Journal: Journal of Cancer

Article Title: Downregulation of miR-375 contributes to ERBB2-mediated VEGFA overexpression in esophageal cancer

doi: 10.7150/jca.63836

Figure Lengend Snippet: Expression of miR-375 and its target genes in EC . A . Expression of miR-375, ERBB2 and VEGFA in EC cell lines by qRT-PCR. Expression of miR-375 in EC cell lines are significantly decreased compared to HET-1A, a non-cancerous epithelial esophageal cell line. B and C. An inverse correlation between miR-375 and ERBB2/VEFGA expression pattern were observed in EC cell lines. D. Decreased miR-375 in pure population of tumor and dysplasia cells microdissected from FFPE tissue. The level of miR-375 expression was significantly decreased in 11 of 14 ESCC compared to their adjacent dysplasia. E. The level of miR-375 expression was significantly reduced in 23 of the 26 in ESCC compared with the adjacent normal tissues. F. Expression of miR-375 in normal, dysplasia and EC cells. Values represent the mean ± S.D. from three independent experiments. (*p <0.05).

Article Snippet: The following antibodies were used: anti-rabbit HER-2 (PA5-14635, ThermoFisher), anti-rabbit VEGF (PA5-16754, ThermoFisher), anti-mouse Phospho-AKT1 (44621G, ThermoFisher), anti-rabbit beta actin (PA1-183, ThermoFisher).

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of Cancer

Article Title: Downregulation of miR-375 contributes to ERBB2-mediated VEGFA overexpression in esophageal cancer

doi: 10.7150/jca.63836

Figure Lengend Snippet: miR-375 inhibits cell proliferation that rescued by re-expression of ERBB2 . A. miR-375 inhibits cell proliferation. The EC cell lines were transfected with mock control, miR-375 mimic, inhibitor control and miR-375 inhibitor; and cell viability was measured by MTT assay. Transfection of miR-375 mimic significantly reduced the proliferation compared to that of mock control in EC cell lines. Values represent the mean ± S.D. from three independent experiments. (** p < 0.01, *p <0.05). B . Rescue experiment showed that the anti-proliferative effect of miR-375 is reversed by ERBB2 restoration. The pHAGE-ERBB2 cDNA expression vectors were transfected into EC cells after 48h of miR-375 mimic or inhibitor transfection. The effect of ERBB2 restoration on cell proliferation in miR-375 transfected cell lines was measured by MTT. The cell proliferation was significantly increased after transfection of ERBB2 into miR-375 transfected or cells compared to that of pHAGE empty control in KYSE-70, FLO-1 and JHU-ad1 cells and KYSE-180, although the change was not statistically. Values represent the mean ± S.D. for three independent experiments. (** p < 0.01, *p <0.05).

Article Snippet: The following antibodies were used: anti-rabbit HER-2 (PA5-14635, ThermoFisher), anti-rabbit VEGF (PA5-16754, ThermoFisher), anti-mouse Phospho-AKT1 (44621G, ThermoFisher), anti-rabbit beta actin (PA1-183, ThermoFisher).

Techniques: Expressing, Transfection, MTT Assay

Journal: Journal of Cancer

Article Title: Downregulation of miR-375 contributes to ERBB2-mediated VEGFA overexpression in esophageal cancer

doi: 10.7150/jca.63836

Figure Lengend Snippet: miR-375 inhibits invasive ability of EC cell lines that rescued by re-expression of ERBB2. Transwell assays were performed for the invasive activity of EC cells transfected with either miR-375 mimic or the inhibitor with their mock controls. Overexpression of miR-375 significantly reduced cell invasion in ESCC cell lines KYSE-70 and KYSE-180 and in EAC cell line FLO-1 but not significant in JHU-ad1 cells. Re-expression of ERBB2 resulted in significantly increased invasive ability compared to the pHAGE empty vector transfected one in EC cell lines. Invasive ability of the cells was displayed as a percentage of the absolute cell numbers (bottom). Results are displayed as mean data ± SE. (** p < 0.01, *p <0.05). Five fields of unit area on each membrane or whole membrane were counted for cell numbers, and the experiments were repeated three times with triplicates.

Article Snippet: The following antibodies were used: anti-rabbit HER-2 (PA5-14635, ThermoFisher), anti-rabbit VEGF (PA5-16754, ThermoFisher), anti-mouse Phospho-AKT1 (44621G, ThermoFisher), anti-rabbit beta actin (PA1-183, ThermoFisher).

Techniques: Expressing, Activity Assay, Transfection, Over Expression, Plasmid Preparation

Journal: Journal of Cancer

Article Title: Downregulation of miR-375 contributes to ERBB2-mediated VEGFA overexpression in esophageal cancer

doi: 10.7150/jca.63836

Figure Lengend Snippet: miR-375 regulates ERBB2 and VEGFA in EC . A. Expression of miR-375 after transfection. B. Forced expression of miR-375 in EC cell lines resulted in decreased ERBB2 and VEGFA expression. C. Map of the plasmids pEZX-MT04 containing miR-375, and pEZX-MT05 containing 3'-UTR of ERBB2 to illustrate the binding site of miR-375 at the 3'-UTR of ERBB2, and its mutant control sequence. D. Dual luciferase reporter assay. Co-transfection with pEZX-miR-375 and pEZX-ERBB2 3' UTR wild type significantly decreased the luciferase activities compared to that with pEZX-miR-375 and ERBB2 3' UTR mutant/miR-375 scrambled control and ERBB2 3' UTR wild type sequence in ESCC cell lines. The data were reported as mean ± S.D. from three independent experiments (** p < 0.01, *p <0.05)

Article Snippet: The following antibodies were used: anti-rabbit HER-2 (PA5-14635, ThermoFisher), anti-rabbit VEGF (PA5-16754, ThermoFisher), anti-mouse Phospho-AKT1 (44621G, ThermoFisher), anti-rabbit beta actin (PA1-183, ThermoFisher).

Techniques: Expressing, Transfection, Binding Assay, Mutagenesis, Sequencing, Luciferase, Reporter Assay, Cotransfection

Journal: Oncogene

Article Title: Epidermal growth factor regulates Mcl-1 expression through the MAPK-Elk-1 signalling pathway contributing to cell survival in breast cancer

doi: 10.1038/onc.2010.616

Figure Lengend Snippet: Activated Elk-1 correlates with increased levels of Mcl-1 in breast tumour samples. A TMA containing samples from 255 ERα negative breast tumours was stained by immunohistochemistry with antibodies specific for Elk-1 when phosphorylated at Ser383, Erk1/2 when phosphorylated at Thr202/Tyr204, ErbB1, ErbB2 and Mcl-1. TMAs were scored by two blinded independent observers. Tumours were assessed using the H-score method. An intensity score of 0–3 was multiplied by the percentage of tumour cells stained. Bars for all graphs represent median scores for each category. P -values were calculated by Mann–Whitney test. ( a ) H-scores for phospho-Elk-1 were separated into two groups based on the median Mcl-1 H-score of 120. ( b ) H-scores for phospho-Erk1/2 were separated into two groups based on the median Mcl-1 H-score of 120. ( c ) H-scores for phospho-Erk1/2 were separated into two groups based on the median phospho-Elk-1 H-score of 100. ( d ) H-scores for Mcl-1 were separated into two groups based on the median ErbB1 H-score of 85. ( e ) H-scores for Mcl-1 were separated into two groups based on the median ErbB2 H-score of 50.

Article Snippet: The following antibodies were used: rabbit anti-Mcl-1 (M8434 Sigma-Aldrich), rabbit anti-Elk-1 (ab32106 Abcam, Cambridge, MA, USA), mouse anti-SRF (MAB4369 Millipore, Billerica, MA, USA), rabbit anti-phospho-Elk-1 (Ser383) (9181, Cell Signaling, Boston, MA, USA), mouse anti-phospho-P44/42 MAPK (Thr202/Tyr204) (9106, Cell Signaling), rabbit anti-P44/42 MAPK (9102, Cell Signaling), rabbit anti-NF-κB p65 (ab7970 Abcam), mouse anti-α-tubulin (T6074 Sigma-Aldrich), rabbit-anti-Stat-3 (9132, Cell Signaling), rabbit anti-Her-2 (Dako, Mississauga, ON, Canada) and mouse anti-EGFR (Ventana, Tucson AZ, USA).

Techniques: Staining, Immunohistochemistry, MANN-WHITNEY