Fig 2: IGF1R, IL-6R and IL-6 are direct targets of let-7c/miR-99a/miR-125b. (a) The positive correlation among miR-99a, let-7c and miR-125b, correlation analysis was determined using the Pearson product–moment correlation. (b) Schematic representation of the interaction of an miRNA with the 3' UTR of its corresponding target. Each predicted MRE 3' UTR was inserted into a psiCHECK-2 vector immediately downstream from the Renilla luciferase gene. Luciferase reporter assays analyzing the putative targets of the three miRNAs. HEK-293T cells were co-transfected with psiCHECK-2 plasmids with the 3' UTR of the target genes or the target genes with mutated miRNA-binding sites (Mut) and miRNA mimics or negative control mimics (NC) using Lipofectamine 2000. Firefly luciferase activity was normalized to Renilla luciferase activity, and the results were expressed relative to the control. (c) QBC939, MZ-cha-1 and SK-cha-1 cells were infected by lentivirus overexpressing miRNA or negative controls, and cells lysates were prepared for western blotting with antibodies against IGF1R, IL-6R or IL-6. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression served as a loading control, and densitometric ratios were recorded. Experiments were performed in triplicate, and all indicated P-values were determined using t-test. *P<0.05; **P<0.01.

Fig 3: Insulin and IGF-1 action on chimeric IR/IGF1R receptors. VSMCs were cultured from aortas of mice which had both IRflox/flox and IGF1Rflox/flox alleles. Then IRflox/floxIGF1Rflox/flox VSMCs were infected with AdCre to knockdown both IR and IGF1R. The IR and IGF1R double knockdown VSMCs were further infected with AdIR-ECD/IGF1R-ICD or AdIGF1R-ECD/IR-ICD or both to overexpress chimeric IR-ECD/IGF1R-ICD or IGF1R-ECD/IR-ICD receptors. a, e indicate representative western blotting images. b–d, f show summarized quantification data (b, n = 3 for WT and DKO, n = 4 for AdIR/IGF1R; c, n = 3 for each group; d, n = 4 for WT, AdIR/IGF1R, and AdIGF1R/IR, n = 3 for DKO; f, n = 3 for each group). The data are mean ± SEM. Two-way ANOVA with a post hoc test. Source data are provided as a Source Data file

Fig 4: The IL-6/STAT3 pathway is regulated by let-7c/miR-99a/miR-125b in CCA cell lines and clinical samples. (a) Treatment of QBC939, MZ-cha-1 and SK-cha-1 cells with IL-6 for 0.5, 1, or 2 h, respectively, induced and maintained STAT3 phosphorylation; and exogenous let-7c, miR-99a and miR-125b eliminated the activity of STAT3. (b) Downregulation of let-7c/miR-99a/miR-125b in clinical CCA samples resulted in the upregulation of IL-6R and IGF1R. GADPH expression served as a loading control and densitometric ratios were recorded. Experiments were performed in triplicate.

Fig 5: (A,B) WB of pIGF-1R/pIR in rat thyroid tissue 60 min after injection of varying doses of HI and glargine. Histogram showing changes in IGF-1Rbeta/IR beta phosphorylation levels in thyroid cells 60 min after injection of 1–200 U/kg of HI (gray) and glargine (black), as calculated from WB results (n = 5–7, compared to blank group, *P < 0.05, **P < 0.01, ***P < 0.001; Compared to HI group, #P < 0.05, ###P < 0.001); (C,D) Detection of pIR after immunoprecipitation of IR in rat thyroid cells 60 min after injection of different doses of HI and glargine. Histogram showing changes in IR beta phosphorylation in rat thyroid cell 60 min after injection of 1–200 U/kg HI (gray) and glargine (black) calculated from IP and WB results (n = 5–8, compared to blank group, *P < 0.05, **P < 0.01, ***P < 0.001; Compared to HI group, #P < 0.05, ###P < 0.001); (E,F) WB of pIGF-1R following immunoprecipitation of IGF-1R from rat thyroid tissue 60 min after injection with different doses of HI and glargine. Histogram showing changes in IGF-1R beta phosphorylation in thyroid cells of rats injected with 1–200 U/kg HI (gray) and glargine (black) as calculated from IP and WB results (n = 5–7, compared to blank group, *P < 0.05, **P < 0.01, ***P < 0.001; compared to HI group, #P < 0.05, ###P < 0.001).