Long non-coding RNA KCNQ1OT1 promotes the progression of gastric cancer via the miR-145-5p/ARF6 axis

Abstract

Background: Long non-coding RNA KCNQ1 opposite strand/antisense transcript one gene (KCNQ1OT1) has been reported to be involved in the progression of many types of human cancer, whereas its role in gastric cancer (GC) remains unknown. The present study aimed to investigate the role of KCNQ1OT1 in GC.

Methods: In total, 25 GC tissues and adjacent normal tissues were collected. The expression of KCNQ1OT1, miR-145-5p and ARF6 in GC tissues and cell lines was detected by quantitative reverse transcriptase-polymerase chain reaction or western blotting. Bioinformatics analysis and a dual luciferase reporter assay were performed to determine the relationship between KCNQ1OT1 and miR-145-5p or miR-145-5p and ARF6. Gain- and loss-of function of KCNQ1OT1 and miR-145-5p were achieved to confirm their roles in GC cells. Cell counting kit-8, colony formation and flow cytometry assays were used to evaluate cell viability, proliferation and apoptosis. A xenograft tumor model was established with BGC803 tumor cells transfected with sh-KCNQ1OT1 or empty vector to determine the role of LINC01089 in vivo.

Results: The expression levels of KCNQ1OT1 were markedly elevated in GC tissues and cells. Knockdown of KCNQ1OT1 inhibited GC tumor growth, reduced GC cell viability and colony formation, and induced GC cell apoptosis. The expression levels of miR-145-5p were significantly decreased in GC cells and correlated with the expression of KCNQ1OT1 in GC tumors. Moreover, KCNQ1OT1 directly binds with miR-145-5p, which is targeting ARF6. Knockdown of KCNQ1OT1 increased the expression levels of miR-145-5p. Inhibition of miR-145-5p increased the expression levels of KCNQ1OT1 and also attenuated the effects of knockdown of KCNQ1OT1 on the viability, proliferation and apoptosis of GC cells. In addition, overexpression of miR-145-5p reduced GC cell viability and colony formation and induced GC cell apoptosis, whereas overexpression of ARF6 attenuated the effects of overexpression of miR-145-5p on GC cell viability, colony formation and apoptosis.

Conclusions: KCNQ1OT1 can promote GC progression through the miR-145-5p/ARF6 axis. KCNQ1OT1 may serve as a therapeutic target and a diagnostic biomarker of GC.

Keywords: ARF6; KCNQ1OT1; gastric cancer; miR-145-5p.

FIGURE 1

The role of KCNQ1OT1 in GC tumorigenesis. (A) The expression of KCNQ1OT1 in GC tissues and matched normal gastric tissue. (B) The expression of KCNQ1OT1 in GC cells and human normal gastric cells. (C) Tumor volumes were measured every week and growth curves are shown. (D) Tumor growth was measured (five tumors are shown in each group). (E) Tumor weight (g) was measured. (F) TUNEL staining is shown. (G) Ki‐67 staining is shown. (H) The expression of KCNQ1OT1 in tumor tissues. (I) The expression of miR‐145‐5p in tumor tissues after transfection with shNC or shKCNQ1OT1. (J) Protein expression of ARF6 in tumor tissues transfected with shNC or shKCNQ1OT1. *p < 0.05, n = 5

FIGURE 2

Knockdown of KCNQ1OT1 increased cell apoptosis and reduced cell proliferation in vitro. (A) mRNA expression of KCNQ1OT1 in cells transfected with shRNA and shKCNQ1OT1. (B) A CCK‐8 assay was used to detect the cell viability of BSG803 cell line. (C) Western blotting was used to detect the expression of cleaved‐caspase 3, caspase 3, Bcl2 and Bax in cells transfected with shNC or shKCNQ1OT1. (D) Flow cytometry was used to detect cell apoptosis. (E) A colony formation experiment was used to detect cell proliferation. *p < 0.05, n = 3

FIGURE 3

miR‐145‐5p was a target of KCNQ1OT1 in gastric cancer cells. (A) Expression of miR‐145‐5p in GC cell lines and the normal gastric cell line. (B) The expression of KCNQ1OT1 was negatively correlated with miR‐145‐5p expression. (C) Expression of miR‐145‐5p in BSG803 cells transfected with miR‐NC, miR‐145‐5p‐mimic, inhibitor NC and miR‐145‐5p‐inhibitor. (D) The shared binding sequences between KCNQ1OT1 and miR‐145‐5p were predicted by TarBase, version 7.0. (E) The relative luciferase activities of BSG803 cells were detected after co‐transfection with miR‐NC or miR‐145‐5p‐mimic and control (ctrl), KCNQ1OT1‐WT or KCNQ1OT1‐MUT. (F) The expression of miR‐145‐5p in BSG803 cells was detected after transfection with shNC or shKCNQ1OT1. (G) The expression of KCNQ1OT1 in BSG803 cells was detected after transfection with miR‐NC, miR‐145‐5p‐mimic, inhibitor NC and miR‐145‐5p‐inhibitor. *p < 0.05, n = 3

FIGURE 4

The effects of KCNQ1OT1 were mediated by miR‐145‐5p in GC cells. (A) A CCK‐8 assay was used to detect the cell viabilities in GC cells transfected with control (ctrl), shKCNQ1OT1 or shKCNQ1OT1 + miR‐145‐5p‐inhibitor. (B) Western blotting was used to detect the protein levels of cleaved‐caspase 3, Bcl2, Bax and GAPDH in BSG803 cells transfected with control (ctrl), shKCNQ1OT1 or shKCNQ1OT1 + miR‐145‐5p‐inhibitor. (C) Flow cytometry was used to detect cell apoptosis in BSG803 cells transfected with control (ctrl), shKCNQ1OT1 or shKCNQ1OT1 + miR‐145‐5p‐inhibitor. (D) A colony formation experiment was used to detect the cell proliferation in BSG803 cells transfected with control (ctrl), shKCNQ1OT1 or shKCNQ1OT1 + miR‐145‐5p‐inhibitor. *p < 0.05, n = 3

FIGURE 5

ARF6 was a target of miR‐145‐5p. (A) The shared common binding sequences were predicted between miR‐145‐5p and ARF6. (B) Luciferase activities were detected in GS cells co‐transfected with control, ARF6‐WT or ARF6‐MUT. (C) Western blotting was used to detect the protein expression of ARF6 in GC cells transfected with miR‐NC, miR‐145‐5p‐mimic, inhibitor NC or miR‐145‐5p‐inhibitor. (D). Western blotting was used to detect the protein expression of ARF6 in cells transfected with shNC or shKCNQ1OT1. (E) mRNA expression of ARF6 was detected in GSE‐1, HS‐746 T, BSG823, MKN‐28, 9811, BGC803, MGC 803 and BGC823. (F) The expression of ARF6 was detected in adjacent tissues and cancer tissues. *p < 0.05, n = 3

FIGURE 6

ARF6 mediated KCNQ1OT1 regulation in GC cells. (A) mRNA expression of ARF6 was detected in cells transfected with pc‐NC or pc‐ARF6. (B) Cell viability (left) and apoptosis rate (right) in cells were detected after transfection with miR‐NC, miR‐145‐5p mimic or miR‐145‐5p mimic + pc‐ARF6. (C) Flow cytometry was used to detect the cell apoptosis in cells transfected with miR‐NC, miR‐145‐5p mimic or miR‐145‐5p + pc‐ARF6. (D) A colony formation assay was used to detect the proliferation of cells transfected with miR‐NC, miR‐145‐5p mimic or miR‐145‐5p mimic + pc‐ARF6. *p < 0.05, n = 3