Knockdown of Long Non-Coding RNA HOTAIR Suppresses Cisplatin Resistance, Cell Proliferation, Migration and Invasion of DDP-Resistant NSCLC Cells by Targeting miR-149-5p/Doublecortin-Like Kinase 1 Axis

Abstract

Background: Long non-coding RNA (lncRNA) HOTAIR has been reported to be associated with cisplatin (DDP) resistance in different human cancers including non-small cell lung cancer (NSCLC). However, the mechanism of HOTAIR in cisplatin resistance of NSCLC remains largely undefined.

Materials and methods: Expression of HOTAIR, miR-149-5p and doublecortin-like kinase 1 (DCLK1) was detected using real-time quantitative PCR (RT-qPCR) and Western blotting. Cisplatin resistance was determined with cell counting kit (CCK)-8 assay and transwell assays in vitro, and xenograft tumor models in vivo. The target binding between miR-149-5p and either HOTAIR or DCLK1 was predicted on Diana Tools website, and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation.

Results: Expression of HOTAIR was upregulated in DDP-resistant NSCLC tumor tissues and cell lines (A549/DDP and H1299/DDP). Knockdown of HOTAIR decreased the acquired cisplatin resistance of A549/DDP and H1299/DDP cells, as evidenced by attenuated 50% inhibitory concentration (IC50) of DDP, cell proliferation, migration and invasion in vitro, as well as tumor growth inhibition in vivo. Mechanically, HOTAIR negatively regulated miR-149-5p expression via targeting, and DCLK1 was a downstream target for miR-149-5p. DCLK1 was indirectly regulated by HOTAIR in DDP-resistant NSCLC cells as well. Functionally, miR-149-5p deletion could counteract the inhibitory effect of HOTAIR knockdown on cisplatin resistance; contrarily, restoring miR-149-5p exhibited the similar inhibition on cisplatin resistance in DDP-resistant cells in vitro, which was then abated by DCLK1 upregulation.

Conclusion: Knockdown of HOTAIR enhances DDP-resistant NSCLC cells to overcome cisplatin resistance partially via regulating miR-149-5p/DCLK1 axis.

Keywords: HOTAIR; NSCLC; cisplatin resistance.

Figure 1

Expression of lncRNA HOTAIR (HOTAIR) in cisplatin (DDP)-resistant non-small cell lung cancer (NSCLC) tissues and cells. (A) Real-time quantitative PCR (RT-qPCR) assay showed the expression levels of HOTAIR in DDP-resistant and -sensitive NSCLC tissues. (B, C) The 50% inhibitory concentration (IC50) values of DDP were determined by Cell Counting Kit (CCK)-8 assay in A549/DDP and H1299/DDP cells with their parental cells. The cells were exposed to different concentrations (10, 20, 40, 60, 80, and 100 μM) of DDP for 48 h. (D) RT-qPCR assay showed HOTAIR levels in A549/DDP and H1299/DDP cells with their parental cells. *P < 0.05.

Figure 2

Effects of HOTAIR knockdown on cisplatin resistance in NSCLC cells. A549/DDP and H1299/DDP cells were transfected with siRNA against HOTAIR (si-HOTAIR) or its negative control (si-NC). (A) RT-qPCR assay showed HOTAIR expression levels after transfection for 24 h. (B) IC50 values of DDP were determined by CCK-8 assay after treated with different concentrations (10, 20, 40, 60, 80, and 100 μM) of DDP for 48 h. (C, D) CCK-8 assay detected cell viability after transfection for 0, 24, 48 and 72 h. (E, F) Transwell assays measured cell migration and invasion after transfection for 24 h. The cell migration/invasion ability was calculated as % of total cells. *P < 0.05.

Figure 3

Identification of the negative regulatory relationship between HOTAIR and miR-149-5p. (A) RT-qPCR assay measured expression levels of miRNAs in A549/DDP and H1299/DDP cells transfected with si-HOTAIR or si-NC. (B) The predicted hsa-miR-149-5p binding sites in HOTAIR according to DianaTools. The corresponding sequence in the mutated version was shown as well. (C, D) Luciferase activity of HOTAIR wild type (HOTAIR-WT) and mutant (HOTAIR-MUT) was examined by dual-luciferase reporter assay in A549/DDP and H1299/DDP cells when transfected with miR-149-5p mimic (miR-149-5p) or miR-NC mimic (miR-NC). (E) Expression of HOTAIR was detected with RNA immunoprecipitation (RIP) assay in A549/DDP and H1299/DDP cells transfected with miR-149-5p/NC. The enrichment of HOTAIR level was showed as % of input. (F, G) RT-qPCR assay showed the expression levels of miR-149-5p in DDP-resistant NSCLC tissues and cells (A549/DDP and H1299/DDP), compared to DDP-sensitive NSCLC tissues and parental cells. (H) RT-qPCR assay measured miR-149-5p expression levels in A549/DDP and H1299/DDP cells transfected with pcDNA-HOTAIR (HOTAIR), si-HOTAIR or their controls. *P < 0.05.

Figure 4

Influence of miR-149-5p reduction on the inhibitory effect of HOTAIR knockdown on cisplatin resistance. A549/DDP and H1299/DDP cells were transfected with si-HOTAIR or si-NC, and co-transfected with si-HOTAIR and miR-149-5p/NC inhibitor (in-miR-149-5p/NC). (A) Expression levels of miR-149-5p were detected by RT-qPCR after transfection. CCK-8 assay detected (B) IC50 values of DDP after transfection for 48 h and (C, D) cell viability after transfection for 0, 24, 48 and 72 h. (E, F) Transwell assays measured cell migration and invasion after transfection for 24 h. *P < 0.05.

Figure 5

Verification of the target relationship between miR-149-5p and doublecortin-like kinase 1 (DCLK1). (A) The predicted hsa-miR-149-5p binding sites in DCLK1 3ʹ untranslated regions (3ʹUTR) according to DianaTools. The corresponding sequence in the mutated version was shown as well. (B, C) Luciferase activity of DCLK1 3ʹUTR wild type (DCLK1 3ʹUTR-WT) or mutant (DCLK1 3ʹUTR-MUT) in A549/DDP and H1299/DDP cells transfected with miR-149-5p or miR-NC. (D, E) RT-qPCR assay showed the expression levels of DCLK1 mRNA in DDP-resistant NSCLC tissues and cells (A549/DDP and H1299/DDP), compared to DDP-sensitive NSCLC tissues and parental cells. (F, G) Western blotting measured DCLK1 protein expression levels in A549/DDP and H1299/DDP cells when transfected with miR-149-5p, in-miR-149-5p, pcDNA-HOTAIR, si-HOTAIR, or their controls. β-actin was detected as the internal reference. *P < 0.05.

Figure 6

Influence of DCLK1 elevation on the role of miR-149-5p in cisplatin resistance. A549/DDP and H1299/DDP cells were transfected with miR-149-5p or miR-NC, and co-transfected with miR-149-5p and pcDNA-DCLK1 (DCLK1) or pcDNA. (A) Expression levels of DCLK1 were detected by Western blotting after transfection. CCK-8 assay detected (B) IC50 values of DDP after transfection for 48 h and (C, D) cell viability after transfection for 0, 24, 48 and 72 h. (E, F) Transwell assays measured cell migration and invasion after transfection for 24 h. *P < 0.05.

Figure 7

Knockdown of HOTAIR inhibited tumor growth of H1299/DDP cells in vivo. H1299/DDP cells were lentiviral infected with short hairpin RNA against HOTAIR (sh-HOTAIR) or its negative control sh-NC prior to injection into BALB/c nude mice (n=5). Xenograft tumors were exposed to DDP (3.0 mg/kg body weight) or phosphate buffer solution (PBS; pH 7.4) every 7 days from 7th day after transplantation. (A) The volumes were calculated every week and the growth curve was drawn. (B) Tumor weight was recorded on day 35 after transplantation. (C) Expression of HOTAIR, miR-149-5p and DCLK1 was confirmed in xenograft tumors using RT-qPCR. (D) Western blotting evaluated protein levels of DCLK1, Ki-67 and cleaved caspase-3. *P < 0.05.