Journal: Cellular & molecular biology letters

Article Title: LARRPM restricts lung adenocarcinoma progression and M2 macrophage polarization through epigenetically regulating LINC00240 and CSF1.

doi: 10.1186/s11658-022-00376-y

Figure Lengend Snippet: Fig. 1 LARRPM was downregulated and associated with prognosis in LUAD. a Correlation between LARRPM expression and overall survival analyzed using the TCGA LUAD dataset. HR = 0.4167, P = 0.0006, log-rank test. n = 248, median LARRPM expression level was used as the cut-off. b LARRPM expression levels in T1-and T2-stage LUAD tissues analyzed using the TCGA dataset. P = 0.0136, Mann–Whitney test. c LARRPM expression levels in TNM stages-I and -I LUAD tissues analyzed using the TCGA dataset. P = 0.0278, Mann– Whitney test. d Correlation between LARRPM expression and overall survival analyzed using our LUAD cohort data. HR = 0.4743, P = 0.0244, log-rank test. n = 70, median LARRPM expression level was used as the cut-off. e LARRPM expression in 70 pairs of LUAD tissues and matched adjacent lung tissues was detected by qRT-PCR. P < 0.0001, Wilcoxon matched-pairs signed rank test. f LARRPM expression in 16HBE cells and LUAD cells A549, H1299, H1975, and HCC827 was detected by qRT-PCR. Results are shown as mean ± standard deviation (SD) based on three independent experiments. Asterisks indicate a significant difference at ***P < 0.001, ****P < 0.0001, by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. 16HBE Human bronchial epithelial cell line, A549, H1299, H1975, HCC827 human LUAD cell lines, HR hazard ratio, LARRPM a LUAD-related long non-coding RNA, LUAD lung adenocarcinoma, qRT-PCR reverse-transcription quantitative PCR, T1, T2 TNM stages T1, T2, TCGA The Cancer Genome Atlas

Article Snippet: Human LUAD cells A549 (Cat. CCL 185), H1299 (Cat. CRL 5803), H1975 (Cat. CRL5908), HCC827 (Cat. CRL-2868) and human monocyte line THP-1 (Cat. TIB-202) were obtained from the ATCC and cultured in F 12K (A549) or RPMI 1640 (H1299, H1975, HCC827, and THP-1) media (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher Scientific).

Techniques: Expressing, MANN-WHITNEY, Quantitative RT-PCR, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Cellular & molecular biology letters

Article Title: LARRPM restricts lung adenocarcinoma progression and M2 macrophage polarization through epigenetically regulating LINC00240 and CSF1.

doi: 10.1186/s11658-022-00376-y

Figure Lengend Snippet: Fig. 2 LARRPM repressed proliferation, induced apoptosis and inhibited migration and invasion of LUAD cells. a LARRPM expression in A549 and H1975 cells with LARRPM stable overexpression and A549 and H1975 control cells was detected by qRT-PCR. b, c Cell proliferation of A549 and H1975 cells with LARRPM stable overexpression and A549 and H1975 control cells was detected using CCK-8 assays (b) and EdU incorporation assays (c). Scale bar: 100 µm. Red color indicates EdU-positive cells. d, e Cell apoptosis of A549 and H1975 cells with LARRPM stable overexpression and A549 and H1975or control cells was detected using caspase-3 activity assays (d) and TUNEL assays (e). Scale bar = 100 µm. Green color indicated TUNEL-positive cells. f, g Cell migration and invasion of A549 and H1975 cells with LARRPM stable overexpression or A549 and H1975 control cells was detected using transwell migration assay (f) and transwell invasion assay (g). Scale bar = 100 µm. Results are shown as mean ± SD based on three independent experiments. Asterisks indicate a significant difference at *P < 0.05, **P < 0.01, ***P < 0.001, by the Student’s t-test. CCK-8 Cell Counting Kit-8, Ctrl control, EdU 5-ethynyl-2′-deoxyuridine, oe overexpression, Tunel terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling

Article Snippet: Human LUAD cells A549 (Cat. CCL 185), H1299 (Cat. CRL 5803), H1975 (Cat. CRL5908), HCC827 (Cat. CRL-2868) and human monocyte line THP-1 (Cat. TIB-202) were obtained from the ATCC and cultured in F 12K (A549) or RPMI 1640 (H1299, H1975, HCC827, and THP-1) media (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher Scientific).

Techniques: Migration, Expressing, Over Expression, Control, Quantitative RT-PCR, CCK-8 Assay, Activity Assay, TUNEL Assay, Transwell Migration Assay, Transwell Invasion Assay, Cell Counting, End Labeling

Journal: Cellular & molecular biology letters

Article Title: LARRPM restricts lung adenocarcinoma progression and M2 macrophage polarization through epigenetically regulating LINC00240 and CSF1.

doi: 10.1186/s11658-022-00376-y

Figure Lengend Snippet: Fig. 3 LARRPM repressed LUAD development in vivo. a A549 cells with LARRPM stable overexpression or A549 control cells were injected into tail vein of nude mice. Six weeks later, lung metastatic nodules were detected using hematoxyln and eosin (HE) staining. b, c Immunohistohemistry (IHC) staining of the antigens Ki67 (b) and PCNA (c) in lung metastatic nodules formed by A549 cells with LARRPM stable overexpression or A549 control cells. Scale bar: 50 µm. d TUNEL assays using lung metastatic nodules formed by A549 cells with LARRPM stable overexpression or A549 control cells. Scale bar = 50 µm. e Immunofluorescence (IF) staining of F4/80 antigen in lung metastatic nodules formed by A549 cells with LARRPM stable overexpression or A549 control cells control. Scale bar: 50 µm. Results are shown as the mean ± SD based on n = 6 mice in each group. Asterisks indicate a significant difference at **P < 0.01, ***P < 0.001 by the Student’s t-test

Article Snippet: Human LUAD cells A549 (Cat. CCL 185), H1299 (Cat. CRL 5803), H1975 (Cat. CRL5908), HCC827 (Cat. CRL-2868) and human monocyte line THP-1 (Cat. TIB-202) were obtained from the ATCC and cultured in F 12K (A549) or RPMI 1640 (H1299, H1975, HCC827, and THP-1) media (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher Scientific).

Techniques: In Vivo, Over Expression, Control, Injection, Staining, Immunohistochemistry, TUNEL Assay, Immunofluorescence

Journal: Cellular & molecular biology letters

Article Title: LARRPM restricts lung adenocarcinoma progression and M2 macrophage polarization through epigenetically regulating LINC00240 and CSF1.

doi: 10.1186/s11658-022-00376-y

Figure Lengend Snippet: Fig. 4 LARRPM upregulated LINC00240 expression via inducing DNA demethylation. a Subcellular localization of LARRPM in A549 cells was detected using subcellular fractionation followed by qRT-PCR. MALAT1 and GAPDH were used as nuclear and cytoplasmic controls, respectively. b The schematic diagram of the genomic localization of LARRPM and LINC00240. c, d LINC00240 expression in A549 cells with LARRPM overexpression (c) and HCC827 cells with LARRPM depletion (d) was detected by qRT-PCR. e RNA–protein pull-down assays followed by western blot were performed to detect the proteins bound by LARRPM. f RNA immunoprecipitation (RIP) assays followed by qRT-PCR were performed to detect the RNAs bound by TET1. g, h Chromatin immunoprecipitation (ChIP) assays followed by qPCR were performed in A549 cells with LARRPM overexpression (g) and HCC827 cells with LARRPM depletion (h) to detect the DNAs bound by TET1. i, j 5-Hydroxymethylcyctosine (5hmC) levels of CpG43 from A549 cells with LARRPM overexpression (i) and HCC827 cells with LARRPM depletion (j) were measured using the EpiMark 5-hmC Analysis Kit. k, l DNA methylation levels of CpG43 from A549 cells with LARRPM overexpression (k) and HCC827 cells with LARRPM depletion (l) were measured using bisulfate DNA sequencing. Results are shown as the mean ± SD based on three independent experiments. Asterisks indicate a significant difference at **P < 0.01, ***P < 0.001; ns, not significant, by Student’s t-test (c, f, g, i, k) or one-way ANOVA followed by Dunnett’s multiple comparisons test (d, h, j, l). GAPDH Glyceraldehyde 3-phosphate dehydrogenase, LINC00240 a long LUAD-related non-coding RNA, MALATI a long non-coding RNA, TET1 a DNA demethylase

Article Snippet: Human LUAD cells A549 (Cat. CCL 185), H1299 (Cat. CRL 5803), H1975 (Cat. CRL5908), HCC827 (Cat. CRL-2868) and human monocyte line THP-1 (Cat. TIB-202) were obtained from the ATCC and cultured in F 12K (A549) or RPMI 1640 (H1299, H1975, HCC827, and THP-1) media (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher Scientific).

Techniques: Expressing, Fractionation, Quantitative RT-PCR, Over Expression, Western Blot, RNA Immunoprecipitation, Chromatin Immunoprecipitation, DNA Methylation Assay, DNA Sequencing

Journal: Cellular & molecular biology letters

Article Title: LARRPM restricts lung adenocarcinoma progression and M2 macrophage polarization through epigenetically regulating LINC00240 and CSF1.

doi: 10.1186/s11658-022-00376-y

Figure Lengend Snippet: Fig. 5 Depletion of the LINC00240 reversed the roles of LARRPM in proliferation, apoptosis, migration and invasion of LUAD cells. a LARRPM and LINC00240 expression in A549 cells with LARRPM overexpression; LINC00240 depletion was detected by qRT-PCR. b, c Cell proliferation of A549 cells with LARRPM overexpression and LINC00240 depletion was detected using the CCK-8 assay (b) and EdU incorporation assay (c). Scale bar: 100 µm. Red color indicates the EdU-positive cells. d, e Cell apoptosis of A549 cells with LARRPM overexpression and LINC00240 depletion was detected using the caspase-3 activity assay (d) and TUNEL assay (e). Scale bar: 100 µm. Green color indicates TUNEL-positive cells. f, g Cell migration and invasion of A549 cells with LARRPM overexpression and LINC00240 depletion were detected using the transwell migration assay (f) and transwell invasion assay (g). Scale bar: 100 µm. Results are shown as the mean ± SD based on three independent experiments. Asterisks indicate a significant difference at *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant, by one-way ANOVA followed by Dunnett’s multiple comparisons test

Article Snippet: Human LUAD cells A549 (Cat. CCL 185), H1299 (Cat. CRL 5803), H1975 (Cat. CRL5908), HCC827 (Cat. CRL-2868) and human monocyte line THP-1 (Cat. TIB-202) were obtained from the ATCC and cultured in F 12K (A549) or RPMI 1640 (H1299, H1975, HCC827, and THP-1) media (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher Scientific).

Techniques: Migration, Expressing, Over Expression, Quantitative RT-PCR, CCK-8 Assay, Caspase-3 Activity Assay, TUNEL Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: Cellular & molecular biology letters

Article Title: LARRPM restricts lung adenocarcinoma progression and M2 macrophage polarization through epigenetically regulating LINC00240 and CSF1.

doi: 10.1186/s11658-022-00376-y

Figure Lengend Snippet: Fig. 6 LARRPM repressed M2 macrophage infiltration. a Schematic diagram of in vitro co-culture system of phorbol-12-myristate-13-acetate (PMA)-stimulated human monocyte line THP-1 cells and LUAD cells. b THP-1 cells were subjected to transwell migration assays towards A549 cells with LARRPM overexpression or A549 control cells. Scale bar: 100 µm. c THP-1 cells were subjected to transwell migration assays towards HCC827 cells with LARRPM depletion or HCC827 control cells. Scale bar: 100 µm. d Expression of M1 and M2 polarization markers in THP-1 cells co-cultured with A649 cells with LARRPM overexpression or A549 control cells was detected by qRT-PCR. e Expression of M1 and M2 polarization markers in THP-1 cells co-cultured with HCC827 cells with LARRPM depleted or HCC827 control cells was detected by qRT-PCR. f, g CD163 (f) and CD206 (g) IF staining in THP-1 cells co-cultured with A549 cells with LARRPM overexpression or A549 control cells, or THP-1 cells co-cultured with HCC827 cells with LARRPM depleted or HCC827 control cells. Results are shown as the mean ± SD based on three independent experiments. Asterisks indicate a significant difference at *P < 0.05, **P < 0.01, ***P < 0.001, by Student’s t-test (b, d) or one-way ANOVA followed by Dunnett’s multiple comparisons test (c, e). ARG1 Arginase 1, IL interleukin, TNF tumor necrosis factor

Article Snippet: Human LUAD cells A549 (Cat. CCL 185), H1299 (Cat. CRL 5803), H1975 (Cat. CRL5908), HCC827 (Cat. CRL-2868) and human monocyte line THP-1 (Cat. TIB-202) were obtained from the ATCC and cultured in F 12K (A549) or RPMI 1640 (H1299, H1975, HCC827, and THP-1) media (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher Scientific).

Techniques: In Vitro, Co-Culture Assay, Migration, Over Expression, Control, Expressing, Cell Culture, Quantitative RT-PCR, Staining

Journal: Cellular & molecular biology letters

Article Title: LARRPM restricts lung adenocarcinoma progression and M2 macrophage polarization through epigenetically regulating LINC00240 and CSF1.

doi: 10.1186/s11658-022-00376-y

Figure Lengend Snippet: Fig. 7 LARRPM repressed CSF1 expression via inducing DNA methylation. a, b The expression of cytokines related to macrophage recruitment and polarization in A549 cells with LARRPM overexpression (a) and HCC827 cells with LARRPM depletion (b) was detected by qRT-PCR. c The expression of CSF1 in another LARRPM stably depleted HCC827 clone was detected by qRT-PCR. d, e ChIP assays followed by qPCR were performed in A549 cells with LARRPM overexpression (d) and HCC827 cells with LARRPM depletion (e) to detect the DNAs (LINC00240 promoter) bound by TET1. f, g 5hmC levels of the CpG island CpG82 from A549 cells with LARRPM overexpression (f) and HCC827 cells with LARRPM depletion (g) were measured using the EpiMark 5-hmC Analysis Kit. h, i DNA methylation levels of CpG82 from A549 cells with LARRPM overexpression (h) and HCC827 cells with LARRPM depletion (i) were measured using bisulfate DNA sequencing. j, k CSF1 secretion from A549 cells with LARRPM overexpression (j) and HCC827 cells with LARRPM depletion (k) was measured by enzyme-linked immunosorbent assay (ELISA). Results are shown as the mean ± SD based on three independent experiments. Asterisks indicate a significant different at *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant, by Student’s t-test (a–d, f, h, j) or one-way ANOVA followed by Dunnett’s multiple comparisons test (e, g, i, k). CCL2, -3, -4 CC chemokine family, CSF1, CSF2 Colony Stimulating Factor 1, IFN interferon, TGF tumor growth factor

Article Snippet: Human LUAD cells A549 (Cat. CCL 185), H1299 (Cat. CRL 5803), H1975 (Cat. CRL5908), HCC827 (Cat. CRL-2868) and human monocyte line THP-1 (Cat. TIB-202) were obtained from the ATCC and cultured in F 12K (A549) or RPMI 1640 (H1299, H1975, HCC827, and THP-1) media (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher Scientific).

Techniques: Expressing, DNA Methylation Assay, Over Expression, Quantitative RT-PCR, Stable Transfection, DNA Sequencing, Enzyme-linked Immunosorbent Assay

Journal: Cellular & molecular biology letters

Article Title: LARRPM restricts lung adenocarcinoma progression and M2 macrophage polarization through epigenetically regulating LINC00240 and CSF1.

doi: 10.1186/s11658-022-00376-y

Figure Lengend Snippet: Fig. 8 Ectopic expression of LARRPM in LUAD cells attenuated the oncogenic roles of infiltrated M2 macrophages. a–f CM was collected from co-culture of macrophages with LARRPM overexpressed or control A549 cells, or collected from only A549 cells with LARRPM overexpression or A549 control cells. a, b Cell proliferation of A549 cells treated with the CM was detected using the CCK-8 assay (a) and EdU incorporation assay (b). Scale bar: 100 µm. Red color indicates EdU-positive cells. c, d Cell apoptosis of A549 cells treated with the CM was detected using the caspase-3 activity assay (c) and TUNEL assay (d). Scale bar: 100 µm. Green color indicates TUNEL-positive cells. e, f Cell migration and invasion of A549 cells treated with the CM was detected using the transwell migration assay (e) and transwell invasion assay (f). Scale bar: 100 µm. Results are shown as the mean ± SD based on three independent experiments. Asterisks indicate a significant difference at *P < 0.05, **P < 0.01, ns, not significant, by Student’s t-test. CM Conditioned medium

Article Snippet: Human LUAD cells A549 (Cat. CCL 185), H1299 (Cat. CRL 5803), H1975 (Cat. CRL5908), HCC827 (Cat. CRL-2868) and human monocyte line THP-1 (Cat. TIB-202) were obtained from the ATCC and cultured in F 12K (A549) or RPMI 1640 (H1299, H1975, HCC827, and THP-1) media (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher Scientific).

Techniques: Expressing, Co-Culture Assay, Control, Over Expression, CCK-8 Assay, Caspase-3 Activity Assay, TUNEL Assay, Migration, Transwell Migration Assay, Transwell Invasion Assay