High glucose status regulates the proliferation of colon tumor cells through the WIF1-Wnt/β-catenin pathway

 In recent years, the incidence of diabetes and colon cancer has gradually increased. More and more researchers have found that diabetes can increase the risk of colon tumors, but the mechanism by which diabetes increases the risk of colon tumors is still unclear [1-2]. Wnt inhibitor 1 (WIF1) is a secreted protein encoded by the WIF1 gene. It is an inhibitor of the Wnt/β-catenin signaling pathway. It directly binds to the Wnt protein and passes through the frizzled protein on the cell membrane. The receptor family transmits signals to the cell, causing the phosphorylation of β-catenin, a key molecule in the pathway, and promoting the degradation of β-catenin, thereby inhibiting the Wnt/β-catenin signaling pathway [3]. Studies have shown that WIF1 can inhibit tumorigenesis [4]. The Wnt/β-catenin signaling pathway plays an important role in cell proliferation, embryonic development and adult tissue regeneration. Previous studies have shown that the tumor cells of colon cancer patients with diabetes have abnormal activation of the Wnt signaling pathway, and the normal colonic epithelium of diabetic patients has increased β-catenin. The Wnt/β-catenin signaling pathway and its inhibitor WIF1 may be involved in increasing diabetes. Patients are at risk of colon cancer [5-6]. Therefore, this study intends to investigate the effect of high glucose state on the expression of WIF1, and the specific molecular mechanism of WIF1 affecting the proliferation of colon cancer cells under high glucose state.

Materials and Methods

1. Cells

Human colon cancer cell line SW620 cells were donated by the Medical Research Center of Sun Yat-sen Memorial Hospital, Sun Yat-sen University. The cells were cultured in a complete DMEM medium containing 10% fetal bovine serum and 1% double antibody in a 37°C, 5% carbon dioxide cell incubator.

2. Main reagents

DMEM medium (Gibco, USA), cell proliferation-toxicity test kit CCK-8 (APExBIO, USA), TRIzol Reagent (Invitrogen, USA), reverse transcription kit Prime ScriptTM RT reagent Kit (Takara, Japan), qPCR SYBR

TB Green? Premix Ex TaqTM Ⅱ (Takara, Japan), RIPA

Cell lysate (Kangwei Century, China), BCA protein quantification kit (Kangwei Century, China), enhanced chemiluminescence ECL kit (Millipore, USA), WIF1 polyclonal antibody (Cell Signaling Technology, USA), β -catenin polyclonal antibody (Cell Signaling Technology, USA), PCNA polyclonal antibody (Cell Signaling Technology, USA), Ki67 polyclonal antibody (abclonal, China), LGR5 polyclonal antibody (abclonal, China), GAPDH polyclonal antibody ( Cell Signaling Technology, USA), goat anti-rabbit secondary antibody (Cell Signaling Technology, USA), Lipofectamin 3000 (Invitrogen, USA), WIF1 siRNA (RiboBio, China), pcDNA3.1-WIF1 overexpression plasmid (AkiBio , China).

Three, method

1. Cell culture and grouping

SW620 cells were cultured in ordinary DMEM complete medium containing 10% fetal bovine serum. Inoculate SW620 cells in a 6-well plate. After SW620 cells adhere to the wall, they are replaced with serum-free medium. After 24 hours, they are cultured with different sugar concentration medium for 72 hours, and they are divided into 3 groups: 5 mmol/L glucose +25 mmol/L mannitol (low sugar group, LG group), 20 mmol/L glucose +10 mmol/L mannitol (normal sugar group, NG group), 30 mmol/L glucose (high sugar group, HG group). The fluid was changed daily, and samples were collected after 72 h for subsequent experiments.

2. Real-time fluorescent quantitative PCR (qPCR)

Collect each group of cells, discard the culture medium, wash once with phosphate buffered saline (PBS), extract total RNA with TRIzol according to the instructions, determine the RNA concentration and purity, then synthesize cDNA from the RNA according to the reverse transcription kit system. The reaction system was prepared according to the qPCR kit and reacted in the Roche LightCycler 480 qPCR instrument. After the reaction, the Ct value of each sample was obtained, and GAPDH was used as the internal reference to calculate the relative expression of the target gene for statistical analysis.

3. Western blotting

Collect each group of cells on ice, add cell lysate (containing protease inhibitor), mix and lyse on ice for 30 min, centrifuge and collect the supernatant, and determine the protein concentration according to the BCA method. After adding the loading buffer, the protein was denatured by heating at 100°C for 5 min. After electrophoresis on the SDS-polyacrylamide gel, the protein was electrotransferred to the PVDF membrane. The PVDF membrane was blocked in 5% skim milk at room temperature for 1 h, and the corresponding primary antibody (1:1000) was added in a shaker at 4°C overnight, and the HRP-labeled secondary antibody corresponding to the primary antibody (1:2000) was added and incubated at room temperature. 1 h. Expose the target band by ECL method, and analyze the gray value of the band by ImageJ software.

4. Cell proliferation activity detection

Inoculate SW620 cells in a 96-well plate with 3000 cells per well, and continue to culture them in groups. Add 10 μl of CCK-8 solution to each well at 0, 24, 48, 72 h, and incubate at 37°C for 2 h. The multifunctional microplate reader detects the absorbance of each well at a wavelength of 450 nm, and expresses the proliferation activity by absorbance. The mRNA and protein levels of proliferation-related genes, proliferating cell nuclear antigen (PCNA), Ki67, and G protein-coupled receptor rich in leucine repeat units (LGR5) were detected.

5. Cell counting experiment

Inoculate SW620 cells in a 12-well plate with 1×104 cells per well. After processing in groups, discard the culture medium, wash once with PBS, and digest with trypsin at 0, 24, 48, and 72 h to prepare cells. Suspension, add 0.4% trypan blue solution according to 1:1, add dropwise to the hemocytometer, and count under the microscope.

6. Cell plate clone formation experiment

Inoculate 1000 SW620 cells per well in a 6-well plate, and continue to culture for 14 d after grouping, and change the medium every 3 d. Discard the culture medium, wash once with PBS, fix with 4% paraformaldehyde for 30 min, wash once with PBS, add 0.1% crystal violet solution, dye for 30 min, wash with water several times and then dry it under a microscope Count and calculate the clone formation rate.

discuss

More and more studies have shown that diabetes can increase the risk of colon cancer, but the molecular mechanism is not yet clear. This research group explored the effects of different sugar concentrations on the proliferation of colon cancer cell line SW620 cells, the expression of WIF1, and the expression of key factors in the Wnt/β-catenin pathway, as well as further studies on molecular regulation and related mechanisms. The results showed that they were in a high glucose state. The decrease of WIF1 expression may promote the proliferation of colon tumor cells under high glucose conditions by activating the Wnt/β-catenin pathway.

Previous studies have shown that high sugar can promote cell proliferation [7]. This study confirmed that the proliferation ability of SW620 cells increased with the increase of sugar concentration. At the same time, with the increase of sugar concentration, the expression of WIF1 gradually decreased, while the expression of β-catenin gradually increased. Previous studies have shown that there is abnormal activation of the Wnt signaling pathway in colon cancer patients with diabetes [5]. The results of this study show that high glucose inhibits the expression of WIF1 and promotes the activity of the Wnt/β-catenin pathway may be related to the promotion of cell proliferation by high glucose. A number of studies have shown that the expression of WIF1 is reduced in tumors of various systems, such as oral endometrial squamous cell carcinoma, nasopharyngeal carcinoma, etc. The reduced expression of WIF1 in various tumors may be related to its promoter methylation [8-10]. At the same time, some researchers found that up-regulation of WIF1 can inhibit the invasion and migration of colon tumor cells, indicating that WIF1 may be a tumor suppressor gene that affects the growth of colon tumor cells [11]. The Wnt signaling pathway plays a crucial role in cell proliferation, embryonic development, adult tissue regeneration, and cancer stem cell characteristics. The abnormal activation of certain genes in the Wnt/β-catenin pathway can be induced by mutations or expression disorders. Cancer [12-14]. WIF1 directly binds to Wnt protein to inhibit the effect of Wnt protein and Fzd receptor on the cell membrane, and affects intracellular signal transduction. Axin, APC and GSK3β in the cytoplasm form a destructive complex, which leads to phosphorylation of β-catenin and then degradation, causing Downstream target gene transcription is inhibited, inhibiting cell proliferation [15]. This study shows that the expression of WIF1 is negatively correlated with the proliferation ability of SW620 cells and the expression of β-catenin, indicating that WIF1 inhibits cell proliferation by inhibiting the activity of the Wnt/β-catenin pathway. The above research results suggest that high glucose inhibits the expression of WIF1, and the decline of WIF1 expression further promotes the proliferation of colon tumor cells in a high glucose state through the Wnt/β-catenin pathway.

 

In summary, the results of this study suggest that high glucose down-regulates the expression of WIF1, blocking the inhibitory effect of WIF1 on the Wnt/β-catenin pathway, thereby promoting the proliferation of colon tumor cells under high glucose conditions. This discovery may provide a theoretical basis and new intervention targets for diabetes to promote colonic tumorigenesis.