Long non-coding RNA KCNQ1OT1 up-regulates CTNND1 by sponging miR-329-3p to induce the proliferation, migration, invasion, and inhibit apoptosis of colorectal cancer cells

Abstract

Background: Long non-coding RNAs (lncRNAs) have been certified to be involved in the occurrence and growth of diverse cancers, including CRC. The purpose of the research was to explore the effects of lncRNA KCNQ1 overlapping transcript 1 (KCNQ1OT1) on proliferation, migration, invasion, and apoptosis in CRC cells and its mechanism.

Methods: The levels of KCNQ1OT1 and miR-329-3p were examined by quantitative real-time polymerase chain reaction (qRT-PCR) in CRC tissues and cells. The mRNA and protein levels of catenin delta-1 (CTNND1) were measured by qRT-PCR and western blot analysis, respectively. The targets of KCNQ1OT1 and miR-329-3p were predicted by online software and confirmed by luciferase reporter assay. The cell proliferation, migration, invasion, and apoptosis were examined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), transwell, and apoptosis assay. The expression levels of CyclinD1, Bcl-2, MMP9, Cleaved-casp-3, and E-cadherin in SW480 and LS1034 cells were gauged by western blot analysis. Xenograft tumor model was structured to prove the biological role of KCNQ1OT1 of CRC in vivo.

Results: The levels of KCNQ1OT1 and CTNND1 were significantly increased in CRC tissues and cells. Knockdown of KCNQ1OT1 suppressed proliferation, migration, invasion, and induced apoptosis in CRC cells. Conversely, CTNND1 overexpression reversed the impact of KCNQ1OT1 knockdown on CRC cells. Moreover, CTNND1 was verified as a direct target of miR-329-3p, and miR-329-3p could specially bind to KCNQ1OT1. Also, the down-regulation of KCNQ1OT1 triggered the CRC progress by up-regulating CTNND1 expression in CRC cells. Besides, KCNQ1OT1 knockdown inhibited CRC tumor growth through the miR-329-3p/CTNND1 axis in vivo.

Conclusion: Our results indicated that KCNQ1OT1 could positively regulate CTNND1 expression by sponging miR-329-3p, thereby boosting the progression of CRC. Our findings provided the underlying therapy targets for CRC.

Keywords: CTNND1; Colorectal cancer; lncRNA KCNQ1OT1; miR-329-3p.

Fig. 1

High expression of KCNQ1OT1 was a poor prognostic factor for CRC patients. a, b The expression level of KCNQ1OT1 in CRC tissues and cells was analyzed by qRT-PCR. c The correlation between KCNQ1OT1 expression and overall survival of CRC patients was analyzed with Kaplan–Meier method and log-rank test. *P < 0.05, vs. control group

Fig. 2

Knockdown of KCNQ1OT1 suppressed CRC cell proliferation, migration, invasion and promoted cell apoptosis. a The expression of KCNQ1OT1 was detected by qRT-PCR in SW480 and LS1034 cells by transfecting with specific siRNAs. b, c Proliferative ability of SW480 and LS1034 cells transfected with si-KCNQ1OT1#1 or si-KCNQ1OT1#2 was examined by MTT assay. d, e The cell cycle analysis of SW480 and LS1034 cells was detected by flow cytometry. f The cellular apoptosis of SW480 and LS1034 cells was tested by flow cytometry. g, h The migration and invasion of SW480 and LS1034 cells after transfected with si-KCNQ1OT1 were revealed by Transwell assays. i, j The protein levels of EMT markers (E-cadherin, MMP9), CyclinD1, apoptosis-associated proteins (Bcl-2, Cleaved-casp-3) were measured by western blot assays. *P < 0.05, vs. control group

Fig. 3

KCNQ1OT1 targeted miR-329-3p in CRC cells. a Predicted binding sites in KCNQ1OT1 and miR-329-3p were predicted by starBase v2.0. b, c Luciferase activity of SW480 and LS1034 cells was detected by dual-luciferase reporter assay. d The expression levels of miR-329-3p in CRC tissues and corresponding adjacent tissues were examined by qRT-PCR. e The expression level of miR-329-3p in CRC cells (LS1034, SW480) and normal cells (NCM460) was measured by qRT-PCR. f The correlation between miR-329-3p expression and overall survival of CRC patients was analyzed with Kaplan–Meier method. g The correlation between miR-329-3p and KCNQ1OT1 in human CRC samples was analyzed by Spearman’s correlation (P < 0.0001, r = − 0.657). h qRT-PCR was performed to measure miR-329-3p expression after transfected with KCNQ1OT1 or si-KCNQ1OT1 in SW480/LS1034 cells. *P < 0.05, vs. control group

Fig. 4

Silenced miR-329-3p expression reversed partial function of si-KCNQ1OT1 on proliferation, migration, invasion, and apoptosis. a The expression of miR-329-3p was detected by qRT-PCR after transfected with anti-miR-329-3p or anti-miR-329-3p-control (con) into SW480 and LS1034 cells. b, c The proliferative ability was measured after SW480 and LS1034 cells were transfected with si-KCNQ1OT1 or si-KCNQ1OT1 + anti-miR-329-3p. d Cell apoptosis was assessed by staining with annexin V and propidium iodide 48 h. the percentage of apoptotic cells was determined using flow cytometric analysis. e, f The cells migration and invasion were performed by MTT assay after transfected with si-KCNQ1OT1 or si-KCNQ1OT1 + anti-miR-329-3p. g, h The protein levels of EMT markers (E-cadherin, MMP9), CyclinD1, apoptosis-associated proteins (Bcl-2, Cleaved-casp-3) were measured in SW480 or LS1034 cells by western blot analysis. *P < 0.05, vs. control group

Fig. 5

CTNND1 was a direct target gene of miR-329-3p. a The predicted binding sites of miR-329-3p and CTNND1. b, c Thedual-luciferase reporter assay was utilized to determine the luciferase activity of miR-329-3p and CTNND1. d The expression level of miR-329-3p in 30 CRC tissues and corresponding adjacent tissues was confirmed by qRT–PCR. e qRT-PCR was applied to determine the expression level of miR-329-3p in SW480 and LS1034 cells and normal colon NCM460 cell. f Spearman’s correlation analysis examined the correlations between the expression levels of CTNND1 mRNA and miR-329-3p in CRC tissues (P < 0.0001, r = − 0.6814). g, h The expression level of CTNND1 was measured by qRT-PCR after being transfected with miR-329-3p or anti-miR-329-3p for 48 h in SW480 and LS1034 cells. *P < 0.05, vs. control group

Fig. 6

Overexpression of CTNND1 relieved the inhibitory effects of miR-329-3p on cell proliferation, migration, invasion and suppressed apoptosis. SW480 and LS1034 cells were transfected with control RNA or miR-329-3p together with plasmid expressing CTNND1 or empty vector. a The expression of CTNND1 in SW480 and LS1034 cells was tested by western blot. b, c SW480 and LS1034 cell proliferation were presented by MTT assay at 24 h, 48 h, and 72 h. d Flow cytometry was used to evaluate apoptosis of SW480 and LS1034 cells. e, f Cell migration and invasion in SW480 and LS1034 was detected by transwell assay. g, h The protein levels of EMT markers (E-cadherin, MMP9), CyclinD1, apoptosis-associated proteins (Bcl-2, Cleaved-casp-3) in SW480 and LS1034 cells were measured by western blot assays. *P < 0.05, vs. control group

Fig. 7

KCNQ1OT1 up-regulated CTNND1 expression via sponging miR-329-3p. a The correlation of KCNQ1OT1 and CTNND1 in CRC tissues was examined by spearman’s correlation analysis (P < 0.0001, r = − 0.6758). b, c SW480 and LS1034 cells were co-transfected with si-con/si-KCNQ1OT1 and si-KCNQ1OT1 + anti-con/si-KCNQ1OT1 + anti-miR-329-3p for 24 h, and then western blot was performed to check the level of CTNND1. d, e MTT assay was used to determine the viability of SW480 and LS1034 cells transfected with si-KCNQ1OT1 or si-KCNQ1OT1 + pcDNA-CTNND1. f And the cell apoptosis rates were measured by flow cytometry in SW480 and LS1034 cells transfected with si-KCNQ1OT1 or si-KCNQ1OT1 + pcDNA-CTNND1. g, h Transwell assay was conducted to evaluate the migratory and invasive ability of SW480 and LS1034 cells transfected with si-KCNQ1OT1 or si-KCNQ1OT1 + pcDNA-CTNND1. i, j The expression of EMT markers (E-cadherin, MMP9), CyclinD1, apoptosis-associated proteins (Bcl-2, Cleaved-casp-3) were evaluated by western blot in SW480 and LS1034 cells transfected with si-KCNQ1OT1 or si-KCNQ1OT1 + pcDNA-CTNND1. *P < 0.05, vs. control group

Fig. 8

KCNQ1OT1knockdown suppressed CRC tumor growth by regulating of miR-329-3p/CTNND1axis in vivo. a, b Tumor volume and tumor weight were measured in xenografts. c The expression levels of KCNQ1OT1,miR-329-3p and CTNND1 in xenografts were assessed by RT-qPCR. d CTNND1 protein level was detected in xenografts. The protein levels of CyclinD1, Bcl-2, and Cleaved-casp-3 were measured in xenografts. e The expression levels of CyclinD1, apoptosis-associated proteins (Bcl-2, Cleaved-casp-3) and the control (GAPDH) were evaluated by western blot in xenografts transfected with sh-KCNQ1OT1 or sh-control. *P < 0.05, vs. control group

Fig. 9

Schematic diagram showing the effect of the KCNQ1OT1/miR-329-3p/CTNND1 on proliferation, migration, invasion, and apoptosis in CRC cells