Hypoxia-induced lncHILAR promotes renal cancer metastasis via ceRNA for the miR-613/206/ 1-1-3p/Jagged-1/Notch/CXCR4 signaling pathway

Abstract

Hypoxia has been identified as a common contributor to tumor progression, including invasion and metastasis. However, the underlying mechanisms of enhanced invasion and metastasis under hypoxia remain unclear. A hypoxic microenvironment promotes invasion and metastasis of renal cell carcinoma (RCC) by upregulating expression of LOC100506178, which we named hypoxia-induced long non-coding RNA (lncRNA) associated with RCC (lncHILAR). Knockdown of lncHILAR inhibited cell invasion and migration, whereas overexpression of lncHILAR, conversely, facilitated cell invasion and migration of RCC cells. Notably, hypoxic RCC cells secreted exosomes packaged with lncHILAR, which were taken up by normoxic RCC cells and then drove normoxic cell invasion. Mechanistically, lncHILAR elevated RCC invasion and metastasis by acting as a competing endogenous RNA (ceRNA) for miR-613/206/1-1-3p, which led to the upregulation of Jagged-1 and the C-X-C motif chemokine receptor 4 (CXCR4). Activation of the Jagged-1/Notch/CXCR4 axis induced RCC metastasis. lncHILAR promotes RCC cell invasion and metastasis via ceRNA for the miR-613/206/1-1-3p/Jagged-1/Notch/CXCR4 axis. The novel lncHILAR may thus serve as a potential biomarker and therapeutic target in RCC.

Keywords: RCC; exosomes; hypoxia; lncRNA; metastasis.

Graphical abstract

Figure 1

Aberrant epigenetic modification devoted to upregulation of lncHILAR in RCC under hypoxia (A and B) Nude mice were injected in the tail vein with normoxic and hypoxic SN12PM6 cells (1 × 10⁶ cells). The first scan was acquired at the fourth week after injection. Then, bioluminescent images were acquired once every 2 weeks. Representative bioluminescent images (A) and verification of metastases by H&E staining (B) are shown. (C) Expression of 10 lncRNAs was increased more than 4.5-fold in SW839 cells under hypoxia, which was investigated by lncRNA microarray. (D) Expression of 10 selected lncRNAs was verified in ACHN and Caki-1 cells by qRT-PCR. ∗p < 0.05. n = 3. (E) Expression level of lncHILAR in non-metastatic samples (n = 40) and metastatic RCC samples (n = 40) detected by qRT-PCR. lncHILAR expression was normalized to GAPDH (ΔCt) and compared with the maximum ΔCt. Data are presented as ΔΔCt. ∗∗∗p < 0.0001. (F) Expression of lncHILAR in plasma of patients with metastatic RCC was greater than in the plasma of patients with non-metastatic RCC. Expression of lncHILAR was detected by qRT-PCR. lncHILAR expression was normalized to GAPDH (ΔCt) and compared with the maximum ΔCt. Data are presented as ΔΔCt. ∗∗∗p < 0.0001. (G) Overall survival of patients with RCC in the high-lncHILAR-expression group (n = 265) and low-lncHILAR-expression group (n = 265), ∗p< 0.01. Data were acquired from TCGA database. (H and I) Cell invasion and cell migration were determined with the transwell assay system after knockdown and overexpression of lncHILAR in ACHN and Caki-1 cells. Each sample had three replicates, and experiments were repeated three times. n = 3, ∗p < 0.05. (J) H3K4Me1, H3K4Me3, and H3K27ac protein were detected under hypoxia in RCC as shown by immunoblot. (K) H3K4me1 methylation could be inhibited with histone demethylase inhibitor (PFI-2 HCl, 1 nM). Results are presented as immunoblots. HDMT, histone demethylase. (L) Expression of lncHILAR was detected by qRT-PCR after inhibition of H3K4Me1. ∗∗p < 0.0001. (M) H3K27ac acetylation could be suppressed by histone deacetylase inhibitor (C646, 20 μM). Results are presented as immunoblots. HDAC, histone deacetylase. (N) Expression of lncHILAR was detected by qRT-PCR after inhibition of H3K27ac. ∗p < 0.05. (O) ChIP assay revealed that H3K4me1 and H3K27ac were enriched in the promoter area of lncHILAR. ∗p < 0.05, ∗∗∗p < 0.0001.

Figure 2

Exosomes derived from hypoxic RCC cells carry lncHILAR and promote cell invasion by normoxic RCC cells (A) The cellular distribution of lncHILAR RNA by qRT-PCR showed that lncHILAR was predominately localized to the cytoplasm. (B) RNAscope analysis was used to explore the distribution of lncHILAR in RCC samples. (C) qRT-PCR analysis of lncHILAR in the cell medium of ACHN and Caki-1 cells treated with RNase (2 mg/mL) alone or combined with Triton X-100 (0.1%) for 20 min. n = 3. ∗∗∗p < 0.0001. (D) qRT-PCR analysis of lncHILAR in the exosomes of ACHN and Caki-1 cells cultured under normoxia and hypoxia. n = 3. ∗p < 0.05. (E) Exosome uptake assay was confirmed with XPack MSCV-XP-GFP-EF1α-Puro Expression LentiVector. GFP-labeled exosomes were taken up by ACHN cells, shown as the green signal. (F) Cells were incubated with CM for 24 h. qRT-PCR analysis of lncHILAR of ACHN and Caki-1 cells after treatment with different CMs. CM+GW4869, CM collected from cells pretreated with GW4869 (5 μM). n = 3. ∗p < 0.05. (G) For in vitro exosome treatment, exosomes collected from about 5 × 10⁶ producer cells were added to 2 × 10⁵ recipient cells in each plate of a six-well plate. The cells were then incubated with exosomes in CM for 24 h. qRT-PCR analysis of lncHILAR of ACHN and Caki-1 cells after treatment with hypoxic exosomes. NM, normal medium. n = 3. ∗∗∗p < 0.0001. (H) Cell invasion analysis of ACHN and Caki-1 cells treated with different CM. n = 3. ∗p < 0.05. (I) Transwell invasion assay of ACHN cells, Caki-1 cells, ACHN^lncHILAR-KD cells, and Caki-1^lncHILAR-KD cells after treatment with hypoxic exosomes. n = 3. Caki-1 Exo, hypoxic exosomes derived from hypoxic Caki-1 cells; ACHN Exo, hypoxic exosomes derived from hypoxic ACHN cells. n = 3. ∗p < 0.05.

Figure 3

lncHILAR functions as a ceRNA for miR-613/206/1-1-3p to activate Jagged1 (A) Potential binding miRNAs were explored in two databases (AnnoLnc and miRDB). The seed sequences are shown in bold and italic. Putative miR-613/206/1-1-3p MREs (microRNA response elements) in the 3′ UTR of CXCR4 and Jagged1 were analyzed by microRNA and TargetScan. (B) FISH technique was applied to co-localize lncHILAR and miR-613/206/1-1-3p in RCC tissues. The lncRNA probe was conjugated with FAM (green), whereas the microRNA probe was conjugated with Cy3 (red). (C) Luciferase activity of psiCHECK-2 reporters, which contained wild-type or mutant lncHILAR (miR-613/206/1-1-3p MREs) in Caki-1 cells. n = 3. ∗p < 0.0001. (D) Immunohistochemistry analysis of Jagged1 expression at different stages in RCC tissue microarray. (E) Luciferase activity of psiCHECK-2 reporters, which contained wild-type or mutant Jagged1 (miR-613/206/1-1-3p MREs) in Caki-1 cells. n = 3. ∗p < 0.0001. (F) Immunoblot analysis of Notch pathway proteins in lncHILAR knockdown and control Caki-1 cells co-transfected with miR-613/206/1-1-3p inhibitors (50 nM) for 48 h. (G) Immunoblot analysis of Notch pathway proteins in lncHILAR knockdown and control Caki-1 cells treated with CM or with CM and exosome inhibitor (GW4689, 5 μM) for 48 h. (H) Immunoblot analysis of Notch pathway proteins in lncHILAR knockdown and control Caki-1 cells treated with hypoxic exosomes.

Figure 4

MiR-613/206/1-1-3p targets the Jagged1/Notch/CXCR4 axis (A) qRT-PCR analysis of CXCR4 mRNA from ACHN and Caki-1 cells transfected with control or miR-613/206/1-1-3p mimics (50 nM) for 48 h. n = 3. ∗p < 0.0001. (B) Immunoblot analysis of CXCR4 protein expression in ACHN and Caki-1 cells transfected with control or miR-613/206/1-1-3p mimics. n = 3. (C) qRT-PCR analysis of CXCR4 mRNA in ACHN and Caki-1 cells after knockdown of lncHILAR or overexpression of lncHILAR. n = 3. ∗p < 0.001. (D) Immunoblot analysis of CXCR4 protein expression in ACHN and Caki-1 cells after knockdown of lncHILAR or overexpression of lncHILAR. n = 3. (E) Luciferase activity of psiCHECK-2 reporters, which contained wild-type or mutant CXCR4 (miR-613/206/1-1-3p MREs) in Caki-1 cells. n = 3. ∗p < 0.001. (F) Cell invasion was determined by transwell invasion assay after overexpression of lncHILAR and co-transfection with sg-CXCR4. n = 3. ∗p < 0.0001.

Figure 5

lncHILAR induces EMT (A) Gene set enrichment analysis (GSEA) of EMT gene signatures in lncHILAR-overexpressing cells versus control cells. NES, normalized enrichment score. (B) Immunofluorescence microscopy analysis of the expression of EMT markers in Caki-1 cells under normoxia or hypoxia. Scale bars, 100 μM. (C) Immunoblot analysis of the expression of EMT markers in Caki-1 cells transfected with miR-613/206/1-1-3p inhibitors (50 nM) for 48 h under normoxia or hypoxia. (D) qRT-PCR analysis of the expression of EMT markers in Caki-1 cells transfected with miR-613/206/1-1-3p inhibitors (50 nM) for 48 h under normoxia or hypoxia. n = 3. ∗p < 0.0001.

Figure 6

LncHILAR promotes RCC metastasis in vivo (A–F) Nude mice were orthotopically xenografted with Ctrl, lncHILAR, sg-CXCR4, or lncHIALR-sg-CXCR4 cells (1 × 10⁷ cells). Representative bioluminescent images (A and B), H&E staining of metastases (C), representative macroscopic images of primary RCC and metastatic lungs or livers (D), metastasis diagrams (E and F), and survival rates (G) of mice in the indicated groups are shown.