LncRNA HOTAIR regulates HIF-1α/AXL signaling through inhibition of miR-217 in renal cell carcinoma

Abstract

Long non-coding RNA HOTAIR was regarded as an oncogene in multiple cancers. Previous studies have shown that HOTAIR is involved in the proliferation and tumorigenesis of renal carcinoma cells, while microRNA (miR)-217 functions as a tumor suppressor in renal cell carcinoma (Rcc). However, the underlying molecular mechanism of HOTAIR in Rcc, especially in association with miR-217, has not been studied. In this study, we first demonstrated that HOTAIR expression was upregulated, which was correlated with tumor progression, and miR-217 downregulated in Rcc tissues and cells. Importantly, HOTAIR expression was negatively correlated with miR-217 expression in Rcc tissues. Gain- and loss-of-function of HOTAIR revealed that HOTAIR functioned as a ceRNA for miR-217 to facilitate HIF-1α expression and then upregulated AXL level promoting Rcc proliferation, migration, and EMT process, and inhibiting apoptosis. Furthermore, HOTAIR knockdown suppressed tumor growth and reduced the expression of proliferation antigen ki-67, HIF-1α, and AXL, but upregulated the expression of miR-217 in vivo. Finally, with AXL inhibitor BGB324, we confirmed that HOTAIR promoted Rcc activity through AXL signaling both in vitro and in vivo. In conclusion, these results suggest that HOTAIR promotes Rcc tumorigenesis via miR-217/HIF-1α/AXL signaling, which may provide a new target for the diagnosis and therapy of Rcc disease.

Figure 1

The expression of HOTAIR and miR-217 in Rcc tissues and cells and the relationship between HOTAIR and miR-217. qRT-PCR for the expression of HOTAIR (a) and miR-217 (b) in Rcc tissues and adjacent histological normal tissues. Data were analyzed by paired Student’s t-test. The expression levels of HOTAIR (c) and miR-217 (d) were assayed in Rcc cells (769-P and ACHN) and normal kidney cells (HK-2). The expression of HOTAIR and miR-217 was normalized to that in HK-2. The differences between groups were analyzed by unpaired Student’s t-test. (e) Bivariate correlation analysis of the relationship between HOTAIR expression and miR-217 level. (f) The putative miR-217 binding sequence of the wild type and mutation sequence of HOTAIR. (g) Relative luciferase assays. Statistical analysis was conducted using unpaired Student’s t-test. *P<0.05

Figure 2

HOTAIR modulates Rcc proliferation, migration, apoptosis, and EMT via negative regulation of miR-217. Cell viability was determined by MTT assay in ACHN (a) and 769-P (b) cells. The inset graph illustrates the efficiency of si-HOTAIR on HOTAIR expression in 769-P cells. Transwell assays were used to analyze the migration of ACHN (c) and 769-P (d) cells. (e) Flow cytometry for apoptosis in 769-P cells. (f) qRT-PCR assay for the mRNA expression of E-cadherin, Vimentin, and Snail in ACHN cells. (g) The mRNA levels of EMT-related genes in 769-P cells. (h) The protein levels of EMT-related genes in Rcc cells. *P<0.05 versus NC group, ^#P<0.05 versus HOTAIR or si-HOTAIR group, ^△P<0.05 versus TGF-β group. Data were analyzed using One-way ANOVA

Figure 3

HOTAIR functions as a ceRNA for miR-217 to facilitate HIF-1α expression. (a) The putative miR-217 binding 3′UTR of HIF-1α (HIF-1α-WT) and HIF-1α mutation sequence (HIF-1α-MUT). The protein (b) and mRNA (c) levels of HIF-1α in ACHN and 769-P cells. *P<0.05 versus NC group. (d) Luciferase activity assay. *P<0.05 versus NC-mimic group. (e) The expression of miR-217 in 769-P cell transfected with si-NC or si-HOTAIR. *P<0.05 versus NC group. Data were analyzed by unpaired Student’s t-test. (f) RIP assay of the enrichment of Ago2 on HOTAIR and HIF-1α transcripts relative to IgG in cells transfected with pLVX-HOTAIR or si-HOTAIR. (g) Luciferase activity of pGL3 reporters which contained wild type or mutant HIF-1α 3′UTR with indicated treatment in Rcc cells. (h) qRT-PCR analysis for HIF-1α mRNA expression in ACHN and 769-P cells. *P<0.05 versus pLVX group, ^#P<0.05 versus HOTAIR group in ACHN cells; *P<0.05 versus si-NC group, ^#P<0.05 versus si-HOTAIR group in 769-P cells. Statistical analysis was conducted using One-way ANOVA

Figure 4

HOTAIR regulates Rcc activity through AXL signaling in vitro. The mRNA (a) and protein (b) expression of AXL in ACHN and 769-P cells. Statistical analysis was conducted by unpaired Student’s t-test. (c) ACHN cells stably transfected with HOTAIR were treated with BGB324 (0.1, 0.5, 1 nM) for 72 h to examine the cell viability. Data were analyzed using One-way ANOVA. (d) The protein expression of E-cadherin, Vimentin, and Snail in ACHN cells stably transfected with HOTAIR and treated with different concentrations of BGB324 were assayed by western blot. The mRNA expression of HIF-1α (e) and AXL (f) in 86 paired Rcc tissues and adjacent histological normal tissues. Data were analyzed paired Student’s t-test. *P<0.05 versus control group, ^#P<0.05 versus HOTAIR treated only group

Figure 5

HOTAIR promotes Rcc cell growth via AXL signaling in vivo. (a) Tumor volume was evaluated for 12–30 days. The inset graph illustrates the efficiency of si-HOTAIR on HOTAIR expression. (b) Representative ki-67 staining (× 100). mRNA expression of miR-217 (c), HIF-1α (d), and AXL (e) in tumor tissues. Statistical analysis was conducted by unpaired Student’s t-test. *P<0.05 versus si-NC group (f) Western blot analysis for the protein levels of HIF-1α and AXL in tumor tissues. (g) Mice were subcutaneously injected with ACHN cells stably transfected with HOTAIR and orally administrated with BGB324 twice daily. Tumor volume was examined. (h) The expression of E-cadherin, Vimentin, and Snail was determined by Western blot. Data were analyzed using One-way ANOVA. n=6 per group, *P<0.05 versus pLVX group, ^#P<0.05 versus HOTAIR group. (i) Schematic of the proposed mechanism of HOTAIR in Rcc. HOTAIR functions as a ceRNA to ‘sponge’ miR-217 and upregulates the expression of HIF-1α as well as the HIF-1α downstream effector that promotes Rcc progression