Long Noncoding RNA KCNQ1OT1 Promotes the Progression of Non-Small Cell Lung Cancer via Regulating miR-204-5p/ATG3 Axis

Abstract

Purpose: Non-small cell lung cancer (NSCLC) is the first leading cause of cancer-related death globally. Long noncoding RNA KCNQ1 overlapping transcript 1 (KCNQ1OT1) was involved in the progression of multiple cancers by sponging target miRNA. We aimed to explore the pathological mechanism of KCNQ1OT1 in NSCLC progression.

Methods: The expression of KCNQ1OT1, miR-204-5p and autophagy-related gene 3 (ATG3) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry assay were conducted for the detection of cell proliferation and apoptosis, respectively. Western blot assay was performed to examine the protein levels of B-cell lymphoma-2 (BCL-2), BCL2-Associated X (Bax), cleaved caspase-3, cleaved caspase-9 and LC3Ⅱ/LC3Ⅰ and P62. The interaction between miR-204-5p and KCNQ1OT1 or ATG3 was validated by dual-luciferase reporter system and RNA immunoprecipitation (RIP) assay. Murine xenograft assay was conducted to explore the function of KCNQ1OT1 in vivo. Immunohistochemistry (IHC) staining assay was used for the analysis of ki67-positive cell percentage.

Results: The expression of KCNQ1OT1 and ATG3 was up-regulated whereas miR-204-5p was down-regulated in NSCLC tumors and cells. MiR-204-5p was inversely correlated with KCNQ1OT1 or ATG3. In addition, KCNQ1OT1 knockdown facilitated apoptosis, inhibited autophagy and proliferation of NSCLC cells in vitro and blocked tumor growth in vivo. However, the miR-204-5p inhibitor reversed the effects. More importantly, ATG3 was a target gene of miR-204-5p and ATG3 overexpression restored the effect of miR-204-5p on NSCLC cell progression.

Conclusion: KCNQ1OT1 promotes cell proliferation and autophagy and inhibits cell apoptosis via regulating miR-204-5p/ATG3 axis, providing a promising target for NSCLC therapy.

Keywords: ATG3; KCNQ1OT1; NSCLC; miR-204-5p.

Figure 1

KCNQ1OT1 knockdown repressed proliferation and autophagy and induced apoptosis in NSCLC. (A, B) KCNQ1OT1 expression in 35 pairs of NSCLC tumor tissues and normal tissues. (C) KCNQ1OT1 expression in NSCLC cell lines (HCC827, H1299, A549, H460) and human bronchial epithelial cell BEAS-2B. (D–K) A549 and H460 cells were stably transfected with sh-KCNQ1OT1 or sh-NC. (D) KCNQ1OT1 expression in stably transfected A549 and H460 cells. (E, F) Cell viability of transfected A549 (E) and H460 cells (F). (G) Cell apoptosis of transfected A549 and H460 cells. (H, I) The expression of apoptosis-related protein cleaved caspase-3, cleaved caspase-9, Bax and anti-apoptosis protein BCL-2 in transfected A549 (H) and H460 cells (I). (J, K) Protein expression of autophagy markers LC3 and P62 in transfected A549 (J) and H460 cells (K). *P<0.05, ***P<0.001.

Figure 2

KCNQ1OT1 directly interacted with miR-204-5p. (A) The putative binding sites between KCNQ1OT1 and miR-204-5p. (B, C) Luciferase activity of A549 (B) and H460 cells (C) co-transfected with WT-KCNQ1OT1 or MUT-KCNQ1OT1 and miR-204-5p or miR-NC. (D) The enrichment of miR-204-5p in Anti-Ago2 immunoprecipitation complex in A549 and H460 cells transfected with KCNQ1OT1 or pcDNA. (E, F) The expression of miR-204-5p in A549 (E) and H460 cells (F) transfected with KCNQ1OT1, sh-KCNQ1OT1, pcDNA or sh-NC. (G, H) The expression of miR-204-5p in NSCLC tumor tissues and normal tissues. (I) The expression of miR-204-5p in NSCLC cell lines (HCC827, H1299, A549, H460) and human bronchial epithelial cell BEAS-2B. (J) The correlation between expression of KCNQ1OT1 and miR-204-5p (r=−0.7785, P<0.0001). *P<0.05, ***P<0.001.

Figure 3

miR-204-5p inhibitor attenuated the effects of KCNQ1OT1 silencing on cell proliferation, apoptosis and autophagy in NSCLC. A549 and H460 cells were transfected with sh-KCNQ1OT1+anti-miR-204-5p, sh-KCNQ1OT1, sh-KCNQ1OT1+anti-miR-NC or sh-NC. (A) The expression of miR-204-5p in transfected A549 and H460 cells. (B, C) Cell viability of transfected A549 (B) and H460 cells (C). (D) Cell apoptosis of transfected A549 and H460 cells. (E, F) The expression of apoptosis-related protein cleaved caspase-3, cleaved caspase-9, Bax and anti-apoptosis protein BCL-2 in transfected A549 (E) and H460 cells (F). (G, H) Protein expression of autophagy markers LC3 and P62 in transfected A549 (G) and H460 cells (H). *P<0.05.

Figure 4

ATG3 is a target of miR-204-5p. (A) The putative binding sites between ATG3 and miR-204-5p. (B, C) Luciferase activity of A549 (B) and H460 cells (C) co-transfected with ATG3 3ʹUTR-WT or ATG3 3ʹUTR-MUT and miR-204-5p or miR-NC. (D, E) Protein expression of ATG3 in A549 (D) and H460 cells (E) transfected with miR-204-5p, anti-miR-204-5p, anti-miR-NC or miR-NC. (F, G) The expression of ATG3 in NSCLC tumor tissues and normal tissues. (H) The correlation between ATG3 and miR-204-5p (r=−0.6014, P<0.0001). (I, J) The expression of ATG3 mRNA (I) and protein (J) in NSCLC cell lines (HCC827, H1299, A549, H460) and human bronchial epithelial cells BEAS-2B. *P<0.05, ***P<0.001.

Figure 5

ATG3 abrogated miR-204-5p mediated acceleration on cell apoptosis and suppression on cell proliferation and autophagy in NSCLC. A549 and H460 cells were transfected with miR-204-5p+ATG3, miR-204-5p+pcDNA, miR-204-5p or miR-NC. (A) The expression of ATG3 protein in transfected A549 and H460 cells. (B, C) Cell viability of transfected A549 (B) and H460 cells (C). (D) Cell apoptosis of transfected A549 and H460 cells. (E, F) The expression of apoptosis-related protein caspase-3, caspase-9, Bax and anti-apoptosis protein BCL-2 in transfected A549 (E) and H460 cells (F). (G, H) Protein expression of autophagy markers LC3 and P62 in transfected A549 (G) and H460 cells (H). *P<0.05.

Figure 6

KCNQ1OT1 silencing suppressed tumor growth in vivo. Sh-NC or sh-KCNQ1OT1 transfected A549 cells or H460 cells were injected into the nude mice. (A, B) Tumor volume of xenograft mice was measured every 4 d from day 8. (C, D) Tumor weight was measured when xenograft mice were sacrificed after 28 days. (E, F) The expression of KCNQ1OT1, miR-204-5p, ATG3 in xenograft mice tumor tissues. (G) Protein expression of ATG3, LC3, P62, BCL-2, cleaved caspase-3, cleaved caspase-9 and Bax in xenograft mice tumor tissues. (H) The percentage of KI67-positive cells in xenograft mice tumor tissues. *P<0.05.