The New Mechanisms Of Snail Involved In Colorectal Cancer: The Regulatory Effects Of Snail On PRL-3 And Nanog

Colorectal cancer (CRC) is a common malignant tumor leading to death in the world. The incidence rate of CRC in china is increasing fast during the past decades. Metastasis is the main cause affecting the therapeutic efficacy and leading to the death of cancer patients. It is urgent to elucidate molecular mechanisms of metastasis and find out the preventive and therapeutic strategies.Snail is an important transcription factor which plays an important role in the process of epithelial mesenchymal transition (EMT) in the tumor. It is studied that Snail is involved in the occurrence of EMT through regulating the expression of E-cadherin. This has been confirmed in several types of tumors such as colon cancer, breast cancer, pancreatic cancer, malignant melanoma, oral squamous cell carcinoma and et al.Our previous work by bioinformatic analysis showed that Snail binding sites were present in the PRL-3 gene promoter region. As an important regulatory factor involved in the process of tumor metastasis, the regulatory role of Snail involved in PRL-3 gene expression is still unknown. Therefore, it is necessary to clarify the relationship between Snail and PRL-3 gene.Embryonic stem cells (ESCs) are the cells of inner cell mass when the fertilized egg develops into the blastula, which has the characteristics of unlimited proliferation, self-renewal and multi-potentiall differentiation. ESCs are a kind of highly undifferentiated cells, which possess developmental totipotency and can differentiate into all tissues and organs of animals, including reproductive cells. Research about ES cells is one of most excited filelds of bio-engineering. Induced embryonic stem cells (iPS cells) are cells which were reprogrammed to embryonic stem cell state from somatic cells through forced induction of a kind of specific combinations of genes. They possess similar functions of embryonic stem cells (ES cells) and can generate many kinds of tissue cells. For example, when we imported the four kinds of genes (OCT4, Nanog, SOX2, LIN28, or OCT3/4, SOX2, C-MYC, KLF4) into human somatic cells, differentiated cells are reversed to the embryonic stem cell state through genetic reprogramming. It has been reported that these embryonic stem cell genes were also expressed in a number of malignant tumors. However, their roles in development, progress and metastasis of cancer are still not very clear. In this study, we preliminary uncover the relationship between Snail transcriptional repressors and induced embryonic stem cell gene (Nanog) in colorectal cancer and the effects of Nanog on the development of colorectal cancer.Methods1. The effects of Snail gene on the biological behaviors of colorectal cancer (CRC) cells.In order to know the effects of Snail gene on the biological behaviors of colorectal cancer (CRC) cells, we knocked down Snail expression in Lovo cells and upreguted Snail expression in SW480 cells. Proliferation, adhesion and migration capacities of CRC cells using MTT, adhesion experiments, Transwell chamber and plate colony formation assay were analyzed.2. The effects of Snail gene on the luciferase activities of the PRL-3 gene promoter and PRL-3 protein expression in CRC cells.The luciferase activities of the PRL-3 gene promoter fragments (-642bp to-383bp and-754bp to 455bp) was studied after expression of Snail was upregulated in SW480 cells or downregulated in Lovo cells. Meanwhile, PRL-3 protein expression was detected using Western blotting after expression of Snail was upregulated in SW480 cells or downregulated in Lovo cells.3. Snail was involved in the regulation of Nanog gene in CRC.Protein expressions of Snail and Nanog in fresh CRC tissues were detected using Western blotting to preliminarily determine the possible correlation between Snail and Nanog genes. Nanog protein expression was detected using Western blotting after Snail was upregulated or down-regulated in CRC cells. The effects of TGF-β1 on Nanog protein expression in CRC cells were also determined. After Nanog was upregulated, mRNA expressions of a-catenin,β-catenin,γ-catenin, E-cadherin, vimentin, snail, slug, and CK were studied by fluorescence quantitative PCR assay.4. Immunohistochemical evaluation of Nanog protein expression in CRCImmunohistochemical staining of Nanog protein expression in 175 human tissue samples of CRC was done using a Dako EnVision System. Rabbit anti-human Nanog polyclonal antibody from Biosynthesis Biotechnology Co., LTD (Beijing, China) was used for immunohistochemical staining of Nanog protein expression in human tissue samples of CRC. The specificity of this antibody was determined by western blot and compared with that of mouse anti-Nanog monoclonal antibody from Abnova (Taiwan). The cellular localization of Nanog was studied by indirect immunofluorescence and confocal microscopy in SW480 cells. The staining intensity was scored on a scale of 0 to 3 as negative (0), weak (1), medium (2) or strong (3). The extent of the staining, defined as the percentage of positive staining areas of tumor cells in relation to the whole tumor area, was scored on a scale of 0 to 4:0 (0%),1 (1-25%),2 (26-50%),3 (51-75%) and 4 (76-100%). An overall protein expression score (overall score range,0-12) was calculated by multiplying the intensity and positivity scores. For statistical analysis, a final staining score of≥4 was considered to be high expression of Nanog protein.5. The effects of Nanog gene on the biological behaviors of colorectal cancer (CRC) cells.Proliferation, adhesion and migration capacities of CRC cells using MTT, adhesion experiments, Transwell chamber and plate colony formation assay were analyzed in the stable nanog-overexpression SW480 cells.Results1. Effect of Snail gene on the biological behaviors of CRC cells.A significantly increased proliferation was found after Snail expression in SW480 cells was upregulated by in vitro MTT assay. A significantly decreased proliferation was found after Snail expression in Lovo cells was knocked down by in vitro MTT assay.Snail overexpression had a significant enhanced ability to form colonies in plates in SW480, and Snail downregulation had a significant inhibtory ability to form colonies in plates in Lovo cells.Using Transwells migration assay, we observed that Snail overexpression had a significant enhanced migratory ability in SW480, and Snail downregulation had a significant inhibtory migration ability in Lovo cells.Results of adhesion assay showed Snail promoted adhesion of human CRC cells.2. PRL-3 was regulated by Snail in CRC cells.Luciferase activity of PRL-3 promoter fragments (-642bp to-383bp and-754bp to 455bp) decreased significantly after Snail expression was knocked down. Luciferase activity of PRL-3 promoter fragments (-642bp to-383bp) increased significantly after Snail expression was up-regulated. Consistent with the changes of PRL-3 promoter activity, we found that PRL-3 protein expression was significantly reduced after after Snail expression was knocked down in Lovo cells and PRL-3 protein expression was significantly increased after Snail expression was up-regulated in SW480 cells.3. Snail was involved in regulation of Nanog gene expression in CRCWe observed that expression of Nanog protein was significantly induced after TGFβ1 stimulation in HT-29 and colo205 cells. Knockdown of Snail, a key gene involved in EMT process, led to inhibitory expression of Nanog and overexpression of Snail resulted in increasing expression of Nanog in SW480 cells. The results suggest that Nanog may participate in EMT process initiated by TGFβ1 and also can be regulated by Snail.Interestingly, semi-quantitative real-time PCR analyses demonstrated that Nanog significantly induced the transcription of Snail, Slug andγ-catenin in at least two Nanog-overexpression clones.4. Relationship between clinicopathologic features and nanog expression in CRCWestern blots results showed that Nanog protein expression in CRC tissues was higher than that in normal colorectal tissues. Nanog protein was localized mainly in the cytoplasm of cancer cells. Nuclear accumulation of Nanog was only observed in a small fraction of cancer cells Cytoplasmic and perinuclear distribution of Nanog protein was found in SW480 cells by means of cytoimmunofluorescent labeling and confocal microscopy. No significant associations were found between Nanog expression and age, gender, tumor size, tumor site, differentiation and invasion of CRC patients (p>0.05). Interestingly, we observed that Nanog expression was positively correlated with lymph node status (p=0.024) and Dukes classification (p= 0.049) of patients. Using Kaplan-Meier analysis method, we found that the protein expression of Nanog in CRC was significantly correlated with overall survival (p= 0.000) of CRC patients. Using a public microarray expression profiling data,11 we also found the expression level of Nanog mRNA was significantly correlated with recurrence-free survival of stage II CRC (p=0.038). These results indicated that the high expression of Nanog was correlated with a shorter survival or recurrence-free survival.To determine whether the expression of Nanog was an independent prognostic factor of outcomes, multivariate survival analysis including invasion, differentiation, Dukes classification and Nanog expression, was done. Results showed that the expression of Nanog protein was a potential independent prognostic factor of outcomes of CRC patients.5. Nanog promotes proliferation, invasion and migration of human CRC cells.A significantly increased proliferation was found in SW480/Nanog clone2, SW480/Nanog clone3 compared with that in Mock cells by in vitro MTT assay. Nanog overexpression had a significant enhanced ability to form colonies in plates. Concordant with these results, Nanog-overexpression clones especially in clone 2 and clone 3 showed a significantly increased number of S phase (PI) by cell cycle analysis of flow cytometry.We also observed that three stable Nanog-overexpression clones displayed significant increase in invasive ability compared with Mock cells). In vitro wound healing migration assay indicated that cells in three stable Nanog-overexpression clones filled in from 50% to 80% of the scratched area after 60 h, whereas Mock cells filled in only about 20% of the scratched area after 60 h.Conclusions1. Snail promotes proliferation, adhesion, invasion and migration of CRC cells.2. Snail is involved in progression of CRC by regulating PRL-3 expression.3. Nanog gene is a potential new target of Snail in colorectal cancer.4. Nanog promotes proliferation, adhesion, invasion and migration of CRC cells.5. Nanog functions as an inducer and also a recipient of EMT in tumor progression of CRC. Nanog expression level is correlated with lymph node status and Dukes stage. Nanog protein expression in CRC patients can be considered as a potential independent prognostic factor.