In this study, we used the DMH-induced CRC mouse model that shows phenotypic and genotypic features similar to those observed in human sporadic CRCs and, thus, can be applied to study the prevention of CRC 
. In this study, we found that the incidence of CRC decreased significantly in mice fed a high calcium diet, indicating a clear role of calcium in the prevention of CRC. Our results are consistent with many other studies. Pence 
have found that rats on the high calcium diet have a decreased CRC incidence compared to the low calcium diet (86% vs. 53%), Salas 
also observed a significant diminution in the number of tumors and a decrease in the CRC incidence in the group fed high calcium diet (97% vs. 64%). However, some studies have observed different outcomes. Sitrin 
found that neither calcium supplementation alone nor supplemental calcium conjunction with vitamin D deficiency altered the incidence of CRC induced by DMH, Mølck 
even found an increased incidence in the rats fed high calcium diet. We think that the differences in the animal strain, animal age, rearing environment, basis diet, DMH doses and duration of injection, calcium concentration, calcium intervention time may be accounted for the differences in the outcome of various studies using high calcium. The confirmed preventive effectiveness of CRC with high calcium diet could not yet be contained from those studies. We think it is may be due to the small doses of calcium in those studies. So we used higher calcium content(3%) in the feed to test the preventive effect of CRC and the mice' tolerance. In fact, this high calcium feed showed good preventive effect of CRC in our experiment and no obvious adverse effects was found in mice. In addition, no tumor was found in the Calcium groups. Growth and development of the mice in the Calcium group were not significantly affected, and the pathological examination of their kidneys, hearts, lungs, livers, and spleens revealed no obvious abnormalities, the expression of most of the selected genes confirmed by real-time PCR in the Calcium group was equal to that in the Control group. These findings indicate that this high calcium diet is safe for the mice.
Next, we used microarray gene expression profile analysis to determine the mechanism of calcium-mediated prevention of CRC. To the best of our knowledge, this is the first investigation to use microarray technology for studying the role of high calcium diet in the prevention of CRC.
When the FC was set to ≥1.5, the changes in 549 genes that were the result of DMH treatment could be reversed with dietary calcium. S100a9
is the most downregulated gene; its product is a cytoplasmic Ca2+
-binding protein. Although extracellular S100a9 could induce apoptosis by binding to a yet unknown receptor and cause dimerization of Bax and Bak and their translocation to the mitochondria leading to mitochondrial damage 
, its expression in epithelial cells could indeed induce cell proliferation by activating the phosphoinositide 3-kinase (PI3K)–Akt–NF-κB survival pathway, which is associated with tumorigenesis 
. In addition, S100a9 is an important proinflammatory cytokine involved in innate immunity, and its expression was found to be elevated in numerous pathological conditions associated with inflammation 
. Many other inflammatory cytokines, such as Defa, were also significantly downregulated [members of the defensin family were downregulated at different levels: Defa-rs2
−8.16)]. Tumor necrosis factor (Tnf
) and its receptors were also downregulated at different levels: Tnf
−2.27). Several other studies performed using the DMH or azoxymethane (AOM)-induced CRC model also found high expression levels of S100a9 and Defa in tumor tissues 
. Inflammatory cytokines play important roles in the innate and adaptive immunity; however, those elevated cytokines are also involved in tumor initiation, promotion, and invasion 
, and thus, play an important role in inflammation-associated carcinogenesis 
MMPs are a family of matrix metalloproteinases that can play important roles in the tumor microenvironment 
. Many members of the MMP family were significantly downregulated in the DMH + Calcium group [MMP10
−7.07), and MMP11
−1.56)]. Other metalloproteinases were also downregulated, such as Adam8
, which were downregulated by 2.08 and 1.6 times, respectively. Recent studies have shown that many members of the MMP and ADAM family could play various roles in tumorigenesis. For example, MMP7
could cleave Fas ligand, thereby lowering the impact of chemotherapy on the tumor by abrogating apoptosis 
may trigger the release of soluble epidermal growth factor (EGF), mediate the shedding of E-cadherin, translocating β-catenin to the nucleus, and thus, driving cell proliferation 
. However, the most important role of the members of the MMP and ADAM family is promoting tumor invasion and metastases by degrading extracellular matrix 
. These findings indicate that a high calcium diet may play an important role in inhibiting invasion and metastases of CRC. Because in vitro
studies showed that, in CRC cells, E-cadherin expression could be induced by increasing the extracellular Ca2+
, calcium may be considered to suppress the epithelial-mesenchymal transition (EMT) to inhibit CRC metastases.
was the most upregulated gene in our study, belonging to the period genes. Those genes are core elements of the transcriptional/translational feedback loops that generate the endogenous circadian rhythm, and Per3
participates in timekeeping in the pituitary and lung 
. Recent studies have suggested that circadian genes participate in the growth and development of various cancers, and the period genes have now been linked to DNA damage response pathways, inhibition of the growth of cancer cells, and increase in the apoptotic rate 
. Oshima's study has shown that the expression of Per3
in CRC tissues was significantly lower than that in the adjacent normal mucosa, suggesting that Per3
may function as a tumor suppressor gene 
was the second most upregulated gene in our study. It belongs to the PAR-domain basic leucine zipper (PAR bZip) transcription factors and is involved in detoxification and drug metabolism 
and in the maintenance of cell shape in fibroblasts 
. It also serves as a pro-apoptotic gene by promoting the expression of bcl-gs or bik 
. Many other genes were also upregulated, e.g., Rnf152
is a novel RING finger protein and has been shown to have pro-apoptotic activity 
is a member of the PRDX family, associated with functions such as cell proliferation, differentiation, and apoptosis, thus executing anti-cancer activity 
By using pathway analysis, we found that the Wnt pathway was significantly affected. Although the expression of the Wnt inhibitory factor wif1
was significantly downregulated, that of many genes associated with the Wnt signaling pathway and their target genes was downregulated [i.e., Tcf7
, cyclin d1
), and Mmp7
at FC of −1.53, −1.61, −1.62, and −7.07, respectively]. The protein expression of β-catenin was significantly reduced in the DMH + Calcium group, suggesting that the Wnt pathway was downregulated in this group. This is consistent with previous findings 
. Interestingly, the cell cycle pathway was also significantly inhibited in the DMH + Calcium group, with many genes in this pathway being consistently downregulated, i.e., the cyclins Ccnb1
were downregulated by 1.79 and 1.62 times, respectively, and the expression of the cyclin-dependent kinase Cdk1
was reduced by 1.66 times.
Among the metabolic pathways, the arachidonic acid pathways were significantly inhibited in the DMH + Calcium group. Many enzymes in this pathway showed reduced expression, e.g., Ptgs2, also known as cyclooxygenase-2 (COX-2), was reduced by 3 times. The arachidonic acid pathway is activated during inflammation, leading to the synthesis of prostaglandin and thromboxane, which play an important role in inflammatory response 
. Furthermore, the key enzyme in this pathway, Ptgs2 (COX-2), plays an important role in the initiation of CRC. COX-2 overexpression was shown to be correlated with carcinogenesis in more than 80% of CRCs 
. In animal models, COX-2 expression was found to be sufficient to induce tumorigenesis 
. The mechanism may be related to the activation of inducible nitric oxide synthase (iNOS) and VEGF signaling to promote formation of new blood vessels 
. On the basis of the finding that the expression of the inflammatory cytokines S100a9, Defa, TNF, and TNF receptors was also significantly downregulated in the DMH+Calcium group, we speculated that calcium may play an important role in reducing inflammation-related tumorigenesis.
Among the Pathway Studio-derived hub genes, which may play key roles in the calcium-mediated prevention of CRC, FoxM1
, etc., were also involved. FoxM1 belongs to a family of evolutionarily conserved transcriptional regulators that were characterized by the presence of a DNA-binding domain known as the forkhead box domain. Higher expression of FoxM1 was noted in many types of human cancers 
. FoxM1 may play important roles in cell proliferation and apoptosis 
. The molecular mechanism underlying FoxM1 signaling-mediated induction of tumor growth has not been completely elucidated. However, multiple oncogenic pathways such as PI3K/Akt, NF-κB, extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK), VEGF, reactive oxygen species (ROS), c-myc, and hypoxia-inducible factor (HIF)-1 have been reported to interact with FoxM1 signaling; this suggests that the interaction between FoxM1 signaling and other signaling pathways may play important roles in tumorigenesis 
. It should be noted that the expression of many genes regulated by NF-κB showed altered expression in the DMH + Calcium group, e.g., TNF
, ETS-related gene 1
, and lymphoid enhancer factor 1
), suggesting that NF-κB may also serve as a hub gene in the calcium-mediated prevention of CRC. Although the expression of NF-κB did not change significantly at the transcriptional level, it may have changed at the translational or posttranslational level. The expression pattern of NF-κB in the DMH + Calcium group and its specific mechanism needs to be further elucidated.
We also found that the gene expression levels of most of the above selected genes in mice from the DMH+Calcium group with tumors was between those from the same group without tumors and those from the DMH group, thus confirmed the importance of those genes in the calcium's prevention of CRC. Interestingly, the expression of some calcium transporters, such as Trpv6, Trpv3 and PMCA, was different among the mice from the DMH + Calcium with/without tumors. One of the biological functions of these calcium transporters is to mediate transcellular Ca2+
movement across epithelial cells 
. Therefore, the differential expression of these calcium transporters disrupted the distribution of intracellular and extracellular calcium, thereby activating the proliferation-related pathways 
. Hence, we speculated that the differential expression of these calcium transporters may partially account for the different expression levels of the selected genes.
It is necessary to mention the limitations of this study. First, we used colorectal tissues instead of colorectal mucosa in our microarray study. There are many different types of cells in the colorectal tissues, so it is hard to determine the cellular origin of the identified genes in this study. However, more and more evidences have shown that the stroma and immune cells play important roles in the tumor initiation. In our studies, we have found that there are many immune cells infiltrated into the colorectal tissues by pathological examination and the expression of many inflammation cytokines is significantly upregulated in the DMH group compared with Control group. These cells can not only cause local inflammation, but also significantly promote tumorigenesis 
. We also have found that high calcium diet could reduce the expression of many inflammation cytokines, suggesting that calcium may play an important roles in the prevention of inflammation-related tumorigenesis. Therefore we believe that using colon tissues instead of colon mucosa could reflect the overall and more realistic changes in the gene expression. To our knowledge, colon tissues are widely used in the study of gene expression associated with CRC 
. Second, since we did not use semi-purified diets in our animal experiment, our results may be biased. Third, the calcium content in our study was rather high. Although this may account for the drastic effectiveness in the prevention of CRC and no obvious adverse effect was found in the mice in our study, it is difficult to apply it for the prevention of sporadic CRC in humans, because exceedingly high calcium and vitamin D concentrations in the diet may pose a risk for developing hypercalcemia and kidney stones. However, a meta-analysis found that a calcium intake of more than 1000 mg/day will have a better preventive effect 
. Another clinical trial found that a lower calcium intake may have no apparent preventive effect 
. Therefore, further study is necessary to confirm the optimal calcium content in the prevention of CRC.
In conclusion, we confirmed the effectiveness of calcium to prevent CRC in the DMH-induced CRC mouse model. We also clarified the comprehensive mechanisms of calcium-mediated prevention of CRC. Our results showed that calcium exerted its protective effect mainly by downregulating the expression of oncogenes such as S100a9, Mmp10, Adam8, and Ptgs2, by inhibiting the activity of tumor-associated pathways such as the Wnt and cell cycle pathway, and by negatively regulating cell proliferation, cell division, cell invasion, and angiogenesis. In particular, calcium is speculated to play important roles in reducing inflammation-associated tumors and suppressing tumor invasion and metastases. However, the mechanism of the calcium-mediated regulation of these genes remains to be studied.