Fig 1: LINC01569 functions in GMD through binding to YBX1.(A) Stretched HSFBs were treated with TA and transfected with the control or LINC01569 siRNAs. Forty-eight hours after transfection, cells were UV cross-linked, and the cytosolic fraction was isolated. IP was performed using an anti-GR antibody or a rabbit IgG as negative control. The immunoprecipitated RNA was extracted and subjected to reverse transcription. The mRNA levels of EGR1, CITED2, and BMP7 were evaluated. (B and C) Cells as described in (A) were subjected to qRT-PCR (B) and Western blotting (C) analyses. qRT-PCR data were normalized to GAPDH mRNA expression. (D) Schematic diagram of predicted YBX1 binding sites within the LINC01569 RNA. (E) Cell lysate of HSFBs in the absence of TA were subjected to an RNA pulldown assay using biotin-labeled LINC01569, followed by Western blotting analysis for the indicated proteins. Proliferating cell nuclear antigen (PCNA) was used as the negative control. (F) Cells as described in (A) were lysed and subjected to RIP using an anti-YBX1 antibody or IgG control. The precipitated RNAs were determined using qRT-PCR primers specific for LINC01569, with hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) as the negative control. (G) HSFBs were transfected using empty vectors or those overexpressing LINC01569 and/or YBX1, followed by treatment with ACD (5 µg/ml). At the indicated time points, RNA was extracted, and mechanosensor expression was measured using qRT-PCR and normalized to the level of GAPDH. For (A), (B), and (G), data are presented as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by Student’s t test for two groups or ANOVA for more than two groups. n.s., not significant.
Fig 2: LINC01569 recruits YBX1 to target mRNAs in the GMD complex.(A) Stretched HSFBs were treated with TA and transfected with the control or LINC01569 siRNAs. The cells were used in an IP experiment performed using an anti-YBX1 antibody (rabbit IgG as a negative control). EGR1, CITED2, and BMP7 mRNA levels were evaluated. (B) Schematic representation of the predicted binding sites of LINC01569 on the UTRs of EGR1, CITED2, and BMP7. (C) TA-treated and stretched HSFBs were transfected with empty vectors or those expressing wild-type or truncated LINC01569 and were further treated with ACD. The levels of indicated mRNAs were measured using qRT-PCR. (D) TA-treated and stretched HSFBs were transfected with the constructs for wild-type LINC01569 or LINC01569 harboring mutations (mut) in the sites responsible for binding to the corresponding mRNAs. RIP was performed using an anti-YBX1 antibody. The immunoprecipitated RNA was extracted and subjected to reverse transcription. (E) HSFBs were transfected with empty vectors or those overexpressing wild-type LINC01569 or LINC01569 harboring mutations in the mRNA binding sites, followed by treatment with ACD. The mRNAs of mechanosensors were measured using qRT-PCR. (F) Schematic representation of the UTR regions of the mechanosensors cloned into the luciferase plasmid. (G) Luciferase vectors containing wild-type or mutated LINC01569-binding sequences of EGR1, CITED2, and BMP7 UTRs and siRNAs were introduced into TA-treated and stretched HSFBs. pCDNA expressing EGR1, CITED2, and BMP7 UTRs were cotransfected as competitors on binding to LINC01569 with luciferase transcripts containing EGR1, CITED2, and BMP7 UTRs. Luciferase mRNA expression was determined by qPCR 48 hours after transfection. Data were normalized to GAPDH mRNA expression. For (A), (C), (D), (E), and (G), data are presented as means ± SD. *P < 0.05 and **P < 0.01, by Student’s t test for two groups or ANOVA for more than two groups.
Fig 3: A schematic diagram showing that glucocorticoid counteracts mechanical cellular responses via LINC01569-guided GMD of mechanosensors.Mechanosensors including EGR1, CITED2, and BMP7 sense stretch and are stably expressed, leading to cellular mechanical responses of HSFBs, including increased collagen, increased proliferation, and decreased apoptosis. However, glucocorticoid up-regulates LINC01569, which mediates GMD of the mechanosensors, and counteracts the cellular mechanical responses of cells. Mechanistically, LINC01569 binds directly with the GMD protein factor YBX1 and guides it to the UTRs of EGR1, CITED2 and BMP7 mRNAs through lncRNA-mRNA interaction, thereby contributing to the successful assembly of the GMD complex that triggers GMD of the mechanosensors. Glucocorticoids and lncRNA-guided GMD system thus hold out promise for effective prevention and treatment of clinical disorders associated with mechanical stretch.
Fig 4: Mechanosensors including EGR1, CITED2, and BMP7 participate in cellular mechanical responses.(A) HSFBs treated with TA were seeded in chambers and subjected to mechanical stretch. Paired HSFBs in the control groups were treated with solvent and subjected to mechanical stretch. Microarray analysis (GSE151240) of differentially expressed genes was performed. Red shading indicates increased gene expression, whereas blue shading indicates decreased expression. The mRNAs that were up-regulated by at least 2-fold (red bars) or down-regulated by at least 0.5-fold (blue bars) were included in the cluster analysis. (B) The bubble chart provides an overview of the most enriched pathways ranked as the top eight altered pathways according to Cytoscape analysis. Bubble size represents the number of genes in each pathway. The bubble color indicates the P value of each pathway. PI3K, phosphatidylinositol 3-kinase. (C) GO annotation using DAVID (Database for Annotation, Visualization, and Integrated Discovery) bioinformatic classification of gene categories from fig. S3. Biological process shows the signaling pathways that were most markedly changed by TA treatment of stretched HSFBs. (D and E) qRT-PCR (D) and Western blotting (E) analyses using HSFBs undergoing stretching and/or treatment with TA. The results of qRT-PCR were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, and in the Western blotting analysis, β-actin was used as an internal loading control. Data are presented as means ± SD. *P < 0.05, by Student’s t test. (F) Correlation analysis of candidate genes involved in the indicated biological processes based on qRT-PCR assays of gene expression in (C). Red indicates positive correlations, and blue indicates negative correlations. The brightness is proportional to the strength of the correlation. (G) Masson’s trichrome staining for collagen using serial sections of clinical hypertrophic scar tissues before or after treatment with TA for 28 days. Immunohistochemical analysis of EGR1, CITED2, and BMP7 proteins in TA-treated hypertrophic scar tissues and paired control tissues is shown.
Fig 5: LINC01569 mediates glucocorticoid repression of cellular mechanical responses via regulation of mechanosensors.(A and B) HSFBs were infected with control lentiviruses or those expressing LINC01569 (Lv-LINC) or LINC01569-targeted short hairpin RNAs (shRNAs) (sh-LINC). Cells were subjected to mechanical TA and stretch treatment, followed by qRT-PCR (A) and Western blotting (B) analyses. (C) Hypertrophic scar tissues from patients were treated with TA and collected for qRT-PCR assay. The results were plotted and subjected to correlation analysis for the expression of the indicated genes. (D) HSFBs were infected with control or sh-LINC lentiviruses or further transfected with EGR1-targeted siRNAs. Collagen I and collagen III produced by cultured HSFBs were measured using ELISA. (E) HSFBs were infected with control or sh-LINC lentiviruses or further transfected with CITED2-targeted siRNAs. Expression of MMP1 and MMP3 were measured using qRT-PCR. (F) HSFBs were infected with control or sh-LINC lentiviruses or further transfected with BMP7-targeted siRNAs. Cells were then subjected to CCK-8 assays. (G to I) HSFBs were infected with control or sh-LINC lentiviruses and further transfected with the indicated siRNAs, followed by Western blotting analysis for indicated proteins. For (B), (E), and (F), data are presented as means ± SD. *P < 0.05 and **P < 0.01, by ANOVA for more than two groups. For (C), *P < 0.05, by Tukey’s post hoc test.
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