| Objective: To study the expression of lnc RNA FEZF1-AS1 in colon cancer tissues and cells its effect on the proliferation,colony formation,migration and invasion of colon cancer cells.Methods:1.RT-qPCR was performed to detect the expression of FEZF1-AS1 in colon cancer tissues and nontumor tissues from patients.The correlation between the expression FEZF1-AS1 and clinical pathological parameters of patients with colon cancer was analyzed.2.RT-qPCR was performed to detect the relative expression of FEZF1-AS1 in the five colon cancer cell lines and NCM460 cell line was used as control.3.MTS,colony formation,wound-healing,transwell and Matrigel assays were performed to detect the proliferation,colony formation,migration and invasion abilities of colon cancer cells with FEZF1-AS1 low-expression.Results:1.RT-qPCR assay showed that the transcript level of FEZF1-AS1 was significantly increased in colon cancer tissues compared with that in the corres-ponding nontumor colon cancer tissues.The increased expression of FEZF1-AS1 was significantly correlated with T stage and TNM stage of tumor.2.Compared with those in NCM460 cells,the expression of FEZF1-AS1 in SW480,HCT116,DLD-1 and HT29 cells were significantly higher.There was no significant difference was found in the expression of FEZF1-AS1 in RKO cell line.3.Compared with the NC group,the proliferation capacity of SW480 and HCT116 cells were significantly inhibited by FEZF1-AS1 si RNA transfection.4.Compared with the NC group,the colony formation activity of SW480 and HCT116 cells were significantly inhibited by FEZF1-AS1 si RNA transfection.5.Compared with the NC group,the migration capability of SW480 and HCT116 cells were significantly inhibited by FEZF1-AS1 si RNA transfection.6.Compared with the NC group,the invasion and migration capability of SW480 and HCT116 cells were significantly inhibited by FEZF1-AS1 si RNA transfection.Objective: To study whether FEZF1-AS1 may regulate the occurrence and development of EMT by modulating OTX1.Methods:1.RT-qPCR and Western-blot assays were performed to detect the expression of OTX1 protein in colon cancer cells with FEZF1-AS1 low-expression.Western-blot assays were performed to detect the expression of EMT related proteins E-Cadherin?N-Cadherin and Vimentin in colon cancer cells with FEZF1-AS1 low-expression.2.Western-blot assays were performed to detect the expression of EMT related proteins E-Cadherin,N-Cadherin and Vimentin in cells with FEZF1-AS1 knockdown and OTX1 over-expression.3.An immunohistochemical assay was performed to detect the expression of OTX1 in colon cancer tissues and their normal counterparts,the correl-ation between the expression of FEZF1-AS1 and OTX1 was analyzed.Results:1.Compared with control,the expression of OTX1 at protein level,but not at at m RNA level,was inhibited by FEZF1-AS1 knockdown in colon cancer cells.The EMT markers of vimentin and N-cadherin were downregulated,whereas epithelial markers E-cadherin was upregulated by FEZF1-AS1 knockdown.2.The inhibited expression of vimentin and N-cadherin and the promoted E-cadherin expression caused by FEZF1-AS1 knockdown were partially rescued by the over-expression of OTX1.3.OTX1 expression in colon cancer tissues was significantly higher than those in the corresponding nontumor colon cancer tissues,and OTX1 was positively correlated with FEZF1-AS1 expression.Objective: To study whether FEZF1-AS1 may achieve its effects on colon cancer by competitive binding to miR-30a-5p.Methods:1.The bioinformatics tool Starbase was used to predict miRNAs that could be bound to FEZF1-AS1,RT-qPCR was performed to detect the expression of four miRNAs in cells with FEZF1-AS1 knocking down.miR-30a-5p was selected,and the expression of FEZF1-AS1 was detected with miR-30a-5p knock-down or over-expression.The binding sites of FEZF1-AS1 and miR-30a-5p were verified by dual-luciferase reporter assay.2.The proliferation and migration abilities of SW480 and HCT116 cells with miR-30a-5p over-expression were analyzed by MTS and transwell assays.3.The expression of miR-30a-5p in colon cancer tissues and nontumor tissues from patients was detected by RT-qPCR.The correlation between the expression level of FEZF1-AS1 and FEZF1-AS1 was analyzed.4.Putative cotargets genes of miR-30a-5p were searched by Three algorithms,Target Scan,Target Miner and miRDB.The binding sites of miR-30a-5p and NT5 E were verified by dual-luciferase reporter assay.5.The protein expression of NT5 E was checked in colon cancer cells with miR-30a-5p knockdown or over-expression by Western-blot.6.The proliferation and migration abilities of SW480 and HCT116 cells with dual-knockdown of FEZF1-AS1 and miR-30a-5p were detected by MTS and transwell assays.7.The expressions of NT5 E in SW480 and HCT116 cells with dualknockdown of FEZF1-AS1 and miR-30a-5p were checked by Western-blot.8.The expression of NT5 E in colon cancer tissues and their normal counterparts was checked by immunohistochemical staining,the correlation between the expression of NT5 E,miR-30a-5p and OTX1 were analyzed.Results:1.There were four miRNAs: miR-610,miR-4443,miR-107,and miR-30a-5p were found by Starbase and were detected by RT-qPCR.Only miR-30a-5p was found increased significantly by FEZF1-AS1 knocking down.The expression of FEZF1-AS1 was suppressed by miR-30a-5p overexpression,and was increased by miR-30a-5p inhibition.A dual-luciferase reporter assay indicated that FEZF1-AS1 fragment B might be a target binding site of miR-30a-5p.2.The proliferation and migration of HCT116 and SW480 cells were significantly inhibited by miR-30a-5p overexpression.3.miR-30a-5p expression was significantly downregulated in colon cancer tissues compared with that in the corresponding normal colon cancer tissues,the negative correlation between FEZF1-AS1 and miR-30a-5p was found in colon cancer tissues.4.NT5 E was found to be a target of miR-30a-5p.A dual-luciferase reporter assay showed that the miR-30a-5p bind to the 3'UTR region of NT5 E.5.The expression of NT5 E was downregulated by miR-30a-5p mimics transfection,while was upregulated by miR-30a-5p inhibiton.6.The inhibition of cell proliferation and migration in SW480 and HCT116 cells caused by FEZF1-AS1 knockdown was partially rescued by cotransfected with miR-30a-5p inhibitor and si-FEZF1-AS1.7.NT5 E protein expression was inhibited by FEZF1-AS1 knockdown and was rescued by the inhibition of miR-30a-5p in SW480 and HCT116 cells.8.The NT5 E expressions in colon cancer tissues were higher than those in the corresponding nontumor colon cancer tissues.There was a positive correlation between NT5 E and FEZF1-AS1,whereas there was a negative correlation between NT5 E and miR-30a-5p.Objective: To investigate the effect of FEZF1-AS1 on the proliferation of colon cancer cells in nude mice.Methods:1.HCT116 cells were stably transfected with lentiviral contained with sh-FEZF1-AS1-HCT116.And subcutaneously transplanted tumor model in nude mice was established.2.The growth of transplanted tumor was observed and the volume and weight of transplanted tumor were measured3.The expression of FEZF1-AS1,OTX1 and NT5 E in the transplanted tumor tissue were checked by RT-qPCR and immunohistochemical staining.Results:1.The colon cancer cell line HCT116 with stable knockdown of FEZF1-AS1 was successfully constructed.2.Compared with the NC group,the weights and sizes of tumors from the sh-FEZF1-AS1-HCT116 group were significantly smaller.3.The expression of FEZF1-AS1,OTX1 and NT5 E expression were all decreased in the transplanted tumor tissue of nude mice.Conclusion: The expression of FEZF1-AS1 was significantly upregulated in colon cancer tissues,and the expression of FEZF1-AS1 was significantly associated with T stage and TNM stage.The proliferation,colony fomation,migration and invasion capacities were inhibited in colon cancer cells with FEZF1-AS1 si RNA transfection.FEZF1-AS1 may affect EMT-related protein expression by regulatig OTX1.FEZF1-AS1 may also promote colon cancer by competitively combine with miR-30a-5p.Downregulation of FEZF1-AS1 might be promising for further tumor targeting therapy. |
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