Long noncoding RNA X‐inactive specific transcript (lncRNA XIST) inhibits hepatic insulin resistance by competitively binding microRNA‐182‐5p

Figures

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  Figure 1. Effects of lncRNA XIST on IGF‐1R, 2‐DG6P, G6Pase, and PEPCK expressions and PI3K/Akt pathway in IR cells. (A) LncRNA XIST expression was detected by qRT‐PCR, and GAPDH was served as the internal reference. (B) The concentration of 2‐DG6P in LX‐2 cells was detected by 2‐DG uptake measurement kit. (C) The mRNA expression levels of G6Pase and PEPCK were determined by qRT‐PCR, and GAPDH was served as the internal reference. (D–E) Western blot was used to detect the G6Pase and PEPCK protein expressions, and GAPDH was served as the internal reference. (F and G) The protein expression of IGF‐1R was also assessed by western blot, and GAPDH was served as the internal reference. (H–J) Western blot was performed again to measure the protein expressions of PI3K, p‐PI3K, Akt, and p‐Akt, and GAPDH was served as the internal reference. All experiments were repeated three times to average. The data were presented as the mean ± standard deviation (SD) of three independent experiments; ***p < .001 versus Blank; ^p < .05; ^^p < .01; ^^^p < .001 versus EVC. 2‐DG6P, 2‐deoxy‐d‐glucose‐6‐phosphate; EVC, empty vector control; G6Pase, glucose‐6‐phosphatase; GAPDH, glyceraldehyde‐3‐phosphate…

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  Figure 2. The target relationship between lncRNA XIST/IGF‐1R and miR‐182‐5p. (A and B) StarBase (http://starbase.sysu.edu.cn/) was used to predict the targeted binding sites between lncRNA XIST/IGF‐1R and miR‐182‐5p. (C and D) Relative luciferase activity of wild‐type lncRNA XIST/IGF‐1R (XIST/IGF‐1R‐WT) and mutant lncRNA XIST/IGF‐1R (XIST/IGF‐1R‐MUT) groups. (E) The enrichment of XIST in the Bio‐NC, Bio‐miR‐182‐WT, and Bio‐miR‐182‐MUT groups was determined by RNA‐biotin pull‐down assay. (F) The enrichment of XIST and miR‐182‐5p in the Anti‐IgG and Anti‐Ago2 groups was detected by RNA immunoprecipitation assay. All experiments were repeated three times to average. The data were presented as the mean ± standard deviation (SD) of three independent experiments; ***p < .001 versus MC; ^^^p < .001 versus Bio‐miR‐182‐MUT; +++ p < .001 versus Anti‐IgG. Ago2, Argonaute 2; Bio, biotin; IGF‐1R, insulin‐like growth factor‐1 receptor; MC, mimic control; XIST, X‐inactive specific transcript.

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  Figure 3. Effects of miR‐182‐5p on IGF‐1R, 2‐DG6P, G6Pase and PEPCK expressions and PI3K/Akt pathway in IR cells. (A) MiR‐182‐5p expression was detected by qRT‐PCR, with U6 as the internal reference. (B) The miR‐182‐5p expression level in EVC + MC, XIST + MC, EVC + M, and XIST + M groups were detected by qRT‐PCR, with U6 serving as the internal reference. (C) The 2‐DG6P content was detected by 2‐DG uptake measurement kit. (D) After transfection, the mRNA expressions of G6Pase and PEPCK were determined by qRT‐PCR, with GAPDH serving as the internal reference. (E–H) The protein expressions of G6Pase, PEPCK, and IGF‐1R were measured by western blot, with GAPDH serving as the internal reference. (I–K) The PI3K/Akt pathway‐related proteins were also detected by western blot, with GAPDH serving as the internal reference. All experiments were repeated three times to average. The data were presented as the mean ± standard deviation (SD) of three independent experiments; + p < .05 versus Blank; *p < .05; ***p < .001 versus EVC + MC; ^^p < .01; ^^^p < .001 versus XIST + MC; ### p < .001 versus EVC + M. 2‐DG6P, 2‐deoxy‐d‐glucose‐6‐phosphate; EVC, empty vector control; GAPDH,…