The lncRNA PVT1 regulates nasopharyngeal carcinoma cell proliferation via activating the KAT2A acetyltransferase and stabilizing HIF-1α

Abstract

Long noncoding RNAs (lncRNAs) play important roles in regulating the development and progression of many cancers. However, the clinical significance of specific lncRNAs in the context of nasopharyngeal carcinoma (NPC) and the molecular mechanisms by which they regulate this form of cancer remain largely unclear. In this study we found that the lncRNA PVT1 was upregulated in NPC, and that in patients this upregulation was associated with reduced survival. RNA sequencing revealed that PVT1 was responsible for regulating NPC cell proliferation and for controlling a hypoxia-related phenotype in these cells. PVT1 knockdown reduced NPC cell proliferation, colony formation, and tumorigenesis in a subcutaneous mouse xenograft model systems. We further found that PVT1 serves as a scaffold for the chromatin modification factor KAT2A, which mediates histone 3 lysine 9 acetylation (H3K9), recruiting the nuclear receptor binding protein TIF1β to activate NF90 transcription, thereby increasing HIF-1α stability and promoting a malignant phenotype in NPC cells. Overexpression of NF90 or HIF-1α restored the proliferation in cells that had ceased proliferating due to PVT1 or KAT2A depletion. Conversely, overexpression of active KAT2A or TIF1β, but not of KAT2A acetyltransferase activity-deficient mutants or TIF1β isoforms lacking H3K9ac binding sites, promoted a PVT1-mediated increase in NF90 transcription, as well as increased HIF-1α stability and cell proliferation. PVT1 knockdown enhanced the radiosensitization effect in NPC cells via inhibiting binding between H3K9ac and TIF1β in a manner. Taken together, our results demonstrate that PVT1 serves an oncogenic role and plays an important role in radiosensitivity in malignant NPC via activating the KAT2A acetyltransferase and stabilizing HIF-1α.

Fig. 1

The PVT1 lncRNA is upregulated in NPC and is associated with a poor prognosis in patients. a Expression of PVT1 mRNA is higher in NPC cell lines compared with the immortalized nasopharyngeal epithelial cell line. b Expression levels of PVT1 mRNA in ten clinical NPC tumors and ten freshly frozen normal nasopharyngeal specimens. c, d Expression levels of PVT1 mRNA in NPC tumors and normal nasopharyngeal specimens. PVT1 mRNA-expression data were obtained from the GSE12452 dataset [21] and the GSE64634 dataset [22] and analyzed. e, f, Kaplan–Meier analysis of relapse-free survival (RFS) and over survival (OS) for patients with high PVT1 mRNA-expression NPC versus low PVT1 mRNA-expression NPC. Error bars ± SD. **P < 0.01. ***P < 0.001. Data are representative from three independent experiments

Fig. 2

Knockdown of PVT1 disrupts NPC cell proliferation and tumor growth. a Scatter plot of 16764 gene expression in HNE-1 cells transfected with PVT1 shRNA (y-axis) compared with the control group (x-axis). Green dots, set of genes exhibiting significant downregulation upon PVT1 knockdown; red dots, set of genes exhibiting significant upregulation upon PVT1 knockdown; black dots, the genes without significant change. b The biological process signaling pathways response upon PVT1 knockdown by Gene GO analyses. c Effects of PVT1 knockdown on NPC cell colony formation. d Quantification of colony formation in c. e Effects of PVT1 knockdown on NPC cell proliferation. f Representative images of PVT1 knockdown-inhibited HNE-1 subcutaneous tumor generation. Tumors were harvested at 3–4 weeks after implantation. Data were from two independent experiments with five mice per group with similar results. g Quantification of tumor weight in f. Error bars ± SD. **P < 0.01. ***P < 0.001. Data are representative from three independent experiments

Fig. 3

PVT1 promotes NPC cell proliferation via HIF-1α. a The signaling pathways response upon PVT1 knockdown by Gene KGEE analyses. Quantitative RT-PCR (b) and WB (c) analyses of effects of PVT1 knockdown on HIF-1α activation. d Quantification of HIF-1α protein in c. e Reduction of HIF-1α mRNA stability in PVT1 knockdown HNE-1 cells compared with control cells. Cells were treated with 1 μg/ml actinomycin D and RNA was extracted at the time of 0, 1, and 2 h. f HIF-1α protein expression assays for HNE-1 and CNE-1 under hypoxia conditions (1% O₂). g Quantification of HIF-1α protein in f. Cell proliferation (h) and colony formation (i) assays for HNE-1 cells under hypoxic conditions. Hypoxia induced the cell proliferation and colony formation of NPC cells. The cell proliferation and colony formation abilities of NPC cells under hypoxic conditions were suppressed by PVT1 knockdown, and these suppressed cell proliferation and colony formation abilities of NPC cells were restored by HIF-1α overexpression. Error bars ± SD. *P < 0.05. **P < 0.01. ***P < 0.001. Data are representative from three independent experiments

Fig. 4

PVT1 binds to KAT2A in NPC cells. a The RIP-qPCR assay of the binding of KAT2A with PVT1 in HNE-1 and CNE-1 cells. b Biotinylated PVT1 was incubated with extracts (HNE-1 and CNE-1 cells), treated with streptavidin beads, and binding proteins were resolved in a gel. The WB assay of the specific binding of KAT2A and PVT1. c Graphic illustration of predicted PVT1 secondary structure (http://www.lncipedia.org), and the truncation of PVT1 in accordance with the stem-loop structure. d Secondary structure regions of PVT1 were treated as in b, and binding KAT2A was detected by the WB assay. e, f, WB detection of KAT2A binding upon secondary structure regions of PVT1 after Flag-MS2bp-MS2bs-based pull-down assay. g Effects of PVT1 knockdown on the specific binding of KAT2A and PVT1. Error bars ± SD. **P < 0.01. ***P < 0.001. Data are representative from two independent experiments

Fig. 5

PVT1 stabilizes HIF-1α via KAT2A. a Effects of PVT1 knockdown on HIF-1α protein in HNE-1 and CNE-1 NPC cells. b Quantification of HIF-1α protein in a. c Effects of PVT1 knockdown on HIF-1α mRNA in HNE-1 and CNE-1 NPC cells. d Overexpression of KAT2A restores PVT1 knockdown-inhibited HIF-1α protein. e Quantification of HIF-1α and KAT2A proteins in d. Overexpression of KAT2A restores PVT1 knockdown-inhibited HIF-1α mRNA (f), HIF-1α mRNA stability (g), NPC cell proliferation (h), and colony formation (i). Error bars ± SD. **P < 0.01. ***P < 0.001. Data are representative from three independent experiments

Fig. 6

KAT2A acetyltransferase activity is required for PVT1-driven HIF-1α stabilization. a Effects of PVT1 knockdown on KAT2A and the acetylation of H3K9 expression in HNE-1 and CNE-1 NPC cells. β-actin and Histone H3 were used as controls. b Effects of overexpression of KAT2A wild-type and acetyltransferase activity-deficient mutant, E570A/D610A, on KAT2A depletion-inhibited HIF-1α expression. c Overexpression of KAT2A wild type but not acetyltransferase activity-deficient mutant, E570A/D610A, restored PVT1 depletion-inhibited HIF-1α expression. Effects of overexpression of KAT2A wild-type and acetyltransferase activity-deficient mutant, E570A/D610A, on NPC cell proliferation (d) and colony formation (e). Inhibition of HIF-1α reduced KAT2A-induced HIF-1α protein expression (f), NPC cell proliferation (g), and colony formation (h). Error bars ± SD. **P < 0.01, ***P < 0.001. Data are representative from three independent experiments

Fig. 7

PVT1 promotes HIF-1α stability via KAT2A -mediated regulation of the TIF1β/H3K9ac complex. a Immunoprecipitation and WB analyses of effects of PVT1 knockdown on H3K9ac association with TIF1β. b KAT2A knockdown inhibited the association of H3K9ac with TIF1β. c Overexpression of KAT2A rescues PVT1 knockdown-inhibited the binding of H3K9ac with TIF1β. d Schematics of TIF1β^WT and various TIF1β deletion mutants. e H3K9ac binds with TIF1β with amino acid residues 512–654. f Overexpression of TIF1β wild type but not K554A/K575A mutant restores PVT1 knockdown-inhibited the binding of H3K9ac with TIF1β. g Overexpression of TIF1β wild type but not K554A/K575A mutant restores TIF1β knockdown-inhibited HIF-1α expression. h Overexpression of TIF1β wild type but not K554A/K575A mutant restores PVT1 knockdown-inhibited HIF-1α mRNA stability. Error bars ± SD. *P < 0.05. **P < 0.01. Data are representative from three independent experiments

Fig. 8

PVT1 promotes TIF1β/H3K9ac complex-mediated NF90 transcriptional activation to regulate HIF-1α stability. Effects of PVT1 knockdown on NF90 protein (a) and mRNA (b) expression in HNE-1 and CNE-1 NPC cells. c Overexpression of KAT2A restored PVT1 knockdown-inhibited NF90 expression. d, e The ChIP-qPCR assay of the association of KAT2A or WDR5 with the NF90 promoter. After using antibodies against KAT2A, WDR5, or control IgG, qPCR assays were conducted using primers corresponding to five different loci of the NF90 promoter. Effects of NF90 knockdown on HIF-1α protein (f) and mRNA (g) expression in HNE-1 and CNE-1 NPC cells. Overexpression of NF90 restored PVT1 (h) and KAT2A (i) knockdown-inhibited NF90 expression. j Effects of overexpression of TIF1β wild-type and K554A/K575A mutant on PVT1 knockdown-inhibited HIF-1α, NF90, and the acetylation of H3K9 expression in HNE-1 cells. k Representation of overexpression of TIF1β wild type but not K554A/K575A mutant on PVT1 knockdown-inhibited the association of KAT2A or WDR5 with the NF90 promoter. Error bars ± SD. **P < 0.01. ***P < 0.001. Data are representative from three independent experiments

Fig. 9

PVT1 suppresses the radiosensitivity of NPC cell lines by increasing the interaction between TIF1β and H3K9ac. a Effects of PVT1 overexpression in HNE-1 and CNE-1 NPC cells. Colony formation assays of PVT1 knockdown-(b) and overexpression-(c) mediated radiosensitivity in HNE-1 and CNE-1 NPC cells. Cell proliferation assays of PVT1 knockdown-(d) and overexpression-(e) mediated radiosensitivity in HNE-1 and CNE-1 NPC cells. f Representative bioluminescence images of PVT1 overexpression-restored radiation-inhibited HNE-1 subcutaneous tumor generation. Mice were imaged at 3–4 weeks after implantation. Data were from two independent experiments with five mice per group with similar results. g Quantification of the bioluminescence activity in f. h Representative bioluminescence images of PVT1 knockdown-induced radiation-inhibited HNE-1 subcutaneous tumor generation. i Quantification of the bioluminescence activity in h. j Overexpression of PVT1 rescues radiation-inhibited HIF-1α and NF90 expression. k The RIP assay of radiation has no effect on the association of KAT2A or WDR5, and PVT1. l Overexpression of PVT1 rescues radiation-inhibited the interaction between TIF1β and H3K9ac. m Effects of PVT1 overexpression on radiation-inhibited the association of KAT2A or WDR5 with the NF90 promoter. Error bars ± SD. *P < 0.05. **P < 0.01. ***P < 0.001. Data are representative from three independent experiments

Fig. 10

A working model for PVT1-regulated NPC tumorigenesis. PVT1 acts as a scaffold of KAT2A and WDR5, and KAT2A promotes H3K9 acetylation and association with TIF1β, leading to increased NF90 transcription and HIF-1α stabilization, thus resulting in enhanced NPC tumorigenesis