 0.01). The expression of βcatenin mRNA in TSCC1 cells was higher than that in HOEC cells (P < 0.01). Similar to previous studies, the expression of β-catenin increased in OSCC tissues and in vitro, supporting the role of βcatenin in promoting progression of OSCC.
Wnt signal pathway is involved in a variety of cellular behaviors, including cell proliferation, stem cell maintenance and differentiation, and the coordination of cell movement [11] . Wnt/β-catenin signal pathway is one of the most widely studied Wnt signal pathways. When the Wnt/β-catenin signal pathway is "off", the cytoplasmic APC/Axin/GSK3β/CK1α destruction complex phosphorylated βcatenin. The phosphorylated β-catenin is recognized and ubiquitinated by β-TrCP, and is degraded by proteasome, preventing the activation of target genes in the nucleus [8][9][10] . When Wnt ligands bind to FZD and LRP5/6 coreceptors, Dsh is recruited to the cell membrane to initiate Wnt/β-catenin signal [8,9,11] . The interaction between FZD and Dsh results in the phosphorylation of Dsh by CK1α and binding to GSK3β. The cytoplasmic APC/Axin/GSK3β/CK1α destruction complex is inactivated, and the β-catenin is accumulated in the cytoplasm and transported to the nucleus [8,10] . The β-catenin entering the nucleus interacts with TCF/LEF to form β-catenin-TCF/LEF complex, activating the transcription of Wnt target gene [9] . As the central transcriptional activator of Wnt/β-catenin signal pathway, the expression of cytoplasmic β-catenin is the key of Wnt/β-catenin signal pathway [17] .
Abberant activation of Wnt/β-catenin signal pathway is observed in many human cancers [9] . There are some somatic inactivation mutation of Wnt/β-catenin signal molecules in head and neck squamous cell carcinoma [31] .
The expression of DKK4 is increased in colorectal cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, ovarian cancer, esophageal cancer and endometrial carcinoma [19] . Pendas-Franco et al.
speculated that the overexpression of DKK4 in colorectal cancer may be the result of the activation of Wnt/β-catenin signal pathway, and DKK4 may be the downstream target gene of β-catenin-TCF/LEF, which is up-regulated due to the activation of Wnt/β-catenin signal pathway [22] . Matsui et al. also thought that DKK4 is the downstream target of Wnt/β-catenin signal pathway, and the overexpression of DKK4 may be a side effect of the activation of Wnt/β-catenin signal [23] . Baehs et al. identified that Wnt/β-catenin signal pathway can fine-tune the activity of β-catenin by regulating the expression of DKK4 under physiological conditions, forming a DKK4-dependent negative feedback loop, which is closed in colorectal cancer, resulting in the growth advantage of colorectal cancer cells [32] . In renal cell carcinoma and ovarian cancer, DKK4 is thought to promote the proliferation, migration and invasion of cancer cells by activating Wnt/JNK signal pathway [27,28] . These are not consistent with the conclusion that the DKK family simply inhibits the Wnt/β-catenin signal pathway [8,9] .
In view of the differences in the expression and function of DKK4 in various cancers and the vague mechanism of Wnt/β-catenin signal in OSCC, we analyzed the expression and function of DKK4 in OSCC and in vitro. It was showed that the expression of DKK4 and β-catenin in the cytoplasm of OSCC tissues was higher than that in NOM tissues. The expression of mRNA and protein of β-catenin The expression of DKK4 and β-catenin mRNA in TSCC1 was higher than that in HOEC (***: P<0.001).