lncRNA H19 binds VGF and promotes pNEN progression via PI3K/AKT/CREB signaling

in Endocrine-Related Cancer
Correspondence should be addressed to C Shao: shaochenghao_czyy@163.com

*(M Ji and Y Yao contributed equally to this work)

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Pancreatic neuroendocrine neoplasms (pNENs) are endocrine tumors arising in pancreas and is the most common neuroendocrine tumors. Mounting evidence indicates lncRNA H19 could be a determinant of tumor progression. However, the expression and mechanism of H19 and the relevant genes mediated by H19 in pNENs remain undefined. Microarray analysis was conducted to identify the differentially expressed lncRNAs in pNENs. H19 expression was analyzed in 39 paired pNEN tissues by qPCR. The biological role of H19 was determined by functional experiments. RNA pulldown, mass spectroscopy and RNA immunoprecipitation were performed to confirm the interaction between H19 and VGF. RNA-seq assays were performed after knockdown H19 or VGF. H19 was significantly upregulated in pNEN tissues with malignant behaviors, and the upregulation predicted poor prognosis in pNENs. In vitro and in vivo data showed that H19 overexpression promoted tumor growth and metastasis, whereas H19 knockdown led to the opposite phenotypes. H19 interacted with VGF, which was significantly upregulated in pNENs, and higher VGF expression was markedly related to poor differentiation and advanced stage. Furthermore, VGF was downregulated when H19 was knocked down, and VGF promoted cell proliferation, migration and invasion. Mechanistic investigations revealed that H19 activated PI3K/AKT/CREB signaling and promoted pNEN progression by interacting with VGF. These findings indicate that H19 is a promising prognostic factor in pNENs with malignant behaviors and functions as an oncogene via the VGF-mediated PI3K/AKT/CREB pathway. In addition, our study implies that VGF may also serve as a candidate prognostic biomarker and therapeutic target in pNENs.

Downloadable materials

  • Supplementary figure 1 Volcano plot of the upregulated and downregulated genes in H19-knockdown cells (a) and VGF-knockdown cells (b).
  • Supplementary figure 2 Gene ontology (GO) enrichment analysis of the two differentially expressed gene sets.
  • Supplementary figure 3 Protein-protein interactions (PPIs) network between the first 300 genes of the two differential gene sets.
  • Supplementary table 1 shRNA sequences of H19 and VGF
  • Supplementary table 2 PCR primers used in the manuscript

 

      Society for Endocrinology

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    High expression of H19 is correlated with poor prognosis in pNEN patients. (A) Heatmap of differentially expressed lncRNAs in 5 pNEN tumor tissue and paired nontumor tissue samples. T, tumor tissues; N, nontumor tissues. (B) Relative H19 expression in tissues (n = 39) identified by qPCR. (C and D) Relative H19 expression in pNEN tissues with no malignant behavior, malignant behavior and liver metastasis using PCR and qPCR assays. 14, 09, 24, 20, 37, 32 and 39 represent the patient number; N represents the adjacent tissues; T represents the tumor tissues, 37liver, 32liver and 39liver correspond to the patient's liver metastatic tumor tissue. (E) Kaplan–Meier survival analysis demonstrated that patients with high H19 expression had poor progression-free survival (PFS) compared to the PFS of those with low H19 expression. (F) OS curve of the high-level and low-level groups divided by H19 expression. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.) A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0552.

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    H19 promotes cell proliferation, migration and invasion in pNENs. (A) Baseline lncRNA H19 levels in eight cell lines (24N, 20N, 37N, 24T, 20T, 37T, QGP-1 and 37 liver) identified by qPCR assay. (B) The H19 knockdown efficiencies were verified by qPCR assay. CT represents cell transfection with control lentivirus; 1A, 2A and 3A represent cells transfected with three kinds of H19 shRNA lentiviruses. (C, D and E) Proliferation of H19-knockdown and control QGP-1 and pNEN primary tumor cells. (F) Colony formation assays of H19-knockdown and control QGP-1 cells. (G) Cell morphology of H19-knockdown and control QGP-1 cells. (H) Transwell assays showed that the invasiveness and migration capabilities of QGP-1 cells and 37T cells were significantly reduced when H19 expression was decreased. (I) The H19 overexpression efficiencies were verified by qPCR assay. NC represents cells transfected with control lentivirus; H19-OE represents cells transfected with H19 overexpression lentivirus. (J, K and L) Proliferation of H19-overexpressing and control QGP-1 cells and pNEN primary tumor cells. (M) Colony formation assays of H19-overexpressing and control QGP-1 cells. (N) Cell morphology of H19-overexpressing and control QGP-1 cells. (O and P) Transwell assays showed that the invasiveness and migration capabilities of QGP-1 cells and 37T cells were significantly increased when H19 was overexpressed. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.) A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0552.

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    H19 promotes pNEN tumor growth in vivo. (A, B, F and G) Representative images of tumors formed in nude mice injected subcutaneously with QGP-1 cells are shown. (A and B: H19 knockdown and control. F and G: H19 overexpression and control). (C and H) The tumor volume was periodically measured for each mouse, and the tumor growth curve was plotted. (D and I) The body weights were periodically measured for each mouse, and the body weight curve was plotted. (E and J) The tumor weights were measured when the tumors were harvested. (K) Representative images (×200) of IHC staining of Ki67 in the tumors. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.) A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0552.

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    lncRNA H19 interacts with VGF and regulates VGF expression in pNENs. (A) Proteins retrieved from the H19-pulldown assay were analyzed by SDS-PAGE. (B) Western blotting confirmed the significant enrichment of VGF with lncRNA H19 using an anti-VGF antibody. (C and D) RIP assay was performed in QGP-1 cells to determine the binding condition between lncRNA H19 and VGF. (E, F, G and H) Assessing VGF expression in H19-knockdown and H19-overexpressing QGP-1 cells with Western blotting and qPCR assays. (I) Representative images (×200) of IHC staining of VGF in H19-knockdown or H19-overexpressing xenograft tumors. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.) A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0552.

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    VGF, which promotes pNEN cell proliferation, migration and invasion, is elevated in pNENs and correlated with pNEN aggressiveness. (A, B and C) VGF expression in 39 paired pNEN tissues was measured by IHC, qPCR and Western blotting. (D) Pearson’s correlation analysis showed a positive correlation between the expression levels of H19 and VGF in pNEN tissues (r = 0.741, P < 0.0001, n = 39). (E) Kaplan–Meier survival analysis demonstrated that patients with high VGF expression had poor progression-free survival (PFS) compared to the PFS of those with low VGF expression. (F) Overall survival curve of the high-level and low-level groups divided by VGF expression. (G and H) The VGF knockdown and overexpression efficiencies were verified by qPCR and Western blotting assays. CT represents cells transfected with control lentivirus; shVGF-a and shVGF-b represent cells transfected with two kinds of VGF shRNA lentivirus. NC represents cells transfected with control lentivirus; VGF-OE represents cells transfected with VGF overexpression lentivirus. (I and J) Cell proliferation of VGF-knockdown, VGF-overexpressing and the corresponding control QGP-1 cells and pNEN primary tumor cells. (K) Transwell assays showed that the invasiveness and migration capability of QGP-1 cells were significantly changed when H19 was knocked down or overexpressed. (L) Overexpression of VGF partially attenuated the decreased cell proliferation caused by H19 knockdown in QGP-1 cells. Blank represents QGP-1 cells with no treatment; CT represents QGP-1 cells transfected with shRNA control lentivirus; NC represents QGP-1 cells transfected with overexpression control lentivirus; 3A represents QGP-1 cells transfected with H19 shRNA-3A lentivirus; 3A + VGF represents QGP-1 cells transfected with H19 shRNA-3A lentivirus and VGF overexpression lentivirus. (M) Overexpression of VGF partially attenuated the decreased cell migration and invasion capacity caused by H19 knockdown in QGP-1 cells. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.) A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0552.

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    H19 interacts with and triggers VGF expression, which could activate the PI3K/AKT/CREB pathway in pNENs. (A) Heatmap of differentially expressed mRNAs in H19-knockdown cells, VGF-knockdown cells and control cells. (B) Enriched KEGG pathways in both differential gene sets. (C and D) Western blotting showing the protein expression of PI3K, P-PI3K, AKT, P-AKT, P-CREB, CREB and CBP in QGP-1 cells and xenografted tumors. (E) Western blotting showing that VGF-OE rescued the inhibitory effect of H19-3A on the expression of P-PI3K, P-AKT, P-CREB, and CBP. A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0552.

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