Journal: Molecular Therapy. Nucleic Acids

Article Title: M2 Macrophage-Derived Exosomal lncRNA AFAP1-AS1 and MicroRNA-26a Affect Cell Migration and Metastasis in Esophageal Cancer

doi: 10.1016/j.omtn.2020.09.035

Figure Lengend Snippet: ATF2 Is a Direct Target Gene of miR-26a and AFAP1-AS1 Absorbs miR-26a to Impact Its Expression (A) Prediction of AFAP1-AS1 subcellular localization by online analysis site. (B) Verification of AFAP1-AS1 subcellular localization by FISH assay (400×; scale bars, 25 μm). (C) Prediction of the binding site of AFAP1-AS1 and miR-26a by RNA22 site. (D) Verification of AFAP1-AS1 binding to miR-26a by dual luciferase reporter gene assay. (E) Detection of miR-26a’s enrichment of AFAP1-AS1 by RNA pull-down assay. (F) Prediction of the targeting site between miR-26a and ATF2 by Targetscan website. (G) Verification of the targeting site between miR-26a and ATF2 by dual luciferase reporter gene assay. ∗p < 0.05. N = 3. The data in the figure were all measurement data expressed as mean ± standard deviation; the t test was used for comparison between two groups, and ANOVA was used for the comparison among multiple groups, after which pairwise comparison was made by Tukey’s multiple comparisons test.

Article Snippet: The synthesized siRNA-NC, AFAP1-AS1-siRNA, mimic-NC, miR-26a mimic, pcDNA-AFAP1-AS1 + mimic-NC, and pcDNA-AFAP1-AS1 + miR-26a mimic (Shanghai GenePharma, Shanghai, China) were transfected into mature M2 macrophages, which were co-cultured with KYSE410 cells 24 h later.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Gene Assay, Pull Down Assay, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: M2 Macrophage-Derived Exosomal lncRNA AFAP1-AS1 and MicroRNA-26a Affect Cell Migration and Metastasis in Esophageal Cancer

doi: 10.1016/j.omtn.2020.09.035

Figure Lengend Snippet: Identification of M2 Macrophages (A) Morphology of M1 and M2 macrophages was observed under an inverted microscope (400×, scale bars, 50 μm). (B) CD68 and CD206 mRNA expression in M1 and M2 macrophages detected by qRT-PCR. (C) Protein bands of CD68 and CD206 in M1 and M2 macrophages. (D) CD68 and CD206 protein expression in M1 and M2 macrophages detected by western blot analysis. (E) AFAP1-AS1, miR-26a, and ATF2 mRNA expression in M1 and M2 macrophages detected by qRT-PCR. (F) Protein band of ATF2 in M1 and M2 macrophages. (G) ATF2 protein expression in M1 and M2 macrophages detected by western blot analysis. ∗p < 0.05 versus M1 macrophages. N = 3. The data were expressed as mean ± standard deviation and analyzed by t test.

Article Snippet: The synthesized siRNA-NC, AFAP1-AS1-siRNA, mimic-NC, miR-26a mimic, pcDNA-AFAP1-AS1 + mimic-NC, and pcDNA-AFAP1-AS1 + miR-26a mimic (Shanghai GenePharma, Shanghai, China) were transfected into mature M2 macrophages, which were co-cultured with KYSE410 cells 24 h later.

Techniques: Inverted Microscopy, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: M2 Macrophage-Derived Exosomal lncRNA AFAP1-AS1 and MicroRNA-26a Affect Cell Migration and Metastasis in Esophageal Cancer

doi: 10.1016/j.omtn.2020.09.035

Figure Lengend Snippet: AFAP1-AS1 and ATF2 Expression Is Upregulated and miR-26a Is Downregulated in M2 Macrophage-Derived Exosomes (A) Exosome morphology observation under TEM (20,000×; scale bars, 100 nm). (B) Protein bands of CD63, CD81, TSG101, and GRP94 in M1 and M2 exosomes. (C) Nanoparticle tracing analysis of size distribution of exosomes. (D) AFAP1-AS1 expression in M1 and M2 exosomes detected by qRT-PCR. (E) miR-26a expression in M1 and M2 exosomes detected by qRT-PCR. (F) ATF2 mRNA expression in M1 and M2 exosomes detected by qRT-PCR. ∗p < 0.05 versus M1 exosome. N = 3. The data were expressed as mean ± standard deviation and analyzed by independent sample t test.

Article Snippet: The synthesized siRNA-NC, AFAP1-AS1-siRNA, mimic-NC, miR-26a mimic, pcDNA-AFAP1-AS1 + mimic-NC, and pcDNA-AFAP1-AS1 + miR-26a mimic (Shanghai GenePharma, Shanghai, China) were transfected into mature M2 macrophages, which were co-cultured with KYSE410 cells 24 h later.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: M2 Macrophage-Derived Exosomal lncRNA AFAP1-AS1 and MicroRNA-26a Affect Cell Migration and Metastasis in Esophageal Cancer

doi: 10.1016/j.omtn.2020.09.035

Figure Lengend Snippet: M2-Exosome Promotes Cell Migration and Invasion as well as In Vitro Lung Tumor Metastasis in EC (A) Observation of uptake of M2-exosome by KYSE410 cells using confocal microscopy (400×; scale bars, 50 μm). (B) Expression of AFAP1-AS1, miR-26a, and ATF2 in KYSE410 cells after co-culture with M2 macrophages. (C) Protein band of ATF2 in KYSE410 cells after co-culture with M2 macrophages. (D) Protein expression of ATF2 in KYSE410 cells after co-culture with M2 macrophages. (E) Wound healing distance in the control and M2-exosome groups. (F) Effect of M2-exosome on wound healing distance of KYSE410 cells. (G) Representative figure for cell migration and invasion by Transwell assay. (H) Effects of M2-exosome on the number of migration and invasion KYSE410 cells. (I) Observation of lung metastasis in nude mice tumor tissue by H&E staining (200×; scale bars, 100 μm). (J) Number of lung metastasis nodules in the control and M2-exosome groups. ∗p < 0.05 versus control group; # p < 0.05 versus M2-exosome group. N = 3. The data are expressed as mean ± standard deviation, and ANOVA was used for the comparison among multiple groups, after which pairwise comparison was made by Tukey’s multiple comparisons test.

Article Snippet: The synthesized siRNA-NC, AFAP1-AS1-siRNA, mimic-NC, miR-26a mimic, pcDNA-AFAP1-AS1 + mimic-NC, and pcDNA-AFAP1-AS1 + miR-26a mimic (Shanghai GenePharma, Shanghai, China) were transfected into mature M2 macrophages, which were co-cultured with KYSE410 cells 24 h later.

Techniques: Migration, In Vitro, Confocal Microscopy, Expressing, Co-Culture Assay, Transwell Assay, Staining, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: M2 Macrophage-Derived Exosomal lncRNA AFAP1-AS1 and MicroRNA-26a Affect Cell Migration and Metastasis in Esophageal Cancer

doi: 10.1016/j.omtn.2020.09.035

Figure Lengend Snippet: M2-Exosomes Upregulate miR-26a or Downregulate AFAP1-AS1 to Reverse the Contributory Impacts of M2-Exosomes on Cell Migration and Invasion, as well as In Vitro Lung Tumor Metastasis in EC (A) AFAP1-AS1, miR-26a, and ATF2 mRNA expression in M2 macrophages of each group detected by qRT-PCR. (B) Protein band of ATF2 in M2 macrophages in each group. (C) ATF2 protein expression in M2 macrophages in each group detected by western blot analysis. (D) AFAP1-AS1, miR-26a, and ATF2 mRNA expression in KYSE410 cells of each group detected by qRT-PCR. (E) Protein band of ATF2 in KYSE410 cells of each group. (F) Protein expression of ATF2 in KYSE410 cells of each group. (G) Representative figure for cell migration by scratch test. (H) Wound healing distance in each group. (I) Representative figure for cell migration and invasion by Transwell assay. (J) Effects of lncRNA AFAP1-AS1 and miR-26a on the migration and invasion of KYSE410 cells. (K) Observation of lung metastasis in nude mice by H&E staining (100×; scale bars, 100 μm). (L) Number of lung metastases in each group. a p < 0.05 versus the siRNA-NC group; b p < 0.05 versus the mimic-NC group; c p < 0.05 versus the pcDNA-AFAP1-AS1 + mimic-NC group; ∗p < 0.05 versus the siRNA-NC-Exo group; # p < 0.05 versus the mimic-NC-Exo group; & p < 0.05 versus the pcDNA-AFAP1-AS1 + mimic-NC-Exo group. N = 3. The data are expressed as mean ± standard deviation, and ANOVA was used for the comparison among multiple groups, after which pairwise comparison was made by Tukey’s multiple comparisons test.

Article Snippet: The synthesized siRNA-NC, AFAP1-AS1-siRNA, mimic-NC, miR-26a mimic, pcDNA-AFAP1-AS1 + mimic-NC, and pcDNA-AFAP1-AS1 + miR-26a mimic (Shanghai GenePharma, Shanghai, China) were transfected into mature M2 macrophages, which were co-cultured with KYSE410 cells 24 h later.

Techniques: Migration, In Vitro, Expressing, Quantitative RT-PCR, Western Blot, Transwell Assay, Staining, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: M2 Macrophage-Derived Exosomal lncRNA AFAP1-AS1 and MicroRNA-26a Affect Cell Migration and Metastasis in Esophageal Cancer

doi: 10.1016/j.omtn.2020.09.035

Figure Lengend Snippet: Primer Sequence

Article Snippet: The synthesized siRNA-NC, AFAP1-AS1-siRNA, mimic-NC, miR-26a mimic, pcDNA-AFAP1-AS1 + mimic-NC, and pcDNA-AFAP1-AS1 + miR-26a mimic (Shanghai GenePharma, Shanghai, China) were transfected into mature M2 macrophages, which were co-cultured with KYSE410 cells 24 h later.

Techniques: Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: M2 Macrophage-Derived Exosomal lncRNA AFAP1-AS1 and MicroRNA-26a Affect Cell Migration and Metastasis in Esophageal Cancer

doi: 10.1016/j.omtn.2020.09.035

Figure Lengend Snippet: Molecular Mechanism of M2 Macrophage-derived Exosomal lncRNA AFAP1-AS1 in EC The mechanistic diagram indicates that M2 macrophage-derived exosomes transferred lncRNA AFAP1-AS1 to downregulate miR-26a and upregulate ATF2, thus promoting cell invasion, migration, and lung tumor metastasis of EC.

Article Snippet: The synthesized siRNA-NC, AFAP1-AS1-siRNA, mimic-NC, miR-26a mimic, pcDNA-AFAP1-AS1 + mimic-NC, and pcDNA-AFAP1-AS1 + miR-26a mimic (Shanghai GenePharma, Shanghai, China) were transfected into mature M2 macrophages, which were co-cultured with KYSE410 cells 24 h later.

Techniques: Derivative Assay, Migration

Journal: Reproduction (Cambridge, England)

Article Title: Estradiol promotes EMT in endometriosis via MALAT1/ miR200s sponge function

doi: 10.1530/REP-18-0424

Figure Lengend Snippet: Primers used in qRT-PCR.

Article Snippet: Two MALAT1-specific siRNAs, the miR200c mimic and the miR200c inhibitor, were synthesized by RiboBio (Guangzhou, China, si-MALAT1–1: No.siB170710110803; si-MALAT1–2: siB121112152859; si-NC: siN05815122147).

Techniques:

Journal: Reproduction (Cambridge, England)

Article Title: Estradiol promotes EMT in endometriosis via MALAT1/ miR200s sponge function

doi: 10.1530/REP-18-0424

Figure Lengend Snippet: (A) The predicted results of the interaction between MALAT1 and miR200s by RNAhybrid (https://bibiserv.cebitec.unibielefeld.de/rnahybrid). (Red: MALAT1. Green: each member of miR200s) (MALAT1 and miR200a, mfe: −22.6 kcal/mol; MALAT1 and miR200b, mfe: −26.8 kcal/mol; MALAT1 and miR200c, mfe: −30.1 kcal/mol; MALAT1 and miR141, mfe: −24.2 kcal/mol; MALAT1 and miR429, mfe: −21.0 kcal/mol.); (B and C) Ishikawa cells were transfected with miR200c mimic or miRNA Negative Control (miRNA NC) and were treated with or without E2 (10−8 mol/L) for 24 h. The expression levels of miR200c and MALAT1 were analyzed by qRT-PCR. (D) Expression levels of EMT-associated markers detected by Western blot after the same treatment for 48 h. (E) The function of miR200c mimic and/or E2 affects the ability of migration (24 h) and invasion (48 h) in Ishikawa cells. Data were evaluated by one-way ANOVA analysis (*P < 0.05, **P < 0.01, ***P < 0.001). Photographs were taken at magnifications of 200×.

Article Snippet: Two MALAT1-specific siRNAs, the miR200c mimic and the miR200c inhibitor, were synthesized by RiboBio (Guangzhou, China, si-MALAT1–1: No.siB170710110803; si-MALAT1–2: siB121112152859; si-NC: siN05815122147).

Techniques: Transfection, Negative Control, Expressing, Quantitative RT-PCR, Western Blot, Migration

Journal: Reproduction (Cambridge, England)

Article Title: Estradiol promotes EMT in endometriosis via MALAT1/ miR200s sponge function

doi: 10.1530/REP-18-0424

Figure Lengend Snippet: (A) The effect of miR200c inhibitor on protein expression levels of down-stream EMT-associated markers for 48 h analyzed by Western blot. (B, C and D) Ishikawa cells were co-transfected with si-MALAT1(−2) or si-NC, and miR200c inhibitor or inhibitor Negative Control (inhibitor NC); (B) Expression levels of Zeb1, Zeb2, E-cadherin and Vimentin after treatment for 48 h analyzed by Western blot; (C) Expression level of MALAT1 after treatment for 24 h analyzed by qRT-PCR; (D) The treatment influences the ability of migration and invasion; (E) Estradiol promotes epithelial– mesenchymal transition in endometriosis via MALAT1/miR200s sponge function. E2-activated ERβ may promote the expression of MALAT1 and mediate EMT in endometriosis. Data were evaluated by one-way ANOVA analysis (*P < 0.05, **P < 0.01, ***P < 0.001). Photographs were taken at magnifications of 200×.

Article Snippet: Two MALAT1-specific siRNAs, the miR200c mimic and the miR200c inhibitor, were synthesized by RiboBio (Guangzhou, China, si-MALAT1–1: No.siB170710110803; si-MALAT1–2: siB121112152859; si-NC: siN05815122147).

Techniques: Expressing, Western Blot, Transfection, Negative Control, Quantitative RT-PCR, Migration