The Effect Of MiR-494-3p/ZEB2 Regulating EMT Signaling Pathway On Invasion And Metastasis Of Colorectal Cancer

Background Colorectal cancer（CRC）is one of the most common malignancies.CRC has been the second leading cause of cancer death,with about the death of 860,000patients worldwide each year.CRC patients have no obvious signs at the early stage,which leads to low diagnostic rate,and most patients are diagnosed in the middle and late stage,losing optimal opportunity for treatment.After systematic treatment,about50%of CRC patients would eventually metastasize,leading to treatment failure and low survival rate.Therefore,the research on the mechanism of CRC invasion and metastasis could achieve early warning and targeted treatment,improve the diagnosis rate and survival rate of patients,and provide new targets and strategies for the diagnosis and treatment.With the development of research,more and more attention has been paid to the role of circulating tumor cell（CTC）in metastasis and recurrence of cancer.CTC would be studied in cell and molecular biology to comprehensively understand the mechanism of invasion,metastasis and recurrence of malignant tumors,and to help us understand the causes of metastasis and recurrence of malignant tumors.Epithelial-mesenchymal transition（EMT）is an important pathophysiological process and closely related to invasion and metastasis of cancer.Previous studies about CTC of our research group found that the number of CTC was related to tumor disease progression.Moreover,CTC had the EMT status（CTC^M）,and the detection rate of CTC^M in patients with metastasis was higher than that in patients without metastasis.EMT is regulated by multi-gene network.Recently,it has been found that micro RNA（miRNA）,as a class of non-coding RNA,could regulate target genes after transcription,which plays a key role in the regulation of EMT and greatly affects the ability of tumor invasion and metastasis.miRNA could induce the degradation or inhibit the translation of m RNA by binding to the 3’-untranslated regions（3’UTR）of the target gene,thereby reducing the protein level of the target gene.Zinc finger E-box-binding homeobox 2（ZEB2）,one of the core transcription factors regulating EMT,plays an important role in the occurrence of EMT.Bioinformatics tools were employed to analyze the core transcription factors of EMT.We found that there was a potential binding site of miR-494-3p in ZEB2 m RNA 3’UTR,suggesting that miR-494-3p might affect the expression of ZEB2.Consequently,we speculate that miR-494-3p may regulate EMT through specific regulation of ZEB2 to enhance the invasion and metastasis ability of tumor cells.According to the above theory and previous research foundation,we would systematically study and analyze the role of miR-494-3p on the invasion and metastasis of CRC and its regulation ZEB2/EMT signaling pathways,through clinical samples,stable transfection cell lines and animal models.We would explore its influence on the occurrence and development of CRC in vitro and in vivo,so as to provide new clues for the early diagnosis and treatment of CRC.Part Ⅰ The expression of miR-494-3p and ZEB2 in colorectal cancer tissues and their correlationObjective To verify the expression of miR-494-3p and ZEB2 in CRC tissues and their correlation.Methods We collected 60 pairs samples of CRC invasive front and matched tumor center tissues from CRC patients in Zhongnan Hospital of Wuhan University from January 2016 to June 2017.Immunohistochemistry,Western blot and q RT-PCR were used to evaluate the expression levels of miR-494-3p and ZEB2 in CRC tissue of invasive front and tumor center.The expression differences and their correlation were analyzed.Meanwhile,the relationship between miR-494-3p and ZEB2 expression level in the CRC invasive front and clinicopathological parameters,CTC/CTM and CTC^Mwere analyzed.Results The expression level of miR-494-3p in the invasive front was significantly lower than that in the tumor center.Moreover,the expression of ZEB2 in the invasive front of CRC tissue was significantly higher than that in the tumor center.Pearson correlation analysis revealed a significantly negative correlation between the expression level of miR-494-3p and ZEB2 in the invasive front tissue（R²=0.444,P<0.001）.Then we analyzed the relationship between miR-494-3p and ZEB2 expression level in the CRC invasive front and clinicopathological parameters,showing that the low expression of miR-494-3p was significantly related to bigger tumor size（P=0.020）,poor differentiation（P=0.004）,lymph node metastasis（P<0.001）,and advanced TNM stage（P=0.008）,and the high expression of ZEB2 was significantly related to deeper invasion depth（P=0.015）,lymph node metastasis（P=0.003）,and advanced TNM stage（P=0.001）.Further analysis revealed that the low expression of miR-494-3p and the high expression of ZEB2 in the CRC invasive front was correlated with CTC/CTM+.CTC^M ratio was negatively related to the expression of miR-494-3p and positively related to the expression of ZEB2.Conclusions The expression level of miR-494-3p was down-regulated in the CRC invasive front tissue,which was negatively correlated with the expression level of ZEB2.They may have a regulatory relationship,affecting the invasion and metastasis of CRC.Part Ⅱ The effect of miR-494-3p on the biological behavior of colon cancer cellObjective To investigate the effect of abnormal expression of miR-494-3p in vitro and in vivo on the biological behavior of colon cancer cell.Methods According to the expression level of miR-494-3p in colon cancer cell,appropriate cell was selected to transfect plasmids and construct stable transfected cell line,investigating the effect of miR-494-3p on the biological function of colon cancer cell.CCK-8 and clonal formation assay were used to detect the effect of miR-494-3p on the proliferation of colon cancer cell.The effect of miR-494-3p on the migration and invasion ability of colon cancer cell was detected by Transwell assay and wound-healing assay.The effect of miR-494-3p on the tumorigenicity and invasion ability of colon cancer cell in vivo was detected by subcutaneous tumorigenesis in nude mice.The effect of miR-494-3p on the ability of colon cancer cell to metastasize in vivo was detected by tail vein in nude mice.Results High expression of miR-494-3p of HCT116 was selected to transfect the plasmid of miR-494-3p inhibitor and NC.Low expression of miR-494-3p of SW480was selected to transfect the plasmid of miR-494-3p and NC.The transfection efficiency of stable transfected cell lines was detected,and the results showed that compared with the control group,the expression level of miR-494-3p was significantly down-regulated in the miR-494-3p inhibitor group,while the expression level of miR-494-3p was significantly up-regulated in the miR-494-3p group.CCK-8 results showed that at 48h and 72h,OD value of HCT116 in miR-494-3p inhibitor group was significantly higher than that in control group.The OD value of SW480 in the miR-494-3p group was significantly lower than that in the control group.The results of clony formation assay showed that the clone formation of HCT116 in miR-494-3p inhibitor group was significantly higher than that in control group.However,the clone formation of SW480 in the miR-494-3p group was significantly less than that in the control group.The results of wound-healing assay showed that compared with the control group,the move ability of HCT116 in miR-494-3p inhibitor group was significantly enhanced after 48h.However,the move ability of SW480 in the miR-494-3p group was significantly inhibited.Transwell assay results showed that compared with the control group,the migration and invasion cells of HCT116 in miR-494-3p inhibitor group was significantly increased.The migration and invasion cells of SW480 in the miR-494-3p group were significantly decreased.The results of subcutaneous tumorigenesis in nude mice showed that,compared with the control group,tumor volume and weight of miR-494-3p inhibitor group were significantly increased from day 20.Further analysis showed that the number of CTC detected by miR-494-3p inhibitor group was significantly increased.The results of tumor formation in the tail vein of nude mice showed that the weight of the miR-494-3p inhibitor group was significantly lower than that of the control group after 25 days.Further analysis showed that liver metastasis rate of miR-494-3p inhibitor group（4/6,66.67%）was increased compared with control group（2/6,33.33%）,and lung metastasis rate of miR-494-3p inhibitor group（3/6,50.00%）was also increased compared with control group（1/6,16.67%）.Conclusion:In vitro,miR-494-3p inhibited could enhance the ability of proliferation,migration and invasion,and overexpression of miR-494-3p could inhibit the ability of proliferation,migration and invasion in colon cancer cell.In nude mice,miR-494-3p inhibited could enhance tumor growth,promote CTC formation,and increase the probability of liver and lung metastasis.Part Ⅲ The mechanism of miR-494-3p targeting ZEB2 to regulate the EMT signaling pathway of colon cancer cellObjective MiR-494-3p could target the 3’UTR of ZEB2 m RNA,inhibit the expression of ZEB2 in colon cancer cell and regulate the EMT signaling pathway.Methods Bioinformatics tool was used to predict whether miR-494-3p could target ZEB2.The luciferase activity of the transfected cell was determined by dual-luciferase reporter assay,and the binding of miR-494-3p to the 3’UTR of ZEB2 m RNA was determined according to the test results.The stable transfected cell lines constructed in the second part were used to detect the expression levels of ZEB2 and EMT related markers in the transfected cell.Immunohistochemistry,Western blot and q RT-PCR were used to detect the expression levels of ZEB2 and EMT related markers in subcutaneous tumor tissues of nude mice.Si RNA was used to interfere with ZEB2expression in stable transfected cell lines to detect the ability of proliferation,migration and invasion in colon cancer cell,as well as the expression level of EMT related markers.Results Bioinformatics tool predicted that the 3’UTR of ZEB2 m RNA had 8 base pairs matching between the 5001-5008 site sequence and miR-494-3p sequence.According to the site of match,we designed the wild type plasmid（pmir GLO-ZEB2-WT）and mutation plasmid（pmir GLO-ZEB2-MUT）,and transfected them with hsa-miR-494-3p mimics and NC into HCT116 and SW480 cells.The results showed that compared with the control group,hsa-miR-494-3p mimics could inhibit luciferase activity in the wild-type plasmid group,but luciferase activity had no statistical difference in the mutant plasmid group.The results of Actinomycin D assay showed that,with the passage of time,the expression level of ZEB2 m RNA in the miR-494-3p group was significantly down-regulated compared with the control group,and the half-life of ZEB2 m RNA in the miR-494-3p group(T_1/2=2.122h)was significantly shortened compared with the control group(T_1/2=4.835h).Expression of ZEB2 and EMT related markers in stable transfected cell line HCT116 were detected,showed that,compared with control group,the expression level of ZEB2 m RNA in miR-494-3p inhibitor group was significantly up-regulated.Western blot results showed that compared with the control group,the expression level of ZEB2 and Vimentin protein was significantly up-regulated and the expression level of E-cadherin protein was significantly down-regulated,while the expression level of ZEB1 protein was not significantly changed in miR-494-3p inhibitor group.The results were reversed in stable transfected cell line SW480.Results of subcutaneous tumor tissue test in nude mice showed that compared with the control group,the expression level of ZEB2,Ki-67 and Vimentin was significantly up-regulated,and E-cadherin expression was significantly down-regulated to induce EMT in miR-494-3p inhibitor group.Si-ZEB2 was transfected into stable transfected HCT116,showed that compared with the control group,the OD value of HCT116 cell was significantly reduced,the clone formation was significantly reduced,and the number of cells migrated and invaded was significantly reduced after ZEB2 silence.Meanwhile,ZEB2 expression was significantly down-regulated,E-cadherin expression was significantly up-regulated,and Vimentin expression was significantly down-regulated after ZEB2 silence.Conclusion:MiR-494-3p could target the 3’UTR of ZEB2 m RNA,affect the stability of ZEB2 m RNA,and inhibit the expression of ZEB2 in colon cancer cell to regulate the EMT signaling pathway.