ZNF217 Gene Contributes Biologicai Behavior Of Colorectal Cancer And Is Regulated By MicroRNA-203

Part ⅠTHE EXPRESSION OF MICRORNA-203 AND ZNF217 IN COLORECTAL CANCER AND THEIR CLINICAL SIGNIFICANCEObjective1. To investigate the expression of ZNF217 in colorectal cancer tissues and their corresponding adjacent normal tissues.2. To evaluate the correlation of clinicopathologic factors with ZNF217 level in colorectal cancer tissues.Such as age, gender, tumor size, histological grade, depth of invasion, lymph node metastasis, distant metastasis, and clinical stage.3. Analysis of the relationship between the expression of ZNF217 and prognosis in colorectal cancer patients.4. To detect the ZNF217 mRNA expression, and to analyse its correlation with miR-203 level in colorectal cancer tissues.Methods1. Immunohistochemistry was used to detect the expression of ZNF217 expression in colorectal cancer tissues and their adjacent nontumor tissues and analysis of the relationship between the expression of colorectal cancer patients ZNF217 and clinicopathological parameters and prognosis.2. qRT-PCR method to detect the expression of ZNF217 mRNA and miR-203 in colorectal cancer tissues, and use Pearson correlation to analyze the correlation between tumor tissue miR-203 and ZNF217 expression.Results1. Immunohistochemistry results showed ZNF217 expression levels in colorectal cancer tissues was significantly higher than the corresponding adjacent control tissues (P<0.05).2. The expression level of ZNF217 in CRC tissues was significantly associated with tumor size (P= 0.042), depth of invasion (P= 0.03)and lymph node metastasis (P = 0.01) However, no significant correlations were found between ZNF217 expression and age, sex, degree of differentiation、metastasis to other organs and clinical stage (P>0.05). The Kaplan-Meier survival analysis showed that CRC patients with high ZNF217 expression had a significantly poorer overall survival rates than those with low ZNF217 expression (P=0.028)..3. Pearson correlation analysis showed that in colorectal cancer tumor tissue relative ZNF217 expression was significantly negatively correlated with the relative expression levels of miR-203 (r= 0.7431, P<0.05).Conclusion1. The expression of ZNF217 in CRC tissues was significantly higher than that in corresponding adjacent non-cancerous tissues, and high ZNF217 expression was associated with tumor size, depth of invasion, lymph node metastasis and prognosis,suggesting that ZNF217 may play an important role in the development of colorectal cancer.2. In colorectal cancer tissues, the expression of miR-203 and ZNF217 were negatively correlated, suggesting that both of them may participate in the pathogenesis of colorectal cancer. associated with tumor size, depth of invasion, lymph node metastasis and prognosis,suggesting that ZNF217 may play an important role in the development of colorectal cancer.2. In colorectal cancer tissues, the expression of miR-203 and ZNF217 were negatively correlated, suggesting that both of them may participate in the pathogenesis of colorectal cancer.Part ⅡEFFECTS OF ZNF217 ON THE PROLIFERATION、 MIGRATION AND INVASION OF COLORECTAL CANCER CELLSObjectiveTo investigate the effects of ZNF217 on CRC cell proliferation migration and invasion ability in vitro that colorectal cancer cell lines were transfected by siRNA-ZNF217Methods1. Transfection of siRNA-ZNF217 and corresponding negative control was carried out in SW480 cells using Lipofect2000 according to the manufacturer’s instructions and test transfection efficiency.2. Before and after transfection siRNA-ZNF217,MTT were performed to test the effects of ZNF217 on CRC cell proliferation ability; Transwell experiments with or without Matrigel were performed to test the effects of ZNF217 on CRC cell migration and invasion ability.ResultsThe low expression of ZNF217 significantly decreased proliferation migration and invasion abilities in colorectal cancer cell line (P<0.05)ConclusionZNF217 gene expression was down regulated can significantly inhibited the proliferation., migration and invasion of colorectal cancer cells. or without Matrigel were performed to test the effects of ZNF217 on CRC cell migration and invasion ability.ResultsThe low expression of ZNF217 significantly decreased proliferation migration and invasion abilities in colorectal cancer cell line (P<0.05)ConclusionZNF217 gene expression was down regulated can significantly inhibited the proliferation., migration and invasion of colorectal cancer cells.Part ⅢSTUDY ON MIR-203 REGULATING ZNF217 IN COLORECTAL CANCERObjective1. Bioinformatics predicted microRNA regulates downstream gene ZNF217 and verified2. Colorectal cancer cell lines were transfected by miR-203 mimics, miR-203 inhibitor,constructed miR-203 over-expression and low-expression model to investigate the abnormal expression effects of miR-203 on CRC cell proliferation migration and invasion ability.3. Explore ZNF217, miR-203 combined with aberrant expression effects of on CRC cell proliferation、migration and invasion ability.Methods1.Transfection miR-203 mimics/miR-203 inhibitor and their corresponding negative control was carried out in colorectal cancer cell lines using Lipofect2000 according to the manufacturer’s instructions and test transfection efficiency.2. Before and after transfection,MTT were performed to test the effects of miR-203 on CRC cell proliferation ability; Transwell experiments with or without Matrigel were performed to test the effects of miR-203 on CRC cell migration and invasion ability.3. Using bioinformatics analysis software TargetScan and micrornaorg predict possible upstream regulatory gene ZNF217 for miR-203; using luciferase reporter gene assay, containing the 3’end of the ZNF217 gene non-coding region sequence (WT-ZNF2173’-UTR) or the mutant sequence (MUT-ZNF2173’-UTR) of the luciferase reporter vector and miR-203, respectively;miR-203 mimics or miR-negative control were co-transfected HEK 293T cells, detect the luciferase activity, verify predictions. In colorectal cancer cell line transfected miR-203 mimics or miR-negative control, using qRT-PCR and Western blot were used to detect the expression level of potential target genes ZNF217 mRNA and protein level.4. The miR-203 mimics or miR-negative control and siRNA-ZNF217 were co-transfected into colorectal cancer cell lines. Then MTT and Transwell assays detected changes in cell proliferation migration and invasion ability before and after transfection. To explore mechanisms of ZNF217 joint miR-203 influence colorectal cancer cell proliferation, invasion and migration ability.Results1. Compared with miR-negative control group, the miR-203 expression of miR-203 mimics transfected was significantly raised, miR-203 inhibitor transfected was significantly decreased.2. The high expression of miR-203 significantly decreased abilities of proliferation migration and invasion abilities in colorectal cancer cell line; the low expression of miR-203 significantly increased abilities of proliferation,migration and invasion (P <0.05).3. Bioinformatics analysis software microrna.org and Targetscan predicted results show ZNF217 containing the 3’-UTR region with miR-203 binding to a complementary sequence. Luciferase reporter gene assay showed that human embryonic kidney 293 T cells were co-transfected when miR-203 mimics stained with luciferase reporter vector containing the WT-ZNF2173’-UTR, cell fluorescence luciferase activity was significantly decreased, significantly lower than the control group (P<0.05);In the SW480 colorectal cancer cells were transfected miR-203 mimics,ZNF217 mRNA and protein levels were significantly decreased (P<0.05).4. Compared with the control group, the abilities of proliferation, migration and invasion in ZNF217-siRNA transfected alone and miR-203 mimics transfected alone colorectal cancer cells ability was decreased. Compared with ZNF217-siRNA transfected alone and miR-203 mimics transfected alone colorectal cancer cells co-transfected ZNF217-siRNA and miR-203 mimics colorectal cancer cells the abilities of proliferation, migration and invasion showed significantly reduced, with significant difference.Conclusion1. The low expression of miR-203 may be significantly enhanced the abilities of proliferation, migration and invasion of colorectal cancer cell lines, while the high expression of miR-203 can significantly inhibited the abilities of proliferation migration and invasion of colorectal cancer cell lines.These indicated that miR-203 /ZNF217 played an important role in the development of colorectal cancer;2. ZNF217 is miR-203 target genes in colorectal cancer and the expression of ZNF217 regulated by miR-203 obviously affected the biological behavior of colorectal cancer cells, participated development of colorectal cancer.