Journal: Biomarker Research

Article Title: LncRNA APCDD1L-AS1 induces icotinib resistance by inhibition of EGFR autophagic degradation via the miR-1322/miR-1972/miR-324-3p-SIRT5 axis in lung adenocarcinoma

doi: 10.1186/s40364-021-00262-3

Figure Lengend Snippet: Significant upregulation of APCDD1L-AS1 in icotinib-resistant LUAD cells. a The icotinib sensitivity in icotinib-resistant LUAD cells and their parental cells treated with different concentrations of icotinib for 96 h was determined by MTT assay. PC9/IcoRL: PC9 low-dose icotinib-resistant cells; PC9/IcoRH: PC9 high-dose icotinib-resistant cells; HCC827/IcoRL: HCC827 low-dose icotinib-resistant cells; HCC827/IcoRH: HCC827 high-dose icotinib-resistant cells. b The cell viability of both the parental cells and their icotinib-resistant cells after treated with icotinib (10 μM) for 24, 48, 72 and 96 h was detected by MTT assay. c The colony formation ability of the parental cells and their icotinib-resistant cells under different concentrations of icotinib was analyzed using colony formation assay. d The subcutaneous tumor mouse models of icotinib-resistant cells and their parental cells were treated with or without icotinib. Average tumor volume for each group was measured ( n = 3). e The level of EGFR expression and phosphorylation in the parental cells and their icotinib-resistant cells was evaluated by western blot. f Four upregulated lncRNAs identified by volcano plots in PC9/IcoRL cells and PC9/IcoRH cells comparing with PC9 cells. g The list of top four upregulated lncRNAs in PC9/IcoRL cells and PC9/IcoRH cells comparing with PC9 cells by transcriptome sequencing. h The expression level of lncRNAs, APCDD1L-AS1, PAX8-AS1, GAS5 and lnc-GSDMD, was determined in the parental cells and their icotinib-resistant cells by qRT-PCR. The mean ± SD of triplicate experiments were plotted, * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not statistically significant

Article Snippet: The icotinib (Betta Pharmaceuticals Co., Ltd., China) resistant lung adenocarcinoma cells PC9/IcoRL (low-dose icotinib-resistant), PC9/IcoRH (high-dose icotinib-resistant), HCC827/IcoRL and HCC827/IcoRH, without T790M mutation and MET amplification confirmed by sequencing, were established with a gradient concentration of icotinib (0.01–20 μM) for over 6 months.

Techniques: MTT Assay, Colony Assay, Expressing, Western Blot, Sequencing, Quantitative RT-PCR

Journal: Biomarker Research

Article Title: LncRNA APCDD1L-AS1 induces icotinib resistance by inhibition of EGFR autophagic degradation via the miR-1322/miR-1972/miR-324-3p-SIRT5 axis in lung adenocarcinoma

doi: 10.1186/s40364-021-00262-3

Figure Lengend Snippet: Contribution of APCDD1L-AS1 to the icotinib resistance of LUAD cells. a The localization and structure of APCDD1L-AS1 on the chromosome in the LNCipedia database. b The coding potentials of lncRNAs (MALAT1, TUG1, APCDD1L-AS1) and mRNAs (GAPDH, ACTB, SDHA) were calculated using CPAT database. c The online software lncLocator was used to predict the subcellular localization of APCDD1L-AS1. d Relative expression of APCDD1L-AS1 in cytoplasm or nucleus of the icotinib-resistant cells (PC9/IcoRH, HCC827/IcoRH) was determined by qRT-PCR. e The localization of APCDD1-AS1 in the PC9/IcoRH cells was detected by RNA-FISH. Blue, DAPI-stained nuclei; Red, Cy3-labeled positive hybridization signals (scale bar, 100 μm). U6 and 18S were used as positive controls. f The effect of APCDD1L-AS1 KD on the IC50 value of icotinib was evaluated in icotinib-resistant cells (PC9/IcoRH, HCC827/IcoRH) by MTT assay. g The level of EGFR and p-EGFR in APCDD1L-AS1-KD or -OE icotinib-resistant cells (PC9/IcoRH, HCC827/IcoRH) was determined by western blot. h Kaplan-Meier analyses of the correlations between APCDD1L-AS1 expression (classified into high and low expression groups according to the median of APCDD1L-AS1 expression) and OS in 672 lung adenocarcinoma patients using Kaplan-Meier Plotter online database. Log rank test was used to calculate P values. i-j The apoptosis of APCDD1L-AS1-KD icotinib-resistant cells (PC9/IcoRL, PC9/IcoRH) induced by icotinib (10 μM) was analyzed using flow cytometry ( i ), and apoptosis-related protein PARP in PC9/IcoRL and PC9/IcoRH cells was detected by western blot ( j ) . GAPDH was used as the internal control. The mean ± SD of triplicate experiments were plotted, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The icotinib (Betta Pharmaceuticals Co., Ltd., China) resistant lung adenocarcinoma cells PC9/IcoRL (low-dose icotinib-resistant), PC9/IcoRH (high-dose icotinib-resistant), HCC827/IcoRL and HCC827/IcoRH, without T790M mutation and MET amplification confirmed by sequencing, were established with a gradient concentration of icotinib (0.01–20 μM) for over 6 months.

Techniques: Software, Expressing, Quantitative RT-PCR, Staining, Labeling, Hybridization, MTT Assay, Western Blot, Flow Cytometry

Journal: Biomarker Research

Article Title: LncRNA APCDD1L-AS1 induces icotinib resistance by inhibition of EGFR autophagic degradation via the miR-1322/miR-1972/miR-324-3p-SIRT5 axis in lung adenocarcinoma

doi: 10.1186/s40364-021-00262-3

Figure Lengend Snippet: MiR-1322/miR-1972/miR-324-3p were sponged by APCDD1L-AS1 and improved the sensitivity of LUAD cells to icotinib. a Potential miRNAs (miR-1322, miR-1972 and miR-324-3p) sponging with APCDD1L-AS1 were predicted by LncBasedatabase and transcriptome sequencing. b Sequence alignment of wild type and mutant type of APCDD1L-AS1 with miR-1322, miR-1972 and miR-324-3p potential targeting sites. c Luciferase reporter assay using miRNA mimics was applied to verify the interaction between APCDD1L-AS1 and miR-1322, miR-1972 or miR-324-3p. d RIP assay was performed using AGO2 antibody, immunoprecipitation of APCDD1L-AS1 and miR-1322, miR-1972 or miR-324-3p were determined by qRT-PCR. IgG was used as a negative control. e The expression level of miRNAs in the parental cells and their icotinib-resistant cells (PC9, PC9/IcoRL and PC9/IcoRH) was determined by qRT-PCR. f The effect of miR-1322, miR-1972 or miR-324-3p mimics on the icotinib sensitivity of PC9/IcoRL cells was determined by MTT assay. g After co-transfection with miR-1322, miR-1972 or miR-324-3p inhibitor and si-RNA APCDD1L-AS1, the viability of PC9/IcoRL cells with the treatment of icotinib for 72 h was determined by MTT assay. h-i The level of EGFR, p-EGFR ( h ) and PARP ( i ) in the icotinib-resistant cells (PC9/IcoRH and HCC827/IcoRH) with the transfection of miR-1322, miR-1972 or miR-324-3p mimics was determined by western blot. The mean ± SD of triplicate experiments were plotted, * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not statistically significant

Article Snippet: The icotinib (Betta Pharmaceuticals Co., Ltd., China) resistant lung adenocarcinoma cells PC9/IcoRL (low-dose icotinib-resistant), PC9/IcoRH (high-dose icotinib-resistant), HCC827/IcoRL and HCC827/IcoRH, without T790M mutation and MET amplification confirmed by sequencing, were established with a gradient concentration of icotinib (0.01–20 μM) for over 6 months.

Techniques: Sequencing, Mutagenesis, Luciferase, Reporter Assay, Immunoprecipitation, Quantitative RT-PCR, Negative Control, Expressing, MTT Assay, Cotransfection, Transfection, Western Blot

Journal: Biomarker Research

Article Title: LncRNA APCDD1L-AS1 induces icotinib resistance by inhibition of EGFR autophagic degradation via the miR-1322/miR-1972/miR-324-3p-SIRT5 axis in lung adenocarcinoma

doi: 10.1186/s40364-021-00262-3

Figure Lengend Snippet: Downregulation of SIRT5 by miR-1322/miR-1972/miR-324-3p through targeting its 3′-UTRs. a The possible target genes of miR-1322, miR-1972 and miR-324-3p were predicted by Targetscan and transcriptome sequencing; the mRNA level of SIRT5 from GEO dataset GSE80344 in gefitinib-resistance cells and their sensitive cells was shown. b Potential binding sites of miR-1322, miR-1972 and miR-324-3p in SIRT5 3′-UTR. c Luciferase reporter assay using miRNA mimics was applied to verify the interaction between SIRT5 3′-UTR and miR-1322, miR-1972 or miR-324-3p. d-e The expression of SIRT5in the parental cells and their icotinib-resistant cells (PC9, PC9/IcoRL and PC9/IcoRH) was detected by qRT-PCR ( d ) and western blot ( e ). f-g The SIRT5 expression in PC9/IcoRL and PC9/IcoRH cells with the transfection of miRNAs mimics or inhibitor was detected by qRT-PCR ( f ) and western blot ( g ). The mean ± SD of triplicate experiments were plotted, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The icotinib (Betta Pharmaceuticals Co., Ltd., China) resistant lung adenocarcinoma cells PC9/IcoRL (low-dose icotinib-resistant), PC9/IcoRH (high-dose icotinib-resistant), HCC827/IcoRL and HCC827/IcoRH, without T790M mutation and MET amplification confirmed by sequencing, were established with a gradient concentration of icotinib (0.01–20 μM) for over 6 months.

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

Journal: Biomarker Research

Article Title: LncRNA APCDD1L-AS1 induces icotinib resistance by inhibition of EGFR autophagic degradation via the miR-1322/miR-1972/miR-324-3p-SIRT5 axis in lung adenocarcinoma

doi: 10.1186/s40364-021-00262-3

Figure Lengend Snippet: Contribution of SIRT5 to icotinib resistance. a The IC50 value of icotinibinSIRT5 KD-icotinib-resistant cells was determined by MTT assay. b The level of EGFR and p-EGFR in SIRT5 KD-icotinib-resistant cells (PC9/IcoRH and HCC827/IcoRH) was detected by western blot. c-d The apoptosis of SIRT5 KD-icotinib-resistant cells with the treatment of icotinib was analyzed by flow cytometry ( c ), and apoptosis-related protein PARP was assessed by western blot ( d ). The mean ± SD of triplicate experiments were plotted, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The icotinib (Betta Pharmaceuticals Co., Ltd., China) resistant lung adenocarcinoma cells PC9/IcoRL (low-dose icotinib-resistant), PC9/IcoRH (high-dose icotinib-resistant), HCC827/IcoRL and HCC827/IcoRH, without T790M mutation and MET amplification confirmed by sequencing, were established with a gradient concentration of icotinib (0.01–20 μM) for over 6 months.

Techniques: MTT Assay, Western Blot, Flow Cytometry

Journal: Biomarker Research

Article Title: LncRNA APCDD1L-AS1 induces icotinib resistance by inhibition of EGFR autophagic degradation via the miR-1322/miR-1972/miR-324-3p-SIRT5 axis in lung adenocarcinoma

doi: 10.1186/s40364-021-00262-3

Figure Lengend Snippet: Inhibition of autophagic degradation of EGFR by SIRT5. a The protein level of EGFR in PC9/IcoRH cells with the treatment of CHX (20 μg/ml) for 0, 2, 4 and 8 h were determined by western blot. b The protein level of EGFR in SIRT5 KD-PC9/IcoRH cells with the treatment of MG-132 (10 μM) for 12 h were determined by western blot. c The level of LC3B and p62 in SIRT5 KD-icotinib-resistant cells (PC9/IcoRH and HCC827/IcoRH) was determined by western blot. d-e The level of EGFR, p-EGFR ( d ) and PARP ( e ) in SIRT5 KD-icotinib-resistant LUAD cells (PC9/IcoRH and HCC827/IcoRH) treated with CQ or 3-MA was determined by western blot. f The icotinib sensitivity in icotinib-resistant LUAD cells (PC9/IcoRL and PC9/IcoRH) with or without rapamycinfor 72 h was determined by MTT assay. The mean ± SD of triplicate experiments were plotted, * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not statistically significant

Article Snippet: The icotinib (Betta Pharmaceuticals Co., Ltd., China) resistant lung adenocarcinoma cells PC9/IcoRL (low-dose icotinib-resistant), PC9/IcoRH (high-dose icotinib-resistant), HCC827/IcoRL and HCC827/IcoRH, without T790M mutation and MET amplification confirmed by sequencing, were established with a gradient concentration of icotinib (0.01–20 μM) for over 6 months.

Techniques: Inhibition, Western Blot, MTT Assay

Journal: Biomarker Research

Article Title: LncRNA APCDD1L-AS1 induces icotinib resistance by inhibition of EGFR autophagic degradation via the miR-1322/miR-1972/miR-324-3p-SIRT5 axis in lung adenocarcinoma

doi: 10.1186/s40364-021-00262-3

Figure Lengend Snippet: Upregulation of SIRT5 by APCDD1L-AS1 through sponging with miR-1322/miR-1972/miR-324-3p. a-d The RNA and protein expression of SIRT5 were detected in PC9/IcoRL and PC9/IcoRH cells with APCDD1L-AS1KD ( a-b ) or OE ( c-d ) by qRT-PCR and western blot. e The level of p-EGFR, EGFR, SIRT5, p62 and LC3B in APCDD1L-AS1 KD-icotinib-resistant LUAD cells co-transfected with the inhibitors of miR-1322, miR-1972 and miR-324-3p was determined by western blot, respectively. The mean ± SD of triplicate experiments were plotted, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The icotinib (Betta Pharmaceuticals Co., Ltd., China) resistant lung adenocarcinoma cells PC9/IcoRL (low-dose icotinib-resistant), PC9/IcoRH (high-dose icotinib-resistant), HCC827/IcoRL and HCC827/IcoRH, without T790M mutation and MET amplification confirmed by sequencing, were established with a gradient concentration of icotinib (0.01–20 μM) for over 6 months.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection

Journal: Biomarker Research

Article Title: LncRNA APCDD1L-AS1 induces icotinib resistance by inhibition of EGFR autophagic degradation via the miR-1322/miR-1972/miR-324-3p-SIRT5 axis in lung adenocarcinoma

doi: 10.1186/s40364-021-00262-3

Figure Lengend Snippet: Enhancement of icotinib sensitivity by APCDD1L-AS1 knockdown through promoting autophagic degradation of EGFR in xenograft mouse model. a-b The tumors isolated from nude mice subcutaneously injected with PC9/IcoRH/Lv-sh-APCDD1L-AS1 or PC9/IcoRH/Lv-NC cells were shown ( a ) ( n = 6). Tumor growth curves were drawn according to the size detected per week ( b ). c-d The expression of APCDD1L-AS1, SIRT5 ( c ), miR-1322, miR-1972 and miR-324-3p ( d ) was analyzed by qRT-PCR assay. e The expression of SIRT5 and EGFR was determined by immunohistochemical staining (scale bar, 50 μm). f The LC3B punctures were observed by immunofluorescence (scale bar, 100 μm). The nuclei were visualized using DAPI. Representative images are presented on the figure. g The cell apoptosis of tumor sections was determined by TUNEL assay (scale bar, 100 μm). The mean gray value represented the status of cell apoptosis (Lv-NC group = 4.39 ± 0.568, Lv-shRNA group = 34.39 ± 2.273). The mean ± SD of triplicate experiments were plotted, ** P < 0.01, *** P < 0.001

Article Snippet: The icotinib (Betta Pharmaceuticals Co., Ltd., China) resistant lung adenocarcinoma cells PC9/IcoRL (low-dose icotinib-resistant), PC9/IcoRH (high-dose icotinib-resistant), HCC827/IcoRL and HCC827/IcoRH, without T790M mutation and MET amplification confirmed by sequencing, were established with a gradient concentration of icotinib (0.01–20 μM) for over 6 months.

Techniques: Isolation, Injection, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Immunofluorescence, TUNEL Assay, shRNA

Journal: Biomarker Research

Article Title: LncRNA APCDD1L-AS1 induces icotinib resistance by inhibition of EGFR autophagic degradation via the miR-1322/miR-1972/miR-324-3p-SIRT5 axis in lung adenocarcinoma

doi: 10.1186/s40364-021-00262-3

Figure Lengend Snippet: The schematic diagram of APCDD1L-AS1 contributing to icotinib resistance in lung adenocarcinoma cells. In this progress, up-regulated APCDD1L-AS1 as a miRNA sponge to decoy miR-1322, miR-1972 and miR-324-3p, promote the expression of SIRT5, inhibit autophagic degradation of EGFR, increase EGFR phosphorylation, inhibit apoptosis and induce icotinib resistance

Article Snippet: The icotinib (Betta Pharmaceuticals Co., Ltd., China) resistant lung adenocarcinoma cells PC9/IcoRL (low-dose icotinib-resistant), PC9/IcoRH (high-dose icotinib-resistant), HCC827/IcoRL and HCC827/IcoRH, without T790M mutation and MET amplification confirmed by sequencing, were established with a gradient concentration of icotinib (0.01–20 μM) for over 6 months.

Techniques: Expressing