Figures & data
Figure 1. GAS5 overexpression improves cardiac function in DCM mice
DCM mouse model was established by injection of STZ. DCM mice were transfected with LV-GAS5 or LV-NC. Normal mice served as control. (a and b) The hemodynamic indexes (LVEF and LVFS) of mice were recorded and analyzed by echocardiography. (c) The expression of GAS5 in heart tissues was detected by qRT-PCR. (d) WB was performed to explore the expression of active forms of NLRP3, caspase-1, IL-1β and IL-18 in heart tissues. (e) HE staining was performed to investigate the levels of myocardial injury. (**P < 0.01, versus Normal; ##P < 0.01, versus LV-NC.)
Figure 2. GAS5 overexpression inhibits NLRP3 inflammasome activation-mediated pyroptosis in vitro.
HL-1 cells were transfected with LV-GAS5 or LV-NC, and then the modified HL-1 cells were incubated with NG or HG. (a) The expression of GAS5 in the HL-1 cells was detected by qRT-PCR. (b) WB was performed to explore the expression of active forms of NLRP3, caspase-1, IL-1β and IL-18 in the HL-1 cells. (c) The caspase-1 activity in the HL-1 cells was estimated. (d) ELISA was performed to explore the levels of IL-1β and IL-18 in the HL-1 cells. (e) LDH release assay was performed to estimate the pyroptosis of the HL-1 cells. (**P < 0.01, versus Ctrl; ##P < 0.01, versus LV-NC.)
Figure 3. GAS5 represses miR-34b-3p expression and promotes AHR expression in vivo and vitro.
DCM mouse model was established by injection of STZ. DCM mice were transfected with LV-GAS5 or LV-NC. Normal mice served as control. (a and b) QRT-PCR and WB were performed to assess the gene or protein expression of miR-34b-3p and AHR in the heart tissues of mice. HL-1 cells were transfected with LV-GAS5 or LV-NC, and then the modified HL-1 cells were incubated with NG or HG. (c and d) QRT-PCR and WB were performed to investigate the gene or protein expression of miR-34b-3p and AHR in the HL-1 cells. (**P < 0.01, versus Normal or Ctrl; ##P < 0.01, versus LV-NC.)
Figure 4. GAS5 regulates AHR expression by sponging miR-34b-3p
(a) The Wt (Mut) GAS5 vector and miR-34b-3p mimic or mimic NC were co-transfected into 293 cells. The relationship between GAS5 and miR-34b-3p was measured by luciferase reporter assay. HL-1 cells were transfected with pcDNA3.1-GAS5, si-GAS5 or its NC. (b) QRT-PCR was performed to assess the expression of GAS5 and miR-34b-3p in the HL-1 cells. (c) The Wt (Mut) 3′ UTR of AHR vector and miR-34b-3p mimic or mimic NC were co-transfected into 293 cells. The relationship between AHR and miR-34b-3p was measured by luciferase reporter assay. HL-1 cells were transfected with miR-34b-3p mimic, miR-34b-3p inhibitor or its NC. (d and e) QRT-PCR and WB were performed to investigate the gene or protein expression of miR-34b-3p and AHR in the HL-1 cells. (**P < 0.01, versus mimic NC; ##P < 0.01, versus Vector; $$P < 0.01, versus Scramble; &&P < 0.01, versus inhibitor NC.)
Figure 5. GAS5 overexpression inhibits NLRP3 inflammasome activation-mediated pyroptosis by inhibiting miR-34b-3p expression
The pcDNA3.1-GAS5 or empty pcDNA3.1 vector and miR-34b-3p mimic or mimic NC were co-transfected into HL-1 cells. (a and b) QRT-PCR and WB were performed to investigate the gene or protein expression of miR-34b-3p and AHR in the HL-1 cells. (c) WB was performed to explore the expression of active forms of NLRP3, caspase-1, IL-1β and IL-18 in the HL-1 cells. (d) The caspase-1 activity in the HL-1 cells was estimated. (e) ELISA was performed to explore the levels of IL-1β and IL-18 in the HL-1 cells. (f) LDH release assay was performed to estimate the pyroptosis of the HL-1 cells. (**P < 0.01, versus Vector+mimic NC; ##P < 0.01, versus Vector+miR-34b-3p mimic.)
Figure 6. GAS5 overexpression inhibits NLRP3 inflammasome activation-mediated pyroptosis by regulating miR-34b-3p/AHR axis
The pcDNA3.1-GAS5 or empty pcDNA3.1 vector and si-AHR or Scramble were co-transfected into HL-1 cells. (a and b) QRT-PCR and WB were performed to investigate the gene or protein expression of AHR in the HL-1 cells. (c) WB was performed to explore the expression of active forms of NLRP3, caspase-1, IL-1β and IL-18 in the HL-1 cells. (d) The caspase-1 activity in the HL-1 cells was estimated. (e) ELISA was performed to explore the levels of IL-1β and IL-18 in the HL-1 cells. (f) LDH release assay was performed to estimate the pyroptosis of the HL-1 cells. (**P < 0.01, versus Vector+Scramble; ##P < 0.01, versus Vector+si-AHR.)
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