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. 2020 Feb 14;11(9):2572-2579.
doi: 10.7150/jca.37959. eCollection 2020.

Upregulation of LncRNA PVT1 Facilitates Pancreatic Ductal Adenocarcinoma Cell Progression and Glycolysis by Regulating MiR-519d-3p and HIF-1A

Junwei Sun  1 , Pingping Zhang  2 , Tao Yin  3 , Feng Zhang  3 , Weixing Wang  1
Affiliations

Upregulation of LncRNA PVT1 Facilitates Pancreatic Ductal Adenocarcinoma Cell Progression and Glycolysis by Regulating MiR-519d-3p and HIF-1A

Junwei Sun et al. J Cancer. .

Abstract

The long, noncoding RNA (lncRNA) PVT1, as an important epigenetic regulator, has a critical role in carcinogenesis. However, its role in pancreatic ductal adenocarcinoma (PDAC) has not been fully investigated. Here, the up-regulated expression of lncRNA PVT1 is found in our PDAC tumor samples. Knockdown of it suppressed PDCA cells growth and glycolysis. An inverse association between miR-519d-3p and PVT1 was found. RIP, RNA pulldown and luciferase assay showed that PVT1 directly targets miR-519d-3p by binding with microRNA binding site. Bioinformatics analysis and study indicated that HIF-1A is a target of miR-519d-3p. Collectively, our findings suggested that PVT1 could act as an oncogenic lncRNA, and promote tumor progression by regulating HIF-1A via competing with miR-519d-3p.

Keywords: HIF-1A; PDAC; PVT1; miR-519d-3p.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
LncRNA PVT1 is often overexpressed in PDAC cancer and correlates with poor prognosis. (A) The level of PVT1 mRNA was measured by qRT-PCR in PDAC tissues and corresponding noncancerous tissues (n = 30). Data are indicated with medians and quartiles. (B) The level of PVT1 mRNA was measured by qRT-PCR in the normal pancreatic cell line H6C7 and various pancreatic cancer cell lines (HPAC, DANG, BXPC3, PANC1, and ASPC-1). Data are shown as the mean ± SD, n=3. (C) Expression levels of PVT1 in the TCGA cohort. Kaplan-Meier analysis of the correlation between PVT1 expression and overall survival in the TCGA cohort. The median level of PVT1 is used as the cutoff. (*P < 0.05, **P < 0.01, ***P < 0.001)
Figure 2
Figure 2
PVT1 knockdown represses PDAC cells proliferation and invasion in vitro. (A) qRT-PCR was used to measure the expression level of PVT1 in HPAC cells that had been transfected with siRNAs against PVT1 or control. (B) Cell viability was determined by MTT assay in PDAC cells transfected with control or si-PVT1#3. (C) A colony formation assay was performed to assess cell proliferation in HPAC cells transfected with control or si-PVT1#3. (D) Representative images of wound healing assays performed using PDAC cells after PVT1 was silenced for 24 h. Magnification 200×, Scale bars = 10 μm. (E) A Transwell assay was performed to assess the invasion of HPAC cells transfected with control or si-PVT1#3. All the results were reproducible in three independent experiments. Data are shown as the mean ± SD, n=3. (*P < 0.05, **P < 0.01, ***P < 0.001)
Figure 3
Figure 3
Knockdown of lncRNA PVT1 minimized glycolysis of PDAC cells. PDAC cells were cultured for 24 h after 48 h of transfection with either control or si-PVT1#3. Glucose uptake (A), lactate secretion (B) and intracellular ATP levels (C) of these cells were quantified and normalized for cell numbers. Shown data are mean ± SD (n = 3). Cell lysates were then analyzed by western blotting by anti-HIF-1A, anti-LDHA, anti-GULT1, anti-HK2 or anti-β-actin antibodies (D). (**P < 0.01, ***P < 0.001)
Figure 4
Figure 4
PVT1 negatively regulates miR-159-3p in PDAC cells. (A) The putative binding site of miR-159-3p and PVT1 was predicted by microrna.org. (B) The luciferase activity of PVT1 was detected using the luciferase report gene assay in HPAC cells co-transfected the wild type (pmirGLO-PVT1-wt) or mutated reporter construct (pmirGLO-PVT1-mut) and miR-159-3p-mimics or NC mimics. (C) Cellular lysates from HPAC cells were used for RIP with an Ago2 antibody and IgG antibody. The levels of miR-159-3p were detected by qRT-PCR. (D) HPAC cells were transfected with biotinylated NC (NC-Bio), biotinylated wild-type miR-519d-3p (miR-519d-3p-Bio) or biotinylated mutant miR-138 (miR-519d-3p-Bio-Mut), and biotin-based miRNA pull-down assays were conducted after 48 h of transfection. The PVT1 were analysed by qRT-PCR. (E) The miR-159-3p were detected in PDAC cells transfected with si-NC, si-PVT1#3, pc-DNA3.1 or pc-DNA3.1-PVT1. The PVT1 were detected in PDAC cells transfected with NC-mimics, miR-159-3p-mimics, inh NC or miR-159-3p-inh. (F) Correlation between PVT1 and miR-159-3p were detected in the cancer tissue of PDAC patients. (** and ## P < 0.01, ***and ### P < 0.001)
Figure 5
Figure 5
PVT1 inhibits miR-159-3p and then positively regulates HIF-1A. (A) Bioinformatics analysis showed the prediction for miR-159-3p binding sites on HIF-1A. (B) The luciferase reporter constructs containing the wildtype (HIF-1A3'UTR-WT) or mutant HIF-1A (HIF-1A3'UTR-MUT) sequence. HIF-1A3'UTR-WT or HIF-1A3'UTR-MUT was co-transfected with miR-159-3p mimics or NC mimics for 48 hours. (C and D) The mRNA and protein levels of HIF-1A transfected with miR-519d-3p-mimic, pc-PVT1 and their controls were measured by qRT-PCR and western blot assays. (E) Correlation analysis between HIF-1A and miR-159-3p were detected in the cancer tissue of PDAC patients. (** and ## P < 0.01, ***, ### and @@@P < 0.001))
Figure 6
Figure 6
The effect of PVT1 on progression and glycolysis is miR-159-3p dependent. Repression of miR-159-3p overcame the inhibitory effects of decreasing PVT1 on cell proliferation by the MTT assay and the colony formation assay and on cell migration by transwell assays and wound healing assay (A, B, C and D). (E) PDAC cells were cultured for 24 h after 48 h of transfection with si-NC, si-PVT1, inh NC or miR-159-3p-inh. Glucose uptake, lactate secretion and intracellular ATP levels were quantified and normalized for cell numbers. (F) Cell lysates were then analyzed by western blotting by anti-HIF-1A, anti-LDHA, anti-GULT1, anti-HK2 or anti-β-actin antibodies. (G, H, I, J and K) A total of 2*106 cells after 48 h of transfection with shRNA or PVT1 shRNA for 48h, were then injected subcutaneously into nude mice. Tumor growth curves were measured after injection (n = 6 for each group). Tumor weights were measured after the tumors were removed. The PVT1 and miR-159-3p were detected by RT-PCR. Immunohistochemistry detection indicated HIF-1A. All values are presented as mean ± SD. (** and ## P < 0.01, ***and ### P < 0.001)
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