PTBP3 contributes to the metastasis of gastric cancer by mediating CAV1 alternative splicing

Fig. 1. PTBP3 is upregulated in gastric cancer tissues and correlates with poor survival. a Dot plots represent PTBP3 expression levels in gastric cancer tissues (n = 375) and normal gastric tissues (n = 32) according to the data from the TCGA database (***P < 0.001). b Kaplan–Meier analysis of overall survival in patients with variable PTBP3 expression according to the data from the TCGA database (P = 0.042). c Representative images and quantitative IHC analysis of PTBP3 expression in normal gastric mucosa tissue, primary gastric cancer tissue and lymph node metastasis of gastric cancer tissue according to the clinical specimens (**P < 0.01). Scale bar, 50 μm. d PTBP3 mRNA levels (log2 intensity) in primary gastric cancer tissue and lymph node metastasis of gastric cancer tissue (*P < 0.05). e Kaplan–Meier analysis of overall survival in lymph node metastasis of gastric cancer patients with variable PTBP3 expression (P = 0.035). f High expression of PTBP3 correlated with the cell migration signalling pathway, as revealed using the TCGA, GSE62245, GSE15459, and GSE57303 datasets. NES normalised enrichment score. Each bar represents the mean ± SD of three independent experiments

Fig. 2. PTBP3 promotes the migration and invasion of gastric cancer cells. a Western blot and real-time PCR analyses of PTBP3 expression in the indicated cells with PTBP3 overexpression (***P < 0.001). b Wound-healing assay showing the migration ability of MKN45 and SGC7901 with PTBP3 overexpression (***P < 0.001). Scale bar, 200 μm. c Transwell assay showing the migration and invasion ability of the indicated cells with PTBP3 overexpression (***P < 0.001). Scale bar, 200 μm. Colony formation assay (d) and growth curve analysis (e) showing that PTBP3 overexpression had no effect on the proliferation rates of MKN45 and SGC7901. Each bar represents the mean ± SD of three independent experiments

Fig. 3. PTBP3 knockdown inhibits the migration and invasion of gastric cancer cells. a Western blot and real-time PCR analyses PTBP3 expression in the indicated cells with PTBP3 knockdown (***P < 0.001). b Wound-healing assay showing the migration ability of MKN45 and SGC7901 cells with PTBP3 knockdown (***P < 0.001). Scale bar, 200 μm. c Transwell assay showing the migration and invasion ability of the indicated cells with PTBP3 knockdown (***P < 0.001). Scale bar, 200 μm. Colony formation assay (d) and growth curve analysis (e) showing that PTBP3 knockdown had no effect on the proliferation rates of MKN45 and SGC7901. Each bar represents the mean ± SD of three independent experiments

Fig. 4. PTBP3 knockdown suppresses axillary lymph node metastasis in vivo. a The weight of tumours derived from the pSilence vector and PTBP3 KD-transfected MKN45 cells were calculated (**P < 0.01). b Immunohistochemical staining shown for LYVE-1 to identify lymphatic vessels (red arrows) at the tumour margin and LYVE-1-stained vessels in three microscopic fields of the highest vessel density were counted (**P < 0.01). c Typical lymph nodes in mice bearing the MKN45-PTBP KD and MKN45-pSilence tumours and lymph node volume were calculated (***P < 0.001). d Histologic staining of lymph node sections from mice with MKN45-PTBP KD and MKN45-pSilence tumours, respectively. Red arrows indicate the tumour cells in the lymph node. Scale bar, 200 μm

Fig. 5. PTBP3 modulates CAV1 alternative splicing via binding to its intronic region. a Co-expression correlation analysis of two pairs of proteins in a clinical cohort of gastric cancer tissue samples (n = 69) by IHC. Images show individual protein staining in gastric cancer tissues from lymph node metastatic patients. Scale bars, 50 μm. Average cell-based staining intensity counts for each individual protein expressed in tumour cells were analysed using inForm software to assess co-expression correlations. b Western blot analysis of PTBP3 and CAV1α expressed in mouse tumours from PTBP3 knockdown and pSilence vector-transfected mice. c Schematic diagram of CAV1 alternative splicing. Real-time PCR (d) and western blot (e) analysis of the expression of PTBP3, CAV1α, and CAV1β in MKN45 cells with PTBP3 overexpression or knockdown (**P < 0.01 compared with pcDNA, ##P < 0.01 and #P < 0.05 compared with pSilence). f RIP experiments were performed using the PTBP3 antibody to immunoprecipitate in total cell extracts of MKN45-WT, MKN45-MU, MKN45-Del cells, and relative enrichment was determined by reverse transcription-PCR (left panel) and quantitative real-time PCR (right panel) (***P < 0.001).…

Fig. 6. PTBP3 knockdown inhibits the migration and invasion of gastric cancer by the upregulation of CAV1α and followed the integrin/Src/FAK pathway. a Western blot analysis of PTBP3, CAV1, and CAV1α expressed in MKN45 cells with PTBP3 siRNA, CAV1 siRNA or CAV1α siRNA treatment. b Inhibition/overexpression of CAV1 affected the cell migratory ability in MKN45 cells (***P < 0.001). Scale bar, 500 μm. c Wound-healing assay showing the migration ability of the indicated cells. MKN45 and SGC7901 cells with PTBP3 knockdown were exposed to CAV1α-siRNA (***P < 0.001). Scale bar, 200 μm. d Transwell assay showing the migration and invasion ability of the indicated cells. MKN45 and SGC7901 cells with PTBP3 knockdown were exposed to CAV1α-siRNA (**P < 0.01, ***P < 0.001). Scale bar, 200 μm. e Western blot analysis with antibodies directed against CAV1α, FAK, p-FAK(Y397), Src, p-Src(Y418), and integrinβ3 was performed. β-Actin was used as a loading control. PTBP3 knockdown induced the reduction of integrinβ3, p-Src, and p-FAK and was abrogated by the knockdown of CAV1α in MKN45-KD cells. Each bar represents the mean ± SD of three independent experiments. f Schematic diagram of PTBP3 knockdown…