LncRNA LINK‐A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

Figures

• 

  Figure 1. LINK‐A overexpression in mice promotes HFD‐induced obesity and insulin resistance a) Venn diagram of differentially expressed lncRNAs in a database of high‐risk diseases in individuals who are obesity (OA: osteoarthritis, GSE111358; GSVV: varicosis of great saphenous vein, GSE51260; RA: rheumatoid arthritis, GSE181614). b) LINK‐A expression was detected by qRT‐PCR in breast tissues of patients in the BMI <25 and BMI≥ 25 groups. Data presented as mean ± SEM, mammary gland (n = 33, 12), Student's t‐test, the data conform to normal or lognormal distribution, *p <0.05. c) Correlation analysis of LINK‐A expression in the breast tissues of patients with patient BMI. Data presented as mean ± SEM, mammary gland (n = 45), Pearson chi‐square test, p = 0.0003. d) Schematic diagram of LINK‐A KI mouse construction strategy. e) The LINK‐A expression levels in different tissues of LINK‐A KI mice were detected by qRT‐PCR. Data presented as mean ± SEM, n = 6, one‐way ANOVA, ****p <0.0001. f) Schematic diagram of the feeding strategy for mice. WT and LINK‐A KI mice received the dietary intervention (ND or HFD) at 8 weeks of age. g) Representative photographs of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI…

• 

  Figure 2. LINK‐A overexpression reduces adaptive thermogenic function in mice under HFD, contributing the HFD‐induced obesity a) Core body temperature of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI during a 6 h cold challenge (fasting at 30 °C for 16 h and then transfer to 4 °C). Data presented as mean ± SEM, per group n = 6, two‐way ANOVA, ns = no significance, *p <0.05, ***p <0.001, ****p <0.0001. b–d) VO2(b), Energy expenditure(c), and RER(d) of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice at cold temperature (4 °C). Data presented as mean ± SEM, per group n = 6, two‐way ANOVA, ns = no significance, ****p <0.0001. e) Representative images of hematoxylin‐eosin (H&E)‐stained sections and immunohistochemistry (IHC) of different adipose tissues. The UCP1 levels were analyzed using ImageJ. Scale bar: scWAT(200 µm), BAT(50 µm), and VAT(50 µm). Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, ***p <0.001, ****p <0.0001. f) Immunoblot analysis of UCP1 protein levels in ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice housed at 4 °C for 48 h. The UCP1 protein was quantified using ImageJ. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, *p <0.05, **p…

• 

  Figure 3. LINK‐A overexpression reduces adaptive thermogenesis in HFD‐fed mice by remodeling the regional inflammatory microenvironment a) Heatmap of inflammatory factor mRNA expression levels in the mammary glands of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice detected by qRT‐PCR. per group n = 6. b–d) Representative images of H&E‐stained sections and IHC of mammary glands(b). The IL‐1β(c) or CXCL16(d) protein levels were analyzed using ImageJ. Scale bar: 50 µm. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, *p <0.05, **p <0.01, ****p <0.0001. e–g) Representative images of H&E‐stained sections and IHC of scWAT(e). The IL‐1β(f) or CXCL16(g) protein levels were analyzed using ImageJ. Scale bar: 50 µm. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, ****p <0.0001. h–i) Mouse plasma concentrations of IL‐1β(h) and CXCL16(i) were measured by ELISA. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, ****p <0.0001. j) The protein levels of UCP1, P‐PKA, PKA, P‐HSL, and HSL in BMSCs‐derived beige adipocytes treatment using IL‐β or/and CXCL16 cytokines by immunoblot analysis, and the protein levels…

• 

  Figure 4. LINK‐A overexpression induces inflammatory factor expression by stabilizing HIF1α through the HFD‐induced HB‐EGF a) Schematic diagram of HRE site at CXCL16 promoters(up) and HIF1α binds the CXCL16 promotor region by HIF1α ChIP‐seq database analysis(down). b) CXCL16 ChIP‐qPCR primer sequences(blue) and HRE site sequences (yellow). c) The interaction between HIF1α and CXCL16 promoter in MCF‐10A was verified by ChIP‐qPCR assay. Data presented as mean ± SEM, pooled data from three independent experiments, unpaired t‐test, ****p <0.0001. d) IL‐1β ChIP‐qPCR primer sequences(blue) and HRE site sequences(yellow). e) The interaction between HIF1α and IL‐1β promoter in MCF‐10A was verified by ChIP‐qPCR assay. Data presented as mean ± SEM, pooled data from three independent experiments, unpaired t‐test, **p <0.01. f) Immunoblot analysis of HIF1α protein levels in the mammary gland of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice. The HIF1α protein was quantified using ImageJ. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, ****p <0.0001. g) qRT‐PCR analysis of Hif1α mRNA in mammary glands of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice. Data presented as mean ± SEM, per…

• 

  Figure 5. HIF1α positively regulated LINK‐A and HB‐EGF to form an activated loop pathway of LINK‐A/HB‐EGF/HIF1α a) HIF1α binds the HB‐EGF promotor region by HIF1α ChIP‐seq database analysis. b) HB‐EGF ChIP‐qPCR primer sequences(blue) and HRE site sequences(yellow). c) The interaction between HIF1α and HB‐EGF in MCF‐10A was verified by ChIP‐qPCR assay. Data presented as mean ± SEM, pooled data from three independent experiments, unpaired t‐test, ****p <0.0001. d) The interaction between HIF1α and HB‐EGF promoter in MCF‐10A was verified by a Dual‐Luciferase reporter assay. The HB‐EGF Pro mut site contains HRE(b). Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ****p <0.0001. e) The protein level of HIF1α in MCF‐10A EV or HIF1α overexpression cells with or without HB‐EGF stimulation was detected by immunoblot analysis, and the HIF1α protein was quantified using ImageJ. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ***p <0.001, ****p<0.0001. f) The mRNA level of HB‐EGF in MCF‐10A EV or HIF1α overexpression cells with or without HB‐EGF stimulation was detected by qRT‐PCR. Data presented as mean ± SEM,…

• 

  Figure 6. Activation of the LINK‐A/HB‐EGF/HIF1α pathway in mammary cells reduces adipocyte thermogenesis a) Schematic representation of bone marrow mesenchymal stem cells (BMSCs)‐derived beige adipocytes treated with MCF‐10A cells conditioned media: LINK‐A overexpressing MCF‐10A and control cells were stimulated with HB‐EGF, and the conditioned media was obtained to treat beige adipocytes. b) The protein levels of UCP1, P‐PKA, PKA, P‐HSL, and HSL in adipocytes treated with different conditioned media by immunoblot analysis, and the protein levels were quantified using ImageJ. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, **p <0.01, ***p <0.001, ****p <0.0001. c–f) The mRNA levels of thermogenic genes in adipocytes treated with different conditioned media by qRT‐PCR. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, **p <0.01, ***p <0.001, ****p <0.0001. g) Schematic representation of BMSCs‐derived beige adipocytes treated with MCF‐10A cells conditioned media: IL‐1β inhibitor or/and CXCL16 inhibitors add to the conditioned media of HB‐EGF‐stimulated LINK‐A…

• 

  Figure 7. ASO drug inhibiting LINK‐A attenuates obesity and metabolic disorders in mice a) Schematic diagram of the mouse mammary gland in situ LINK‐A LNAs injection strategy. b) LINK‐A levels in mice mammary glands injected with LINK‐A LNAs in situ for two weeks by qRT‐PCR. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, **p<0.01. c) Body weight changes in HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment. Data presented as mean ± SEM, per group n = 6, two‐way ANOVA, ns = no significance, ****p <0.0001. d,e) GTT(d) and ITT(e) of HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment. Data presented as mean ± SEM, per group n = 6, two‐way ANOVA, *p <0.05, ***p<0.001. f) The HIF1α protein levels in the mammary gland of HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment were detected by immunoblot analysis, and the HIF1α protein levels were quantified using ImageJ. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, **p <0.01. g,h) The plasma concentrations of IL‐1β(g), and CXCL16(h) in HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment were measured by ELISA. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, **p <0.01. i–k) Representative…

• 

  Figure 8. The LINK‐A expression was positively correlated with the expression of inflammatory cytokines in overweight people a–e) Representative images of H&E‐stained sections and IHC of breast tissue microarrays(a) of patients with overweight, and the inflammatory factor levels were analyzed using ImageJ. Scale bar: 50 µm. Data presented as mean ± SEM, n = 12, chi‐square test, ns = no significance, ****p <0.0001. f–i) Correlation between IL‐1β, CXCL16, LINK‐A and BMI. Data presented as mean ± SEM, n = 45, Pearson chi‐square test. *p <0.05, **p <0.01, ***p<0.001. j–m) Correlation between BMI, IL‐1β, CXCL16, LINK‐A, and HIF1α protein. Data presented as mean ± SEM, n = 24, Pearson chi‐square test. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001. n) Article General Mechanism Diagram.