Fig 1: THBS2 regulates the CUX1/MMP9 cascade. (A) CUX1 directly interacted with the MMP9 promoter. qPCR analysis following ChIP revealed enrichment of the MMP9 promoter sequence in the anti-CUX1-precipitated DNA compared with the IgG group. (B) Luciferase reporter analysis after 293 cells were co-transfected with plasmid, with or without CUX1 and reporter, with or without MMP9 promoter for 48 h. (C) qPCR and (D) western blot analysis demonstrating the expression of CUX1 after THBS2 was overexpressed or knocked-down. (E and F) qPCR analysis of the MMP9 sequences bound with CUX1 after ChIP analysis in NC or (E) THBS2 OE QGP-1 and (F) BON-1 cells. (G and H) The relative luciferase activity was detected after NC or (G) THBS2 OE QGP-1 and (H) BON-1 cells were co-transfected with plasmid with or without CUX1 and reporter with or without MMP9 promoter for 48 h. (I-K) MMP9 expression was detected by qPCR (I and J) and western blot (K) after THBS2 or CUX1 was overexpressed or knockdown. **P<0.01, ***P<0.001 or ****P<0.0001 vs. NC or siNC; ##P<0.01 vs. THBS2 or siTHBS2; ###P<0.001 or ####P<0.0001 vs. CUX1+MMP9 promoter of NC cells; &&P<0.01 or &&&P<0.001 vs. NC of THBS2 OE cells. THBS2, thrombospondin 2; CUX1, CUT-like homeobox 1; MMP, matrix metalloproteinase; qPCR, quantitative PCR; OE, THBS2 lentivirus; NC, negative control; NS, not significant; si, small interfering.
Fig 2: THBS2 is downregulated in pNET. (A) Gene Expression Omnibus dataset (GSE73338) analysis of THBS2 expression in pNET specimens compared to normal controls. (B) The expression of THBS2 in 10 pNET tissues and the paired para-carcinoma tissues were detected by qPCR. (C) Hematoxylin and eosin stain and immunohistochemistry stain of pNET and the paired para-carcinoma tissues Hematoxylin and eosin stain magnification, ×40; immunohistochemistry stain magnification, ×100. (D) The expression of THBS2 in pNET cell lines QGP-1 and BON-1 and normal human cell line 293 and CCL-153 were detected by qPCR. **P<0.01 vs. CTRL; &&P<0.01 and &&&P<0.001 vs. 293 cells; ###P<0.001 vs. CCL-153 cells. THBS2, thrombospondin 2; pNET, pancreatic neuroendocrine tumor; qPCR, quantitative PCR; CTRL, control.
Fig 3: THBS2 was a direct target of miR-744-5p. (A) Venn diagram demonstrating that 12 genes are putative miR-744 targets predicted by TargetScan, miRTarBase and RegRNA2.0 database. (B) The correlation between miR-744-5p and THBS2 in pancreas-adenocarcinoma-other subtype from the LinkedOmics databases. (C) qPCR analysis of the expression of miR-744-5p in BON-1, QGP-1 and 293 cells. (D) Luciferase activity of wt THBS2 reporter and mutant THBS2 reporter constructs in 293 cells following transfection of miR-744-5p mimics. (E) qPCR and (F) western blot analysis the expression of THBS2 following transfection of miR-744-5p mimics or inhibitor. (G) The expression of THBS2 was negatively correlated with miR-744-5p in pNET tissues. $$$$P<0.0001 vs. 293, &&&P<0.001 vs. THBS2 wt+mi-NC, ****P<0.0001 vs. ni-NC, ####P<0.0001 vs. mi-NC. THBS2, thrombospondin 2; miR, microRNA; qPCR, quantitative PCR; wt, wild-type; Mut, mutation; NC, negative control.
Fig 4: THBS2 negatively regulated Akt/NF-?B signaling pathway during OD procedure of VICs. (a–c) Representative images of protein bands during Western blot (a) and the levels of p-Akt/Akt (b) and p-p65/p65 (c) of VICs transfected with shTHBS2 and THBS2 overexpression plasmid were assessed by Western blot after the induction of OD. GAPDH was a loading control. (d) Relative expression of p65 in nucleus of VICs transfected with shTHBS2 and THBS2 overexpression plasmid was assessed by Western blot after the induction of OD. Lamin B1 was a loading control. (e) Relative mRNA expressions of THBS2, Akt, and p65 in VICs transfected with shTHBS2 and treated with LY294002 were tested by qRT-PCR after the induction of OD. GAPDH was a loading control. (f) Representative images of protein bands during Western blot. (g and h) The levels of p-Akt/Akt (g) and p-p65/p65 (h) of VICs transfected with shTHBS2 and treated with LY294002 were determined through Western blot after the induction of OD. GAPDH was a loading control. (i) Relative protein expression of THBS2 in VICs transfected with shTHBS2 and treated with LY294002 was determined through Western blot after the induction of OD. GAPDH was a loading control. (j) and (k) Relative mRNA (j) and protein (k) expressions of p65 in nucleus of VICs transfected with shTHBS2 and treated with LY294002 were detected by Western blot after the induction of OD. Lamin B1 was a loading control. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. vector group; ***p < 0.01 vs. shTHBS2 group; ?p < 0.05; ??p < 0.01; ???p < 0.001 vs. LY294002 group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. Abbreviations: THBS2: thrombospondin 2; OD: osteogenic differentiation; VICs: valve interstitial cells; p-: phosphorylation-; shTHBS2: short hairpin THBS2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR: quantitative reverse transcription-polymerase chain reaction.
Fig 5: A comparison of expression levels of the 10 hub genes in different stages of GC. (A–J) respectively shows the BGN, COL1A2 COL3A1, COL4AI, COL5A1, COL5A2, FN1, SPARC, THBS1 and THBS2 expression level. There were significant variations in the expression levels of BGN (A), COL1A2 (B), COL3A1 (C), COL5A1 (E), COL5A2 (F), SPARC (H), THBS1 (I), and THBS2 (J) in GC patients from different stages.
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