lncRNA KCNQ1OT1 enhances the chemoresistance of oxaliplatin in colon cancer by targeting the miR-34a/ATG4B pathway

Abstract

Purpose: The chemoresistance of colon cancer to oxaliplatin (L-OHP) indicates poor prognosis. Long non-coding RNA (lncRNA) KCNQ1OT1 (KCNQ1 opposite strand/antisense transcript 1) has been shown to participate in the tumorigenesis of several types of cancers. However, little is known about the role of KCNQ1OT1 in the chemoresistance and prognosis of colon cancer.

Materials and methods: Quantitative-PCR and Western blot were used to measure the expression profiles of KCNQ1OT1, miR-34a, and Atg4B in colon cancer tissues and cells. Cell viability assay and flow cytometry were used to examine their effects on cell proliferation and death. Cleavage of LC3 and GFP-LC3 plasmid transfection were used to detect autophagic activity. Double luciferase reporter assay was used to verify the interactions between miRNA and lncRNA or mRNA. Xenograft tumor model was used to verify the effects of KCNQ1OT1 in vivo.

Results: In this study, it is shown that the expression level of KCNQ1OT1 was increased in tumor, which indicated poor prognosis in colon cancer patients. Using colon cancer cell lines HCT116 and SW480, it was demonstrated that knockdown of KCNQ1OT1 decreased the cell viability and increased the apoptosis rates upon L-OHP treatment. Further studies indicated that Atg4B upregulation was partially responsible for KCNQ1OT1-induced protective autophagy and chemoresistance. Moreover, miR-34a functioned as a bridge between KCNQ1OT1 and Atg4B, which could be sponged by KCNQ1OT1, while it could also bind to the 3'-UTR of Atg4B and downregulate its expressions. Finally, we show that the KCNQ1OT1/miR-34a/Atg4B axis regulated the chemoresistance of colon cancer cells in vitro and in vivo.

Conclusion: lncRNA KCNQ1OT1 promoted the chemoresistance of colon cancer by sponging miR-34a, thus upregulating the expressions of Atg4B and enhancing protective autophagy. KCNQ1OT1 might become a promising target for colon cancer therapeutics.

Keywords: Atg4B; KCNQ1OT1; chemoresistance; colorectal cancer; miR-34a; protective autophagy.

Figure 1

The expression profiles of lncRNA KCNQ1OT1 in colon cancer patients and the correlation with prognosis. Notes: (A) The expression levels of KCNQ1OT1 in colon cancer tissues and corresponding adjacent tissues were detected by qRT-PCR. **P<0.01. (B) The expression levels of KCNQ1OT1 in the tumor tissues of stage I/II and stage III/IV patients. ***P<0.0001. (C) The fold changes of KCNQ1OT1 expression level in each patient were measured. (D) Kaplan–Meier curves of overall survival and log-rank test of patients with low and high KCNQ1OT1 expression levels. *P<0.05. (E) HCT116 and SW480 cells were transfected with KCNQ1OT1 siRNA and negative control, and then the cell viability assay was conducted with different concentrations of L-OHP. *P<0.05. (F and G) The apoptosis rates of colon cancer cells were measured by flow cytometry. *P<0.05 and ***P<0.0001. Data are presented as mean ± SEM. Abbreviations: L-OHP, oxaliplatin; lncRNA, long non-coding RNA; PI, propidium iodide; qRT-PCR, quantitative real-time PCR; ns, not significant.

Figure 2

The effects of KCNQ1OT1 on L-OHP-induced protective autophagy and Atg4B expression levels in colon cancer cells. Notes: (A) HCT116 and SW480 cells were treated with L-OHP with the absence and presence of autophagy inhibitor 3-MA or CQ, and then the expression levels of PARP and LC3 were detected by Western blot, with GAPDH as a loading control. *P<0.05, **P<0.01, and ***P<0.0001. (B) HCT116 and SW480 cells were transfected with siKCNQ1OT1 and treated with 10 μmol/L L-OHP for 24 h, and then Western blot was performed. *P<0.05, **P<0.01, and ***P<0.0001. (C) HCT116 and SW480 cells were treated with GFP-LC3 plasmid and then 10 μmol/L L-OHP was added. After treatment, cells were observed in the fluorescence microscope. (D) HCT116 and SW480 cells were co-transfected with siNC/siKCNQ1OT1 and pcDNA/pcDNA-Atg4B plasmids, and then cell viability assay was performed as above. *P<0.01 and **P<0.01. (E) HCT116 and SW480 cells were treated as shown in (D), and then the apoptosis rates were measured by flow cytometry. *P<0.05 and **P<0.01. Abbreviations: CQ, chloroquine; L-OHP, oxaliplatin; 3-MA, 3-methyladenine; ns, not significant.

Figure 3

The mechanisms of miR-34a functioning as the connection between KCNQ1OT1 and Atg4B. Notes: (A) Predicted binding site of miR-34a on the 3′-UTR of Atg4B mRNA base in the Targetscan database. (B) Western blot analysis was conducted to detect the expression levels of Atg4B, transfected with miR-34a mimic and NC. N=3, *P<0.05 and ***P<0.0001. (C) Double luciferase reporter assay results of the interaction between miR-34a and the 3′-UTR of Atg4B mRNA. N=3, *P<0.05. (D) Predicted miR-34a binding sites in KCNQ1OT1 sequence based on the DIANA-LncBase analysis. (E) The expression level of miR-34a was measured by qRT-PCR after being transfected with KCNQ1OT1 siRNA for 48 h. N=3, *P<0.05 and **P<0.01. (F) The double luciferase reporter assay experiments investigating the binding affinity of miR-34a to KCNQ1OT1. *P<0.05 and **P<0.01. (G) Cell viability assay of miR-34a mimic or inhibitor on colon cancer cells. N=3, *P<0.05. (H) HCT116 and SW480 cells were treated as shown in (G), and then apoptosis rates were measured using flow cytometry. N=3, *P<0.05 and **P<0.01. Abbreviations: L-OHP, oxaliplatin; qRT-PCR, quantitative real-time PCR; ns, not significant.

Figure 4

The effects of KCNQ1OT1/miR-34a/Atg4B axis in regulating the sensitivity of colon cancer cells to L-OHP in vitro and in vivo. Notes: (A and B) HCT116 and SW480 cells were cotransfected with siNC/siKCNQ1OT1 and miR-34a NC/miR-34a inhibitors for 24 h, and then L-OHP was added and Western blot performed. N=3, *P<0.05, **P<0.01, and ***P<0.0001. (C and D) Colon cancer cells were treated as shown in (A), and then the cell viability and apoptosis rates were measured by CCK-8 and flow cytometry. *P<0.05. (E) HCT116 and SW480 cells were pre-transfected with KCNQ1OT1 siRNA or siNC, and then the cells were injected subcutaneously to build the xenograft tumor model. **P<0.01 and ***P<0.0001. (F) The expression levels of miR-34a were measured by qRT-PCR in xenograft tumor. *P<0.05 and **P<0.01. (G and H) The expression levels of PARP, Atg4B, and LC3 were detected by Western blot in the xenograft tumor tissues, and the bands were analyzed. **P<0.01 and ***P<0.0001. Abbreviations: CCK-8, Cell Counting Kit-8; L-OHP, oxaliplatin; qRT-PCR, quantitative real-time PCR.