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1) Product Images from "miR‐1‐3p and miR‐206 sensitizes HGF ‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT"
Article Title: miR‐1‐3p and miR‐206 sensitizes HGF ‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT
Journal: Journal of Cellular and Molecular Medicine
doi: 10.1111/jcmm.13629
Figure Legend Snippet: HGF induced gefitinib resistance and down‐regulated expression of miR‐1‐3p and miR‐206 in lung adenocarcinoma PC ‐9 and HCC 827 cells. A‐B, HGF induced gefitinib resistance in PC ‐9 (A) and HCC 827 (B) cells. Tumour cells were incubated with increasing concentrations of gefitinib in the presence/absence of HGF , and cell viability was determined after 72 h of treatment by MTT assay. Data are means of three separated experiments ± SD , ** P < .01 compared with gefitinib group. C‐D, HGF ‐induced down‐regulated expression of miR‐1‐3p and miR‐206 in PC ‐9 (C) and HCC 827 cells (D). Tumour cells were incubated with HGF (50 ng/ mL ) for 72 h, the expression of miR‐1‐3p and miR‐206 were determined by QPCR assay. Data are means of three separated experiments ± SD , ** P < .01 compared with control group
Techniques Used: Expressing, Incubation, MTT Assay
Figure Legend Snippet: miR‐1‐3p and miR‐206 overcame HGF ‐induced gefitinib resistance in PC ‐9 and HCC 827 cells. A, HGF overexpression lentivirus increased the production of HGF in PC ‐9 and HCC 827 cells. The cells were incubated in medium contained lentivirus for 48 h, and culture supernatants were harvested. The level of HGF was determined by ELISA . B, HGF overexpressed PC ‐9 and HCC 827cells increased gefitinib resistance. The PC ‐9/ NC , HCC 827/ NC , PC ‐9/ HGF , HCC 827/ HGF cells were incubated with increasing concentrations of gefitinib for 72 h. Cell viability was measured by MTT assay. Data are means of three separated experiments ± SD , * P < .05, ** P < .01 compared with control group. C, miR‐1‐3p and miR‐206 mimics transfection reversed HGF ‐induced gefitinib resistance. The PC ‐9/ HGF , HCC 827/ HGF cells were transfected with miR‐1‐3p or miR‐206 mimics for 24 h and then treated with increasing concentrations of gefitinib. Cell viability was measured by MTT assay. Data are means of three separated experiments ± SD , * P < .05, ** P < .01 compared with negative control ( NC ) group. PC ‐9/ NC and HCC 827/ NC cells: negative control lentivirus infected cells; PC ‐9/ HGF and HCC 827/ HGF cells: HGF overexpressed lentivirus‐infected cells
Techniques Used: Over Expression, Incubation, Enzyme-linked Immunosorbent Assay, MTT Assay, Transfection, Negative Control, Infection
Figure Legend Snippet: miR‐1‐3p/miR‐206 reversed HGF ‐induced gefitinib resistance by c‐Met. A, c‐Met knockdown mimicked the effect of miR‐1‐3p and miR‐206. Left, c‐Met knockdown was evaluated by western blot assay in PC ‐9 and HCC 827 cells. Right, PC ‐9 and HCC 827 cells with c‐Met knockdown were treated with or without gefitinib (1 μmol/L) in the presence/absence of HGF (50 ng/ mL ) for 72 h, cell viability was determined by MTT assay. Data are means of three separated experiments ± SD , ** P < .01. B, Top, c‐Met overexpression was evaluated by Western blot assay in PC ‐9 and HCC 827 cells. Bottom, c‐Met overexpression attenuated the effects of miR‐1‐3p and miR‐206. PC ‐9/ HGF and HCC 827/ HGF cells were co‐transfected with c‐Met overexpression plasmid and miR‐1‐3p/miR‐206 mimics and then treated with gefitinib (1 μmol/L), cell viability was determined after 48 h of treatment by MTT assay. Data are means of three separated experiments ± SD , ** P < .01
Techniques Used: Western Blot, MTT Assay, Over Expression, Transfection, Plasmid Preparation
Figure Legend Snippet: miR‐1‐3p/miR‐206 suppresses c‐Met/Akt and Erk pathway in HGF ‐mediated gefitinib‐resistant cells. miR‐1‐3p and miR‐206 inhibited Akt and Erk1/2 signalling, even in HGF treated PC ‐9 (A) and HCC 827 (B) cells. PC ‐9 and HCC 827 cells were transfected with miR‐1‐3p or miR‐206 mimics and then treated with gefitinib (1 μmol/L) in the presence/absence of HGF (50 ng/ mL ) for 1 h, and cell extracts were prepared and immunoblotted with the indicated antibodies. GE : gefitinib
Techniques Used: Transfection
Figure Legend Snippet: miR‐1‐3p/miR‐206 inhibits EMT in HGF ‐mediated gefitinib‐resistant cells. A‐B, HGF induced‐transition from epithelial morphology to mesenchymal morphology (A) with increased EMT ‐related molecular markers expression (B). PC ‐9 and HCC 827 cells were treated with HGF (50 ng/ mL ) for 48 h, the morphology was photoed by fluorescence microscope. EMT ‐related molecular markers were detected by Western blot analysis. C‐D, miR‐1‐3p and miR‐206 mimics transfection inhibited HGF ‐induced EMT . PC ‐9 and HCC 827 cells were transfected with miR‐1‐3p or miR‐206 mimics and then treated with HGF (50 ng/ mL ) for 48 h. The morphology was photoed by fluorescence microscope. EMT ‐related molecular markers expressions were detected by Western blot analysis. E‐F, Immunofluorescence stain of EMT markers of E‐cadherin and Vimentin in PC ‐9/ NC , HCC 827/ NC , PC ‐9/ HGF and HCC 827/ HGF cells transfected with or without miR‐1‐3p/miR‐206 mimics. Scale bar: 20 μm
Techniques Used: Expressing, Fluorescence, Microscopy, Western Blot, Transfection, Immunofluorescence, Staining
Figure Legend Snippet: miR‐1‐3p/miR‐206 inhibits HGF ‐mediated gefitinib resistance in vivo . A‐B, PC ‐9/ NC , and PC ‐9/ HGF cells (10 7 ) were inoculated subcutaneously into nude mice on day 0. Mice received oral gefitinib (25 mg/kg/d) and/or locally injected miR‐1‐3p/miR‐206 agomirs, starting on day 10. The tumour size was measured every 3 days, and tumour volumes were calculated as described in . We set three days of stop gefitinib (stop‐ GE ) interval to confirm the effect of gefitinib and combined treatment. Error bars indicate standard errors of 3 mice. B, macroscopic appearances of tumours harvested on day 25 are shown. GE :gefitinib
Techniques Used: In Vivo, Injection
Figure Legend Snippet: Proposed models on the inhibitory role of miR‐1‐3p/miR‐206 in HGF ‐induced gefitinib resistance. As depicted in the model, miR‐1‐3p/miR‐206 overcome HGF ‐induced gefitinib resistance by targeting c‐Met‐Akt/Erk pathway and epithelial‐mesenchymal transition ( EMT ) in lung cancer with EGFR activating mutation
Techniques Used: Mutagenesis
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In addition, we set 3 days of Control:Article Title: miR‐1‐3p and miR‐206 sensitizes HGF ‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT Article Snippet: MiR‐1‐3p and miR‐206 agomirs and their negative control (NC) (2 nmol; Genepharma, Shanghai, China) were given locally by direct injection into the xenografts every 3 days.MiR‐1‐3p and miR‐206 agomirs and their negative control (NC) (2 nmol; Genepharma, Shanghai, China) were given locally by direct injection into the xenografts every 3 days.. 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In addition, we set 3 days of Enzyme-linked Immunosorbent Assay:Article Title: miR‐1‐3p and miR‐206 sensitizes HGF ‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT Article Snippet: MiR‐1‐3p and miR‐206 agomirs and their negative control (NC) (2 nmol; Genepharma, Shanghai, China) were given locally by direct injection into the xenografts every 3 days.MiR‐1‐3p and miR‐206 agomirs and their negative control (NC) (2 nmol; Genepharma, Shanghai, China) were given locally by direct injection into the xenografts every 3 days.. 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In addition, we set 3 days of Immunofluorescence:Article Title: miR‐1‐3p and miR‐206 sensitizes HGF ‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT Article Snippet: MiR‐1‐3p and miR‐206 agomirs and their negative control (NC) (2 nmol; Genepharma, Shanghai, China) were given locally by direct injection into the xenografts every 3 days.MiR‐1‐3p and miR‐206 agomirs and their negative control (NC) (2 nmol; Genepharma, Shanghai, China) were given locally by direct injection into the xenografts every 3 days.. 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In addition, we set 3 days of Mutagenesis:Article Title: miR‐1‐3p and miR‐206 sensitizes HGF ‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT Article Snippet: MiR‐1‐3p and miR‐206 agomirs and their negative control (NC) (2 nmol; Genepharma, Shanghai, China) were given locally by direct injection into the xenografts every 3 days.MiR‐1‐3p and miR‐206 agomirs and their negative control (NC) (2 nmol; Genepharma, Shanghai, China) were given locally by direct injection into the xenografts every 3 days.. 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In addition, we set 3 days of Xenograft Assay:Article Title: miR‐1‐3p and miR‐206 sensitizes HGF ‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT Article Snippet: MiR‐1‐3p and miR‐206 agomirs and their negative control (NC) (2 nmol; Genepharma, Shanghai, China) were given locally by direct injection into the xenografts every 3 days.MiR‐1‐3p and miR‐206 agomirs and their negative control (NC) (2 nmol; Genepharma, Shanghai, China) were given locally by direct injection into the xenografts every 3 days.. In addition, we set 3 days of |
