Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: STAT3/miR-130b-3p/MBNL1 feedback loop regulated by mTORC1 signaling promotes angiogenesis and tumor growth

doi: 10.1186/s13046-022-02513-z

Figure Lengend Snippet: miR-130b-3p promotes angiogenesis via targeting MBNL1. A-D Tsc2 − / − MEFs were infected with lentiviruses harboring control vector (Lv) or MBNL1 overexpression constructs (Lv-MBNL1). A The expression of MBNL1 was determined by western blot. B Cell culture supernatants from the cells were analyzed for VEGF-C by ELISA. (RU, relative unit). C and D The effect on angiogenesis of was detected by tube formation assay ( C , scale bar, 50 μm) and CAM assay ( D ). E–G anti-miR-130b-3p-expressing NTC/T2 null cells were infected with lentivirus harboring shRNAs targeting MBNL1 (shMBNL1-1 and shMBNL1-2) or a control shRNA (shSc). E Cell lysates were subjected to immunoblotting with the indicated antibodies. F and G The angiogenic abilities of the indicated cells were detected by tube formation assay ( F , scale bar, 50 μm) and CAM assay ( G ). Representative images (left panels) and quantifications (right panels). H–K The indicated cells were inoculated subcutaneously into nude mice ( n = 5), and tumor growth was monitored. H Tumor pictures. Scale bar, 1 cm. I Tumor volumes, J Tumor weight. K Tumor tissues were subjected to HE and IHC staining. Scale bar, 50 μm. Data indicate mean ± SD of 3–5 replicates. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The levels of secreted VEGF-C in cell-free supernatant of MBNL1-overexpressing cells and the control cells were quantified using a mouse VEGF-C ELISA Kit (Novus Biologicals, CO, USA) according to the manufacturer’s instructions.

Techniques: Infection, Control, Plasmid Preparation, Over Expression, Construct, Expressing, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Tube Formation Assay, Chick Chorioallantoic Membrane Assay, shRNA, Immunohistochemistry

Journal: International Journal of Oncology

Article Title: OxLDL promotes lymphangiogenesis and lymphatic metastasis in gastric cancer by upregulating VEGF-C expression and secretion

doi: 10.3892/ijo.2018.4648

Figure Lengend Snippet: Plasma oxLDL in patients with gastric cancer is positively correlated with lymph node metastasis. (A) The association between plasma oxLDL and lymph node metastasis in gastric cancer (case numbers: N0=8, N1=4, N2=4, N3a=5 and N3b=7; * P<0.05, compared with N0). (B) The correlation between the plasma oxLDL and the LVD was determined by Pearson’s correlation analysis (n=17; r=0.79 and P<0.05). Plasma oxLDL in patients with gastric cancer was detected via ELISAs. Lymphatic vessels in gastric cancer tissues were stained with D2-40. LVD was calculated using the number of vessels per field. (C) The correlation between the plasma VEGF-C and the LVD. Plasma VEGF-C in patients with gastric cancer was detected by ELISAs (n=17, r=0.87 and P<0.05). (D) Lymphatic vessel in adjacent tissues and gastric cancer tissues were stained with D2-40 by immunohistochemistry. Histograms represent the number of lymphatic tubes per field (for adjacent and gastric cancer tissues n=17). (E) Tube formation of LEC incubated with the condition medium from the culture medium of HGC-27 treated by nLDL, oxLDL or siLOX-1+oxLDL for 24 h ( * P<0.05 vs, Ctrl). A total of 2×10 4 human lymphatic endothelial cells were seeded with 200 µ l conditioned medium in each well and incubated for 12 h, and then the tube formation was observed with microscope. Results are presented as the means ± standard deviations. N0, no regional lymph node metastasis; N1, 1-2 regional lymph node metastases; N2, 3-6 regional lymph node metastases; N3, 7 or more regional lymph node metastases; N3a, 7-15 regional lymph nodes metastases; N3b, >16 regional lymph nodes with metastasis; LVD, lymphatic vessel density; VEGF-C, vascular endothelia growth factor-C; oxLDL, oxidized low-density lipoprotein; Ctrl, control; nLDL, native LDL, siLOX-1, small interfering lectin-like oxLDL.

Article Snippet: Finally, the supernatants were centrifuged (4°C at 300 × g for 10 min), and the concentration of oxLDL and VEGF-C was measured using an ELISA kit specific for VEGF-C (oxLDL kit; cat. no. STA-388; Cell Biolabs, Inc., San Diego, CA, USA; and VEGF-C kit; cat. no. DVEC00; R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Clinical Proteomics, Staining, Immunohistochemistry, Incubation, Microscopy, Control

Journal: International Journal of Oncology

Article Title: OxLDL promotes lymphangiogenesis and lymphatic metastasis in gastric cancer by upregulating VEGF-C expression and secretion

doi: 10.3892/ijo.2018.4648

Figure Lengend Snippet: OxLDL promotes the expression and secretion of VEGF-C. (A) Plasma oxLDL was correlated with plasma VEGF-C in patients with gastric cancer. Plasma oxLDL and VEGF-C levels in patients with gastric cancer were detected using ELISA kits. The number of patients was 23 (r=0.64 and P<0.05). (B) VEGF-C mRNA levels in HGC-27 cells treated with 50 µ g/ml nLDL or oxLDL for 24 h, as measured by reverse transcription-quantitative polymerase chain reaction. A total of three independent experiments were performed. (C) VEGF-C expression levels were upregulated by nLDL and oxLDL in AGS, HGC-27, MGC-803 and SGC-7901 gastric cancer cell lines. (D) OxLDL dose-dependently downregulated VEGF-C expression in gastric cancer cells. (E) OxLDL time-dependently downregulated VEGF-C expression in gastric cancer cells. VEGF-C protein levels in cell lysates were measured by western blotting, with β-actin as the control. (F) To measure the effect of oxLDL on the secretion of VEGF-C by HGC-27 and SGC-7901 gastric cancer cells, VEGF-C was detected in the supernatants using an ELISA kit. * P<0.05, results are presented as the means ± standard deviations. OxLDL, oxidized low-density lipoprotein; Ctrl, control; nLDL, native LDL; VEGF-C, vascular endothelia growth factor-C.

Article Snippet: Finally, the supernatants were centrifuged (4°C at 300 × g for 10 min), and the concentration of oxLDL and VEGF-C was measured using an ELISA kit specific for VEGF-C (oxLDL kit; cat. no. STA-388; Cell Biolabs, Inc., San Diego, CA, USA; and VEGF-C kit; cat. no. DVEC00; R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: International Journal of Oncology

Article Title: OxLDL promotes lymphangiogenesis and lymphatic metastasis in gastric cancer by upregulating VEGF-C expression and secretion

doi: 10.3892/ijo.2018.4648

Figure Lengend Snippet: NF-κB signaling is involved in the regulation of VEGF-C expression by oxLDL. (A) Activation of the NF-κB signaling pathway in HGC-27 cells following oxLDL treatment. IκB/p-IκB, P65/p-P65 and β-actin in gastric cancer cell lysates were measured by western blot analysis. (B) Translocation of NF-κB and P65 following oxLDL treatment; HGC-27 cells were incubated with 50 µ g/ml oxLDL for 6 h. (C) The amount of P65 that was translocated into the nucleus following oxLDL treatment was measured by western blotting. HGC-27 cells were incubated with 50 µ g/ml nLDL and oxLDL for 12 h. (D) The upregulated VEGF-C level was rescued by the NF-κB inhibitor PDTC. The protein levels were measured via western blotting. (E) VEGF-C levels in the supernatants were detected using an ELISA kit. (F) The knockdown effect of P65 siRNA was verified by western blotting. (G) The upregulated VEGF-C level was rescued by P65 siRNA. P65/p-P65 and VEGF-C protein levels in gastric cancer cell lysates were measured via western blotting. * P<0.05, results are presented as the means ± standard deviations. OxLDL, oxidized low-density lipoprotein; Ctrl, control; nLDL, native LDL; VEGF-C, vascular endothelia growth factor-C; NF-κB, nuclear factor-κB; p-, phospho-; si, small interfering; PDTC, pyrrolidine dithiocarbamic acid, ammonium salt; IκB, inhibitor of κB α; NS, non-significant.

Article Snippet: Finally, the supernatants were centrifuged (4°C at 300 × g for 10 min), and the concentration of oxLDL and VEGF-C was measured using an ELISA kit specific for VEGF-C (oxLDL kit; cat. no. STA-388; Cell Biolabs, Inc., San Diego, CA, USA; and VEGF-C kit; cat. no. DVEC00; R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Expressing, Activation Assay, Western Blot, Translocation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Knockdown, Control

Journal: International Journal of Oncology

Article Title: OxLDL promotes lymphangiogenesis and lymphatic metastasis in gastric cancer by upregulating VEGF-C expression and secretion

doi: 10.3892/ijo.2018.4648

Figure Lengend Snippet: OxLDL promotes the activation of the NF-κB signaling pathway, as well as the expression and secretion of VEGF-C, through LOX-1. (A) Validation of siRNA efficacy in HGC-27 cells. (B) The effects of oxLDL on NF-κB activity and VEGF-C expression were blocked following the knockdown of LOX-1. VEGF-C protein levels in the cell lysates were measured via western blotting, (C) supernatant VEGF-C levels were detected using an ELISA kit. (D) The regulatory effects of oxLDL on NF-κB activity and VEGF-C expression were blocked following LOX-1 being inhibited by 250 µ g/ml Poly I. VEGF-C protein levels in the cell lysates were measured via western blotting, (E) supernatant VEGF-C levels were detected using an ELISA kit. (F) Schematic depicting the mechanism by which oxLDL promotes lymph node metastasis in gastric cancer. Results are presented as the means ± standard deviations. NS, non-significant; IKK, IBκ kinase; OxLDL, oxidized low-density lipoprotein; Ctrl, control; nLDL, native LDL; VEGF-C, vascular endothelia growth factor-C; NF-κB; nuclear factor-κB; p-, phospho-; si, small interfering; LOX, lectin-like oxLDL; IκB, inhibitor of κB α; NCsiRNA, non-specific control siRNA; poly I, polyinosinic acid.

Article Snippet: Finally, the supernatants were centrifuged (4°C at 300 × g for 10 min), and the concentration of oxLDL and VEGF-C was measured using an ELISA kit specific for VEGF-C (oxLDL kit; cat. no. STA-388; Cell Biolabs, Inc., San Diego, CA, USA; and VEGF-C kit; cat. no. DVEC00; R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Activation Assay, Expressing, Biomarker Discovery, Activity Assay, Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: British Journal of Cancer

Article Title: Clinical implication of expression of vascular endothelial growth factor-C in metastatic lymph nodes of uterine cervical cancers

doi: 10.1038/sj.bjc.6601963

Figure Lengend Snippet: VEGF-C levels in primary tumour and metastatic lymph node lesions of uterine cervical cancers. VEGF-C levels were determined using a VEGF-C ELISA kit (MBL, Nagoya, Japan). Each level is the mean±s.d. of nine determinations from three parts of the same tissue in triplicate. In the primary tumours and the corresponding metastatic lymph node lesions, alive and deceased cases are numbered in ○ and •, respectively. Bold lines, increased significantly ( P <0.05 vs each primary tumour) from the primary tumour to the corresponding metastatic lymph node lesion. Broken lines, no change between the primary tumour and the corresponding metastatic lymph node lesion.

Article Snippet: VEGF-C levels were determined using a VEGF-C ELISA kit (MBL, Nagoya, Japan).

Techniques: Enzyme-linked Immunosorbent Assay