Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 1 Elevated miR-762 expression is associated with gefitinib resistance in NSCLC cells. a RT-qPCR analysis of miR-762 expression in different NSCLC cells. Relative expression levels of miR-762 were obtained in each sample by normalization of the expressions of miR-762 to that of the U6 snRNA signal. For presentation of data, expression levels of miR-762 from NuLi-1 cells were taken as 100% and the others were normalized accordingly. Each value is a mean ± S.E.M. from three independent experiments. Different superscript letters denote groups that are statistically different (P < 0.05). b The IC50 value (inhibitory concentration to produce 50% cell death) following a 48-h exposure to gefitinib was determined in PC-9 and PC-9/GR cells using MTT assay. c The IC50 value following a 48-h exposure to gefitinib was determined in A549 and A549/GR cells using MTT assay. d-e Characterization of miR-762 expression in different NSCLC cells using RT-qPCR. The value indicates the relative expression levels of miR-762 in the cells (PC-9/PC-9/GR and A549/A549/GR cells) at different batches of gefitinib resistant induction

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay, MTT Assay

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 2 IL-6 serves as a potential upstream regulator of miR-762 induction in NSCLC cells. a-b Characterization of expression levels of different cytokines in different NSCLC cells using RT-qPCR. Each value is a mean ± S.E.M. from three independent experiments. c A549 cells were incubated with different cytokines, including IL-1α (5 ng/ml), IL-1β (10 ng/ml), IL-6 (10 ng/ml) and IL-8 (50 ng/ml) for 24 h, followed by RT-qPCR analysis of miR-762 expression. d A549 cells were stimulated with different doses of IL-6 for 24 h, followed by RT-qPCR analysis of miR-762 expression. e A549 cells were stimulated with 10 ng/ml of IL-6 for different durations as indicated, followed by RT-qPCR analysis of miR-762 expression. f A549 cells were stimulated with 10 ng/ml of IL-6, in the presence or absence or co-treatment with 5 μM of WP1066, for 24 h, followed by RT-qPCR analysis of miR-762 expression. Inhibition of STAT3 activation was verified using immunoblotting analysis of pSTAT3 expression (upper panel). g A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA. 48 h later, knockdown of STAT3 in A549 cells was validated using immunoblotting. h 48 h after siRNA treatment, A549 cells were stimulated with 10 ng/ml of IL-6 for 24 h, followed by RT-qPCR analysis of miR-762 expression

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Expressing, Quantitative RT-PCR, Incubation, Inhibition, Activation Assay, Western Blot, Transfection, Knockdown

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 3 miR-762 upregulation desensitizes NSCLC cells to gefitinib treatment. a 48 h after transfection with miR-762 inhibitors or negative controls (NC), PC-9/GR and A549/GR cells were subjected to RT-qPCR analysis of miR-762 expression. b PC-9/GR and A549/GR cells were treated with different doses of gefitinib (8 μM for PC-9/GR, 60 μM for A549/GR) for 24 or 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm (*P < 0.05 and **P < 0.01). c Cells were treated with different doses of gefitinib (8 μM for PC-9/GR, 60 μM for A549/GR) for 24 or 48 h. Cell apoptosis was assayed using an ApoStrand™ELISA Apoptosis Detection Kit at 405 nm (*P < 0.05 and **P < 0.01). d In vivo gefitinib sensitivity was evaluated using a xenograft model, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 and ** P < 0.01 when comparing Inhibitors + vehicle to Inhibitors + gefitinib). e 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9 and A549 cells were subjected to RT-qPCR analysis of miR-762 expression. f PC-9 and A549 cells were treated with different doses of gefitinib (0.2 μM for PC-9 and 12.5 μM for A549 cells) for 24 or 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm (*P < 0.05 and **P < 0.01). g PC-9 and A549 cells were treated with different doses of gefitinib (0.2 μM for PC-9 and 12.5 μM for A549 cells) for 24 or 48 h. Cell apoptosis was assayed using an ApoStrand™ELISA Apoptosis Detection Kit at 405 nm (*P < 0.05 and **P < 0.01). h In vivo gefitinib sensitivity was evaluated using a xenograft model, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 and **P < 0.01 when comparing Mimics + vehicle to Mimics + gefitinib)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Transfection, Quantitative RT-PCR, Expressing, MTT Assay, Enzyme-linked Immunosorbent Assay, In Vivo

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 4 ABR serves as the direct target of miR-762 in NSCLC cells. a Prediction of putative target genes of miR-762 by Target scan and miRDB programs. b PC-9/GR and A549/GR cells were transfected with miR-762 inhibitors or inhibitors-NC for 48 h, followed by immunoblotting analysis of ABR expression. c PC-9/GR and A549/GR cells were transfected with miR-762 inhibitors or inhibitors-NC for 48 h, followed by RT-qPCR analysis of ABR expression (*P < 0.05 and **P < 0.01). d PC-9 and A549 cells were transfected with miR-762 mimics or mimics-NC for 48 h, followed by immunoblotting analysis of ABR expression. e PC-9 and A549 cells were transfected with miR-762 mimics or mimics-NC for 48 h, followed by RT- qPCR analysis of ABR expression (*P < 0.05 and **P < 0.01). f Predicted miR-762 binding sites in the 3′-UTR of ABR gene. g miR-762 mimics/Mimics- NC (25 pmol/well) and pGL3-ABR 3’UTR-Luc reporter (0.25 μg/well), together with 0.001 μg of the Renilla luciferase reporter (Promega, Beijing, China), were co-transfected into subconfluent proliferating NIH/3 T3 cells for 24 h, followed by measurement of the relative luciferase activity (*P < 0.05)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Transfection, Western Blot, Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay

Journal: BMC cancer

Article Title: miR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer.

doi: 10.1186/s12885-019-6416-4

Figure Lengend Snippet: Fig. 5 ABR overexpression alleviates miR-762-induced gefitinib resistance. a PC-9 and A549 cells that stably expressed the exogenous ABR was established as described in Materials and methods. PC-9/ABR and A549/ABR cells were transiently transfected with miR-762 mimics or mimics-NC for 48 h, followed by immunoblotting analysis of ABR expression. b 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were treated with different doses of gefitinib (0.2 μM for PC-9/ABR and 12.5 μM for A549/ABR cells) for 48 h. Cell viability was assayed using a MTT Assay Kit at 590 nm. c 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were treated with different doses of gefitinib (0.2 μM for PC-9/ABR and 12.5 μM for A549/ABR cells) for 48 h. Cell apoptosis was assayed using an ApoStrand™ ELISA Apoptosis Detection Kit at 405 nm. Different superscript letters denote groups that are statistically different (P < 0.05). d 48 h after transfection with miR-762 mimics or Mimics-NC, PC-9/ABR and A549/ABR cells were subjected to a xenograft model to measure the in vivo gefitinib sensitivity, as described in Materials and methods. Tumor volume was measured and recorded once a week (*P < 0.05 when comparing Mimics + gefitinib + vector to Mimics + gefitinib + pCMV3-ABR)

Article Snippet: To further validate the STAT3dependent regulation of miR-762 by IL-6, A549 cells were transiently transfected with STAT3 siRNA or Ctrl siRNA (Santa Cruz Biotechnology, Shanghai, China) using Lipofectamine® 2000 (Thermo Fisher Scientific) for 48 h. The specificity and effectiveness of the STAT3 siRNA has been validated [11].

Techniques: Over Expression, Stable Transfection, Transfection, Western Blot, Expressing, MTT Assay, Enzyme-linked Immunosorbent Assay, In Vivo, Plasmid Preparation