Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

Figure 1 Expression levels of osteopontin and capacity of proliferation in human gastric cancer cell lines. A: real-time quantitative-reverse transcription analysis of osteopontin (OPN) mRNA levels in the gastric cancer (GC) cell lines (AGS, SGC-7901, HGC-27) and normal human gastric mucosal epithelial cell line (GES-1); B: Western blot assay of OPN protein expression levels in various human GC cell lines; C: Densitometry analysis of the protein bands of OPN proteins; D: The relative proliferation rate at 0, 24, 48 and 96 h in various GC cells by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide assay. Data are shown as the means ± SE (n = 3). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. OPN: Osteopontin.

Figure 2 Osteopontin knockdown inhibits proliferation, invasion and metastasis of gastric cancer cells. A: Fluorescence microscope observation transfection efficiency of SGC-7901 cells after being transfected with osteopontin-short hairpin RNA (shRNA) (× 100); B: 3distinct, sequence-specific OPN shRNAs (OPN-shRNA1; OPN-shRNA2; OPN-shRNA3) and negative control shRNA were designed, and the OPN-shRNA3 has the best interference efficiency of OPN; C: Western blot assay of OPN protein levels in SGC-7901 cells 48  h after transfection; D: Densitometry analysis of the protein bands of OPN proteins in SGC-7901 cells 48  h after they were transfected; E: 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide assay observation the relative proliferation rate at 0, 24, 48 and 96 h in SGC-7901 cells following transfection, compared with control group; F: Microscope observation effect of the invasive ability of OPN knockdown in SGC-7901 cells was assessed by the Transwell matrigel-coated assay (× 100); G: Microscope observation of the migrative ability of OPN knockdown on SGC-7901 cells was assessed by the Transwell assay (× 100); H and I: Cells invading and migrating through the membrane were counted in 3 random fields for respective group. Data are shown as the means ± SE (n = 3). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. OPN: Osteopontin; shRNA: Short hairpin RNA; NC-shRNA: Negative control shRNA.

Figure 3 Osteopontin upregulates matrix metalloproteinase 2 and vascular endothelial growth factor expression. A: Matrix metalloproteinase 2 mRNA level was tested by real-time quantitative-reverse transcription polymerase chain reaction (PCR) and normalized to β-Actin expression; B: Vascular endothelial growth factor mRNA level was detected by quantitative PCR and normalized to β-Actin expression. Data are shown as the means ± SE (n = 3). ^bP < 0.01. MMP-2: Matrix metalloproteinase 2; VEGF: Vascular endothelial growth factor; NC-shRNA: Negative control shRNA.

Figure 4 Osteopontin regulates the matrix metalloproteinase 2 and vascular endothelial growth factor expression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway. A: Western blotting analysis of mammalian target of rapamycin (mTOR), protein kinase B (AKT), phosphorylated AKT (p-AKT) (Ser473), matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF) protein expression levels in SGC-7901 cells after transfected into negative control short hairpin RNA (shRNA) (negative control-shRNA) or osteopontin-shRNA3. β-actin expression was used as a loading control and for normalization; B: Densitometry analysis of the protein bands of mTOR, AKT, MMP-2, p-AKT, p-AKT and AKT, VEGF; C: Western blotting assay of SGC-7901 cells administrated LY294002 inhibitor on mTOR, AKT, p-AKT (Ser473), MMP-2 and VEGF protein expression levels; D: Relative protein expression of mTOR, AKT, MMP-2, p-AKT, p-AKT/AKT and VEGF. Data are shown as the means ± SE (n = 3). ^aP < 0.05; ^bP < 0.01. AKT: Protein kinase B; mTOR: Mammalian target of rapamycin; MMP-2: Matrix metalloproteinase 2; VEGF: Vascular endothelial growth factor; NC-shRNA: Negative control short hairpin RNA; OPN: Osteopontin.