KCNQ1OT1/miR-217/ZEB1 feedback loop facilitates cell migration and epithelial-mesenchymal transition in colorectal cancer

Abstract

Long noncoding RNAs are widely acknowledged as a group of regulatory factors in various diseases, especially in cancers. KCNQ1 overlapping transcript 1 (KCNQ1OT1) has been reported as oncogene in human cancers. However, the role of KCNQ1OT1 in colorectal cancer (CRC) has not been fully explained. Based on the database analysis, KCNQ1OT1 was highly expressed in CRC samples and predicted the poor prognosis for CRC patients. Functional experiments revealed that KCNQ1OT1 knockdown negatively affected the proliferation, migration and epithelial-mesenchymal transition (EMT) in CRC cells. Moreover, we identified the cytoplasmic localization of KCNQ1OT1 in CRC cells, indicating the post-transcriptional regulation of KCNQ1OT1 on gene expression. Mechanism experiments including RNA Immunoprecipitation (RIP) assay and dual luciferase reporter assays verified that KCNQ1OT1 acted as a competing endogenous RNA (ceRNA) in CRC by sponging microRNA-217 (miR-217) to up-regulate the expression of zinc finger E-box binding homeobox 1 (ZEB1). Further mechanism investigation revealed that ZEB1 enhanced the transcription activity of KCNQ1OT1 by acting as a transcription activator. Finally, rescue assays were designed to demonstrate the effect of KCNQ1OT1-miR-217-ZEB1 feedback loop on proliferation, migration, and EMT of CRC cells. In brief, our research findings revealed that ZEB1-induced upregulation of KCNQ1OT1 improved the proliferation, migration and EMT formation of CRC cells via regulation of miR-217/ZEB1 axis.

Keywords: KCNQ1OT1; ZEB1; colorectal cancer; miR-217; migration.

Figure 1.

High expression of KCNQ1OT1 was a poor prognostic factor for CRC patients. (a) Based on TCGA analysis, KCNQ1OT1 was upregulated in CRC samples than that in normal samples. (b) According to the data of TCGA database, the correlation between KCNQ1OT1 expression and overall survival of CRC patients was analyzed with Kaplan Meier method. (c) qRT-PCR determined the expression level of KCNQ1OT1 in CRC cells (HT-29, HCT116, SW480, DLD1) and normal cells (FHC, NCM460). *P < 0.05, **P < 0.01 vs. control group.

Figure 2.

Knockdown of KCNQ1OT1 suppressed CRC cell proliferation. (a) The expression of KCNQ1OT1 was silenced in DLD1 and SW480 cells by transfecting with specific shRNAs. The optimal transfection efficiency was obtained after 48 h. (b,c) MTT assay disclosed that the proliferative ability of DLD1 and SW480 cells which were transfected with KCNQ1OT1-specific shRNAs was reduced. **P < 0.01 vs. control group.

Figure 3.

Silencing of KCNQ1OT1 negatively regulated the migration and EMT progress of CRC cells. (a) Transwell assays revealed that the migration of CRC cells after KCNQ1OT1 was silenced. (b,c) The mRNA and protein levels of EMT markers (E-cadherin, β-catenin, N-cadherin, Vimentin) were measured by qRT-PCR and western blot assays. (d) Immunofluorescence assays were used to detect the expressions of E-cadherin and N-cadherin. **P < 0.01 vs. control group.

Figure 4.

ZEB1 was upregulated by KCNQ1OT1 in CRC cells. (a) The levels of ZEB1 in CRC cells and normal cells were measured by qRT-PCR. (b) The correlation between ZEB1 and KCNQ1OT1 was analyzed. (c,d) The mRNA and protein levels of ZEB1 in response to the knockdown of KCNQ1OT1 were measured by qRT-PCR and western blot. (e) Luciferase reporter assays were utilized to determine the effect of KCNQ1OT1 on the promoter activity of ZEB1. N.S: no significant, **P < 0.01 vs. control group.

Figure 5.

KCNQ1OT1 positively modulated the expression of ZEB1 through sponging miR-217. (a) Nuclear separation experiment was used to determine the location of KCNQ1OT1 in cytoplasm and nucleus. (b) Seven miRNAs bound with ZEB1 were found through searching from bioinformatics websites (PITASites and miRandaSites). (c,d) MS2-RIP assay was performed to validate the interaction between these seven miRNAs and KCNQ1OT1. (e) Ago2-RIP assay further validated the interaction between KCNQ1OT1 and miR-217. (f) Pull down assay was further applied to demonstrate the interaction between KCNQ1OT1 and miR-217. (g) The binding sites harboring the miR-217 for KCNQ1OT1 were obtained from online bioinformatics analysis. (h) Luciferase activity reporter assays were applied to determine the interaction between KCNQ1OT1 and miR-217. N.S: no significance, **P < 0.01 vs. control group.

Figure 6.

ZEB1 is a target of miR-217 in CRC cells. (a) Pull-down assay was used to validate whether ZEB1 can be efficiently pulled down by biotin-labeled miR-217. (b) The binding sites between miR-217 and ZEB1 were predicted and obtained using bioinformatics analysis. (c) Dual luciferase reporter assay was applied to demonstrate the binding of KCNQ1OT1 to miR-217. (d) ZEB1-WT vector and empty vector were inserted into HEK-293T cell by co-transfecting with miR-NC, miR-217 mimics or miR-217 mimics + pcDNA-KCNQ1OT1. (e,f) qRT-PCR and western blot assays were performed to test the levels of EMT markers in DLD1 and SW480 cells which were transfected with miR-217 mimics or co-transfected with miR-217 mimics and pcDNA-KCNQ1OT1. N.S: no significance, *P < 0.05, **P < 0.01 vs. control group.

Figure 7.

ZEB1 transcriptionally activated KCNQ1OT1 in CRC cells. (a) The binding sequences of ZEB1 to the promoter region of KCNQ1OT1 were predicted from JASPAR. (b) Luciferase reporter vectors containing serial truncations of KCNQ1OT1 promoter were constructed and transfected into HEK-293T cells. The luciferase activity of these plasmids was measured. (c) ChIP assay was performed to demonstrate the direct affinity of ZEB1 to the promoter region of KCNQ1OT1 in two CRC cells. (d) ZEB1 was efficiently overexpressed or silenced in DLD1 and SW480 cells by transfecting with sh-ZEB1 and ZEB1 expression vector. (e,f) The expression level of KCNQ1OT1 in DLD1 and SW480 cells was examined in response to the knockdown or overexpression of ZEB1. *P < 0.05, **P < 0.01 vs. control group.

Figure 8.

KCNQ1OT1-miR-217-ZEB1 feedback loop regulated the proliferation, migration and EMT formation of CRC cells. (a,b) MTT assay and colony formation assay were carried out to detect the proliferation ability of DLD1/sh-KCNQ1OT1 cells after transfection with miR-217 inhibitors or co-transfection with miR-217 inhibitors + sh-ZEB1. (c) Transwell assay revealed the metastatic ability of DLD1/sh-KCNQ1OT1 cells after transfection with miR-217 inhibitors or co-transfection with miR-217 inhibitors + sh-ZEB1. (d) The levels of EMT markers were examined in indicated DLD1 cells after transfections. **P < 0.01 vs. control group.