JAG1, Regulated by microRNA-424-3p, Involved in Tumorigenesis and Epithelial-Mesenchymal Transition of High Proliferative Potential-Pituitary Adenomas

Abstract

Pituitary adenomas (PAs) are a neoplastic proliferation of anterior pituitary. Signature of Notch pathway relies upon the histopathological type of PAs. The details of Notch pathway that are involved in the migration and invasion of Pas are still unclear. This paper filters and testifies the relation between Notch signaling pathway and the migration/invasion in subtypes of PAs. The diversity of genes and pathways is investigated based on transcriptome data of 60 patients by KEGG pathway analysis and GSEA. A series of functional experiments demonstrate the role of candidate genes by overexpression and antibody blocking in GH3 cell line. Volcano map and GSEA results exhibit the differential and the priority of Jagged1 canonical Notch Ligand (JAG1) in the Notch pathway combined with clinical features. JAG1 is involved in epithelial-mesenchymal transition (EMT) in PAs by correlation analysis of RNA-seq data. Progression-free survival (PFS) of patients with high JAG1 was shorter than patients with low JAG1 according to follow-up data (P = 0.006). Furthermore, overexpression and antibody blocking experiments in GH3 cell line indicate that JAG1 could promote cell proliferation, migration, and G1/S transition. Double luciferase reporter assay gives manifests that JAG1 is the target gene of miR-424-3p, and mimics or inhibitor of miR-424-3p can regulate the level of JAG1 which, in turn, affects cell proliferation and the levels of MMP2 and VIM in GH3 cell line, respectively. Our study delves into the relation between the Notch signaling pathway and cell proliferation and EMT in PAs, providing a potential treatment through targeting JAG1.

Keywords: Jagged1 canonical Notch Ligand; Notch signaling; epithelial–mesenchymal transition; five-tiered prognostic classification of PitNETs; miR-424-3p; pituitary adenoma.

Figure 1

Transcriptome of 60 PA patients. (A) Heatmap of unsupervised hierarchical clustering of the top 490 most variable genes. Pathological and clinical annotations were provided. (B) KEGG pathway analysis based on the diversity of total productive maintenance (TPM) values (Log₂FC <−1 or >1, P < 0.05). (C) The most Enriched GO Terms. (D) Volcano map of transcriptome. Notch signaling pathway, cell circle signature, EMT signature and MMP family were provided. Log₂FC<−1 or >1, P < 0.05 (E) GSEA showed JAG1’s position in Notch signaling pathway.

Figure 2

Notch signaling pathway involved into the tumorigenic and recurrence of PA. (A) Heatmap of key gene median expression including angiogenesis, high mobility group family and Notch signaling pathway. Pathological and clinical annotations were provided. (B) RNA-seq quantitative levels (TPM) of Notch pathway and genes related to EMT. (C) The correlation analysis between Notch pathway and EMT. (D) The correlation of between JAG1 and Notch receptors, genes related to EMT. Log₂FC<−1 or >1, P<0.01 (E) PFS time according the levels of JAG1, MMP2, SNAI1 and VIM. Log₂FC<−1 or >1, P <0.01. **p < 0.01, ***p < 0.001.

Figure 3

Levels of JAG1 and MMP2 in PA patients. (A) MR images of PA patients. (B) Bands of western blot on group 1/2a and group 1/2b. (C) Immunohistochemistry image of JAG1, MMP2 and Ki-67. (D) mRNA levels of JAG1, MMP2, SNAI1, and VIM. *compared to group 1/2a P<0.05 ***P<0.001. NS, no sense.

Figure 4

Overexpression JAG1 promoted cell proliferation, cell cycle and migration in GH3 cell line. (A) JAG1 promoted the GH3 cell line in the time manner. (B, C) JAG1 increased the percent of S phase of GH3 cell line. (D) Transwell experiment showed that JAG1 increased the number of trans-membrane positive GH3 cells. (E) RT-qPCR experiment showed that JAG1 increased the mRNA levels of MMP2, SNAI1 and VIM in GH3 cells. (F) Western blot experiment showed JAG1 increased the protein levels of MMP2, SNAI1 and VIM in GH3 cells. *compared to Vector group P<0.05 **P<0.01 ***P<0.001. NS, no sense.

Figure 5

Anti-JAG1 antibody on cell proliferation, apoptosis, and migration of GH3 cell line. (A) Anti-JAG1 antibody inhibited the cell viability of GH3 cells in the time and dose manner. (B, C) Anti-JAG1 antibody increased the Annexin V positive and PI positive cells in dose manner after 48 h treatment. (D) Anti-JAG1 antibody (10 μg/ml) reduced the trans-membrane positive cell after 48 h treatment. (E) Western blot experiment showed Anti-JAG1 antibody (10 μg/ml) reduced the protein levels of MMP2, SNAI1, and VIM in GH3 cells. (F) RT-qPCR experiment showed that Anti-JAG1 antibody (10 μg/ml) reduced the mRNA levels of MMP2, SNAI1, and VIM in GH3 cells. * compared to control group P < 0.05 **P < 0.01 ***P < 0.001. NS, no sense.

Figure 6

Inhibition of miR-424-3p on GH3 cells by targeting JAG1. (A) miRNA differential expression targeting JAG1 between pituitary (n = 6) and patients (n = 20). (B) Volcano map of miRNAs showed four down-regulated miRNAs and two up-regulated miRNAs in patients compared to those in normal pituitary. Log₂FC <−2 or >2, P < 0.01 (C) Correlation analysis between JAG1 and down-regulated miRNAs (miR-424-3p r = −0.47, miR-450a-5p r = −036, miR-509-3-5p, r = −0.3, miR-514a-3p, r = −0.28). Log₂FC <−2 or >2, P < 0.01 (D) Pattern diagram showed the binding site of JAG1 and miR-424-3p. (E) The luciferase assay showed WT-JAG1/MT-JAG1 and miR-424-3p co-transfection in GH3 cells. *compared to control group P < 0.05 (F) The effect of mimic and inhibitor of miR-424-3p on f GH3 cells. ***compared to NC group P < 0.001 (G) RT-qPCR experiment showed that mimic and inhibitor of miR-424-3p regulated the mRNA levels of MMP2 and VIM by targeting JAG1 in GH3 cells. *compared to NC group P < 0.05 **P < 0.01 ***P < 0.001 (H) Western blot experiment showed that mimic and inhibitor of miR-424-3p regulated the protein levels of MMP2 and VIM by targeting JAG1 in GH3 cells. NS, no sense.