Upregulation of DKK3 by miR‐483‐3p plays an important role in the chemoprevention of colorectal cancer mediated by black raspberry anthocyanins

It is reported that black raspberry (BRB) anthocyanins could act as a potential chemopreventive agent for colorectal cancer (CRC). However, the underlying mechanism by which BRB anthocyanins inhibits the carcinogenesis of CRC cells has not been elucidated. The abnormal expression of microRNAs (miRNAs) that target important tumor suppressor genes is usually associated with CRC development. In this study, we explored whether BRB anthocyanins could affect the expression of certain miRNAs in an azoxymethane (AOM)/dextran sulphate sodium (DSS)‐induced CRC mouse model and human CRC cell lines. miRNA microarray analysis was used to determine the differences in miRNA expression between AOM/DSS‐induced mice fed with a diet supplemented without or with BRB anthocyanins. The expression of one particular miRNA, miR‐483‐3p, was found to decrease dramatically in AOM/DSS‐induced mice that were fed with a diet supplemented with BRB anthocyanins. Subsequent quantitative real‐time polymerase chain reaction and Western blot analyses showed that the reduced expression of miR‐483‐3p was accompanied by an increased expression of Dickkopf 3 (DKK3), a potential target of miR‐483‐3p as predicted by bioinformatic analysis. The protein and messenger RNA levels of DKK3 were significantly upregulated when the miR‐483‐3p level was reduced by a miR‐483‐3p‐specific inhibitor, suggesting that DKK3 might be the target gene of miR‐483‐3p. In addition, the downstream factors of the DKK3 signaling pathway, which included Wnt/β‐catenin, also played a role in the miR‐483‐3p‐mediated anticancer effect of BRB anthocyanins. Thus, miR‐483‐3p might be a potential target in BRB anthocyanin‐mediated prevention of CRC.

either act as an oncogene or a tumor suppressor, whereas miR-22, 6 miR-490-3p, 7 and miR-93 8 all act as tumor suppressors. In addition, miRNAs also serve as useful markers for the detection of cancer. For example, miR-193a-3p, miR-23a, and miR-338-5p in the blood can be used for the early detection of colorectal cancer (CRC). 9 miR-483-3p plays the role of an oncogene by inhibiting the expression of DPC4/Smad4, thereby promoting cell proliferation and clonality in pancreatic cancer. 10 As more miRNAs are found to have an abnormal expression in CRC, the use of miRNA will become a hotspot in CRC research that centers on diagnosis, targeted therapy, and prognosis. CRC is common cancer and the fourth most common cause of death worldwide. 11 The occurrence and development of CRC involve changes in multiple genes and signaling pathways. 12 The development of CRC is accompanied by the abnormal activation of the Wnt/β-catenin signaling pathway. 13 The Dickkopf (DKK) protein family, which consists of DKK1, DKK2, DKK3, and DKK4, acts as an extracellular inhibitor of the Wnt/β-catenin signaling pathway by interacting with the LRP5/LRP6 receptor to influence the activation of the nonclassic Wnt signaling pathway. 14,15 Long term consumption of black raspberry (BRB) could reduce the risk of cancer as well as heart disease, arthritis, and respiratory diseases. 16,17 The consumption of BRB would result in the production of more secondary metabolites, which can lead to an improvement in health. BRB anthocyanins are among the main metabolites of BRB produced in the body, and they are also a major dietary source of anthocyanins in the diet. 18,19 The most significant beneficial effect of BRB is its potential cancer chemoprevention effect, which has been investigated by several studies. [20][21][22][23] Our previous investigation has found that BRB anthocyanins could regulate gut microbiota and further demethylate the promoter of secreted frizzled-related protein 5, eventually retarding the development of CRC in azoxymethane/dextran sulphate sodium (AOM/DSS)-treated mice. 24 Anthocyanins appeared to be the key anticancer components in the lyophilized BRB powder used in the clinical trial. 25,26 The involvement of miRNAs in BRB anthocyanin-mediated chemoprevention of CRC has been reported in our recent study, where miR-24-1-5p was found to downregulate the Wnt/β-catenin signaling pathway, contributing to its role in the prevention of the CRC development. 27 In this study, we investigated the role of yet another miRNA, miR-483-3p, to determine the mechanism by which it might suppress the development of CRC in an AOM/DSS-induced CRC mouse model following the oral administration of BRB anthocyanins. The role of miR-483-3p was also examined in the human CRC cells following treatment with BRB anthocyanins.

| The acquisition of BRB anthocyanins
BRBs contain various types of chemicals, such as phenolic acids and anthocyanins. The main content of BRB anthocyanins is cyanidin-3-  28,29 Mice from both groups were injected with AOM (10 mg/kg) on the first day of the experiment and then another dose on the 8th day. At the same time, they were also treated with 2% DSS water daily for the first week followed by normal water for the next 2 weeks, and the whole process was repeated three times.
Thus, the whole experiment lasted for 9 weeks, and at the end of the treatment period, they were killed and their colons were removed and frozen in liquid nitrogen. Part of the tissue was used for miRNA array analysis, while the rest was stored at −80°C for further analysis.
During the whole experimental procedure, the mice were housed in cages (3-5 per cage) kept at 22°C ± 1°C and under 50% ± 5% humidity, and a 12 hours light-dark cycle (7:00 AM-7:00 PM). All the procedures were carried out according to the National Institutes of Health regulations for the care and use of animals, and were approved by the Liaoning University of Traditional Chinese Medicine Ethics Committee.

| miRNA microarray
The extracted colon tissues were subjected to microarray analysis.
Microarray (8*60 K, Design ID: 070155) and the basic analysis of the expression of the different genes from the raw data was performed by Agilent (Agilent, Palo Alto, CA).

| Cell culture
Human HCT116 and HT29 CRC cell lines were obtained from the  (Table S1) and amplified using an Applied Biosystems 7500 Real-Time PCR system.

| Protein extraction and Western blot analysis
The cells were first lysed with radioimmunoprecipitation assay buffer

| Wound healing assay
The cells were cultured in a six-well plate until they reached 60% confluence. The cell layer was then scratched with a sterile micropipette to generate a wound, and the medium of the culture was replaced with fresh medium. Afterward, the cells were transfected with miRNAs for 24 hours, and the photomicrograph of the cell layer was then taken. The extent of cell migration was calculated from the photomicrograph.

| 3-(4,5-Dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide assay
A stock solution of BRB anthocyanins was diluted with medium to the appropriate concentrations and then added to a 96-well plate containing the cells grown to 60% confluent. BRB anthocyanins were added to the cells to final concentrations 25 and 50 μg/mL. For the control group, the same volume of medium only was added to the cells instead of BRB anthocyanin. Cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (Sangon Biotech Shanghai Co, Ltd) reagent as previously described. 27 2.10 | Identification of miRNA target gene and its correlation with CRC patient survival   Taken together, the data suggested that DKK3-Wnt/β-catenin might be directly targeted by miR-483-3p, implying that miR-483-3p could probably regulate tumorigenesis-associated genes in vitro.   There is evidence worldwide to suggest that CRC is associated with high morbidity and mortality rates. The development of therapies against CRC and the prognosis of patients with CRC remains undesirable. Therefore, establishing the underlying mechanism of CRC pathogenesis and finding more efficient ways to prevent or cure CRC is of great importance. miRNAs are short and noncoding RNAs that target the 3′-UTR of related mRNAs to repress their translation. miRNAs regulate different kinds of biological processes, including apoptosis, cell angiogenesis, cell migration, and cell proliferation. 35 miRNAs usually serve as tumor suppressor genes or oncogenes by targeting different mRNAs. 35 In this study, we found that miR-483-3p, which acted as an oncogene in a CRC mouse model, was decreased after the animals were given BRB anthocyanins in the diet. High levels of miR-483-3p expression have been found in the plasma of patients with lung or pancreatic cancer. 10,36,37 In CRC, miR-483-3p is also highly expressed, but its specific mechanism of action has not been studied in detail. 38 Reduced DKK3 expression has been detected in CRC. 39 Bioinformatic analysis showed that the expression of DKK3 could influence the survival time of patients ( Figure 2C). DKK3 level in LoVo and SW480 cells was upregulated after the cells were treated with BRB anthocyanins ( Figure 3G,H). Thus, BRB anthocyanins could affect the expression of miR-483-3p and DKK3.

| miR-483-3p influences proliferation and migration of CRC cells
F I G U R E 5 Effect of miR-483-3p the proliferation and colony formation of CRC cells. Human CRC cells, LoVo and SW480 cells were transfected with a miR-483-3p inhibitor and then subjected to the following analyses: A and B, Cell viability was determined by MTT assays; C and D, Cell proliferation was determined by colony formation assay. The plots show the quantitative comparison of the data in C and D. Data are expressed as means ± SEMs. * And ** indicate significantly different from the control (no BRB supplement) at the P < .05 and P < .01 levels, respectively. BRB, black raspberry; CRC, colorectal cancer; MTT, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide [Color figure can be viewed at wileyonlinelibrary.com] DKK3 has been shown to inhibit the proliferation of cancer cells in osteosarcoma, colon, gastric, glioma, prostate, cervix, hepatic, and lung cancer. 39 Moreover, DKK3 is also an inhibitor of the Wnt signaling pathway, where it is involved in the degradation of nuclear β-catenin, preventing the translocation of the protein to the cytosol. 40 Wnt signaling pathway is highly conserved and it plays important roles in embryogenesis, homeostasis, and cancer development. 41 The Wnt/β-catenin signaling pathway is involved in cell invasion, migration, proliferation, and differentiation processes that are important to in the initiation and progression of CRC. 42 and DNMT3B in colon cancer cells. 48 Moreover, in precancerous colon tissue, black raspberries protectively regulate the methylation of genes in the Wnt signaling pathway. 49 We, therefore, speculate that changes in miRNA expression (and possibly activity) in response to BRB anthocyanins could be mediated through the effect of BRB anthocyanins on the epigenetic mechanism. However, whether BRB anthocyanins could influence the expression of miRNAs by regulating the enzymes involved in the epigenetic process is a topic for further work.
Taken together, the data obtained from this study indicated that miR-483-3p could play a vital role in CRC by regulating the Wnt/βcatenin pathway via DKK3, one of the targeted genes of miR-483-3p.
The expression of miR-483-3p could be downregulated by BRB anthocyanins, further demonstrating the potential of BRB anthocyanins as a chemopreventive agent against CRC. Moreover, the silencing of miR-483-3p could significantly inhibit the migration and proliferation of CRC cells, suggesting that specifically targeting the expression of miR-483-3p may also be considered as a therapeutic approach for the treatment of CRC in the future.

ACKNOWLEDGMENTS
The work was financially supported from National Natural Science Foundation of China to Bi, X #1 (grants #81272333 and #81472821); #2 LN Revitalization Talents Program (#XLYC1807058); and in part from #3 Shenyang Youth Science and Technology Innovation Project.
We thank Dr. Alan K Chang for revising the language of the manuscript.