LncRNA LINK‐A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

Abstract High‐fat diet (HFD)‐induced obesity is a crucial risk factor for metabolic syndrome, mainly due to adipose tissue dysfunctions associated with it. However, the underlying mechanism remains unclear. This study has used genetic screening to identify an obesity‐associated human lncRNA LINK‐A as a critical molecule bridging the metabolic microenvironment and energy expenditure in vivo by establishing the HFD‐induced obesity knock‐in (KI) mouse model. Mechanistically, HFD LINK‐A KI mice induce the infiltration of inflammatory factors, including IL‐1β and CXCL16, through the LINK‐A/HB‐EGF/HIF1α feedback loop axis in a self‐amplified manner, thereby promoting the adipose tissue microenvironment remodeling and adaptive thermogenesis disorder, ultimately leading to obesity and insulin resistance. Notably, LINK‐A expression is positively correlated with inflammatory factor expression in individuals who are overweight. Of note, targeting LINK‐A via nucleic acid drug antisense oligonucleotides (ASO) attenuate HFD‐induced obesity and metabolic syndrome, pointing out LINK‐A as a valuable and effective therapeutic target for treating HFD‐induced obesity. Briefly, the results reveale the roles of lncRNAs (such as LINK‐A) in remodeling tissue inflammatory microenvironments to promote HFD‐induced obesity.


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Figures S1 to S5

Figure S1 .
Figure S1.LINK-A overexpression in mice promotes HFD-induced obesity a) The stacked graph for LINK-A conserved regions of sequences in various species.To be specific, the sequence of MSA is depicted as an uninterrupted, semi-opaque line.Each line represents a sequence.A: red, C: blue, T: green, G: yellow.The Sequence logo visualization method calculates the frequency of characters in each column in the MSA, and scales and stacks the characters in each column according to the frequency.b) LINK-A expression was assessed by qRT-PCR in scWAT(left) and VAT(right) tissues of patients in the BMI < 25 and BMI ≥ 25 groups.Data presented as mean ± SEM, n=14, 6, Student's t-test, the data conform to normal or lognormal distribution, *p<0.05,****p<0.0001.c) Correlation analysis of LINK-A expression in the in scWAT(left) and VAT(right) tissues of clinical patients with patient BMI.Data presented as mean ± SEM, n=20, Pearson chi-square test, p=0.0019(scWAT), p=0.0154(VAT).d) Identification of mouse genotypes by pulsed electric field gel electrophoresis.e-h) Representative images of H&E-stained sections of different adipose tissues(e).The distribution of adipocyte size of scWAT(f), BAT(g), and VAT(h) of ND-WT, ND-KI, HFD-WT, and HFD-KI mice were analyzed using ImageJ.Scale bar: 50µm.Data presented as mean ± SEM, per group n=6, chi-square test, ns=no significance, *p<0.05,***p<0.001,****p<0.0001.i) Food intake of WT or LINK-A KI mice fed ND or HFD.Data presented as mean ± SEM, per group n=6, one-way ANOVA, ns=no significance, *p<0.05,**p<0.01.j-k) Line graphs(left) and bar charts(right) of RER in WT and LINK-A KI mice fed ND(j) or HFD(k).Data presented as mean ± SEM, per group n=6, two-way ANOVA, ns=no significance, **p<0.01.

Figure S2 .
Figure S2.LINK-A overexpression reduces adaptive thermogenesis in HFD-fed mice by remodeling the regional inflammatory microenvironment a-g) Inflammatory factor mRNA expression levels in the mammary glands of ND-WT, ND-KI, HFD-WT, and HFD-KI mice were detected by qRT-PCR.Data presented as mean ± SEM, per group n=6, one-way ANOVA,

Figure S3 .
Figure S3.LINK-A overexpression induces inflammatory factor expression by stabilizing HIF1α through the HFD-induced HB-EGF a) The HIF1α protein levels in MCF-10A EV or HIF1α overexpression cells were detected by immunoblot analysis, and the HIF1α protein levels were quantified using ImageJ.Data presented as mean ± SEM, pooled data from three independent experiments, unpaired t-test, **p<0.01.b) The HIF1α protein levels in MCF-10A shControl (shCtrl) or shHIF1α (shHIF1α#1, shHIF1α#2) cells by immunoblot analysis, and the

Figure S4 .
Figure S4.ASO drug inhibiting LINK-A attenuates obesity and metabolic disorders in mice a-b) The LINK-A levels in the MDA-MB-231(a) and MCF-10A(b) treated with different concentrations of LINK-A LNAs were detected by qRT-PCR.Data presented as mean ± SEM, pooled data from three independent experiments, one-way ANOVA, ns=no significance, *p<0.05,***p<0.001,****p<0.0001.c) The HIF1α protein levels in the MDA-MB-231 and MCF-10A with or without LINK-A LNAs treatment were detected by immunoblot analysis, and the HIF1α protein levels were quantified using ImageJ.Data presented as mean ± SEM, pooled data from three independent experiments, two-way ANOVA, *p<0.05,***p<0.001.d) The mRNA expressions of HIF1α or HIF1α downstream genes were detected by qRT-PCR.Data presented as mean ± SEM, pooled data from three independent experiments, two-way ANOVA,

Figure S5 .
Figure S5.The expression levels of HIF1α protein in clinical breast samples.a) The HIF1α protein level in the breast tissue of clinical patients was detected by immunoblot analysis, and the HIF1α protein was quantified using ImageJ.n=24.