Fig 1: Characterization of SNP-regulated IL-6/IL-6-AS1 expression upon inflammatory stimuli. (a) Promoter and enhancer signatures at the IL-6/IL-6-AS1 locus. RNA-seq, DNase I, and H3K4me3-seq profiles of the risk-associated SNPs in the IL-6/IL-6-AS1 locus. The position of the 3 promoter SNPs and 2 intronic SNPs are indicated, and the Caucasian-common SNPs are shown in gray. (b and c) mRNA levels of IL-6-AS1 (b) and IL-6 (c) following LPS treatment at the indicated times in PBMCs with WT or variant genotypes. Each assay was performed for each donor (WT/WT, n = 5; Var/Var, n = 5) in technically triplicate. (d and e) mRNA expression of IL-6-AS1 (e) and IL-6 (f) upon SeV infection in PBMCs. Each assay was performed for each donor (WT/WT, n = 2; Var/Var, n = 2) in technical triplicate. Data represent means ± SD per group. Data were analyzed by multiple t test with Bonferroni correction for multiple comparisons. *, P < 0.05; ***, P < 0.001. (d) Genetic polymorphisms of IL-6 in Asian populations underlie vulnerability to COVID-19. (Left) Viral infections stimulate bidirectional transcription of the IL-6/IL-6-AS1 locus in individuals carrying the wild-type alleles at rs1800796, rs1524107, and rs2066992 loci. Upregulated IL-6-AS1 acts in cis to further enhance the transcription of IL-6. High levels of IL-6 exacerbate the excessive inflammatory response and are linked to worse clinical outcome of COVID-19. (Right) The variant allele disrupts CTCF binding at the enhancer element of IL-6-AS1, leading to transcriptional inactivation of IL-6-AS1 and reduced upregulation of IL-6 in response to an acute SARS-CoV-2 infection. The motif-altering SNP rs2066992 is marked by red (ancestral allele) or blue (derived allele).
Fig 2: Association between IL-6 polymorphisms and the expression of IL-6 and its antisense transcript lncRNA IL-6-AS1. (a) Expression quantitative trait locus (eQTL) analysis of association trends for rs1800796, rs1524107, rs2066992, and IL-6-AS1 expression in lung tissues. All data were collected from the GTEx project. (b) Pearson correlation analysis between IL-6-AS1 and IL-6 mRNA expression in several cell lines. r, Pearson correlation coefficient. (c to e) mRNA expression of IL-6-AS1 and IL-6 in HeLa (c and d) and A549 (e) cells. Cells were treated with TNF-a (c) or transfected with poly(I·C) (d and e) for 12 h. One-way ANOVA with Bonferroni correction for multiple comparisons was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (f and g) Time course expression of IL-6 and IL-6-AS1 following poly(I·C) transfection into HeLa and A549 cell line. Data represent means ± SD per group.
Fig 3: Cytokine concentration and neurosupportive potential of astrocyte-conditioned medium (ACM) from healthy control and sporadic ALS astrocytes. (a–) ELISA-determined concentrations, pg/ml, of (a) IL1ß (b) IL6, and (c) TNFa of ACM. (d) MTT-determined viability of motor neurons treated with fresh medium, normal medium, control astrocyte ACM, patient astrocyte ACM, and cytokine medium. (e) IF staining of motor neurons for the markers ChAT (choline acetyltransferase), TUJ (ß-tubulin III), and DAPI. * represents p < 0.05, ** represents p value < 0.01, and *** represents p < 0.001. ELISA: enzyme-linked immunosorbent assay, MTT: 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide.
Fig 4: Haplotypes of rs1800796, rs1524107, and rs20669992 affect the enrichment of CTCF in the IL-6/IL-6-AS1 locus. (a) Genomic locations of rs1800796, rs1524107, and rs20669992 in IL-6 and its antisense lncRNA IL-6-AS1. Exons are marked with boxes, in which untranslated regions are shown as open boxes, and coding regions are marked with black boxes. (b) The susceptibility SNPs rs1800796, rs1524107, and rs2066992 reside in or close to the CTCF-binding regions of the IL-6/IL-6-AS1 locus. The ChIP-PCR fragments used in panel d are marked with lines below the gene. (c) The location of CTCF-binding sites in the IL-6 gene. Human CTCF binding motif logo was from JASPAR2020, and the 12-bp consensus sequences of CTCF-binding sites are in boxes. The wild-type allele is in blue and variant allele in red. Phylop basewise conservation score derived from 100 vertebrate species is shown. (d) ChIP-PCR in PBMCs of a donor with wild-type genotype at the candidate SNPs using anti-CTCF antibody or control IgG. H19 was used as a positive control. MW, molecular weight; ddH2O, double-distilled water. (e) CTCF binding intensity at IL-6/IL-6-AS1 locus in LCL cell lines with different genotypes at the loci of interest. (f) ChIP-qPCR assay of CTCF binding at two target regions of IL-6 locus in PBMCs of healthy donors with different genotypes. Each assay was performed for each donor (WT/WT, n = 2; WT/Var, n = 2; Var/Var, n = 2) in technical triplicate. Data represent means ± SD per group. One-way ANOVA with Bonferroni correction for multiple comparisons. (g) Basal level of IL-6-AS1 in PBMCs of donors as in panel f. Each assay was performed for each donor in technical triplicate. Relative expression of IL-6-AS1 is normalized to GAPDH. Data represent means ± SD per group. One-way ANOVA with Bonferroni correction for multiple comparisons was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not statistically significant.
Fig 5: ELISA detected (a, b) the contents of IL-6 and TNF-a in the supernatant of HK-2 cells transfected and treated with LPS (1 µg/mL, 6 h). Data are expressed as the mean ± standard deviation (n = 3), ##P < 0.01 versus the blank group; *P < 0.05, **P < 0.01 versus the control group. (c–e) WB detection of transfected FXR overexpression or interfering RNA and protein expression of HK-2 cells after stimulation with 10% concentration drug-containing serum and treatment with TGF-ß (20 ng/mL, 24 h). Data are expressed as the mean ± standard deviation (n = 3), *P < 0.05, **P < 0.01. WB result (f–h) observation of protein expression after transfection of siFXR or sia-SMA and treatment with TGF-ß (20 ng/mL, 24 h). **P < 0.01 versus the control group; ns: not statistically significant.
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