Figures & data
Table 1 siRNAs, miRNA-675-3p Mimics and Inhibitors Used in This Study
Table 2 q-PCR Primers Used in This Study
Figure 1 Successful establishment of murine asthma model. (A) Protocol of murine asthma model by using OVA. (B) The number of leukocytes, neutrophils, eosinophils, lymphocytes and monocytes in BALF of murine asthma group and control group. (C) The levels of IgE, IFNγ and IL-5 in BALF were determined by Elisa analysis. (D) HE (haematoxylin and eosin) and PAS (periodic acid-Schiff stain) analysis of lung tissues in murine asthma group and control group, and histopathology score (PA) and periodic acid-Schiff stain score (PAS). The study was repeated for three times. **P<0.01; ***P<0.001, ****P<0.0001.
Figure 2 Decreased lncRNA H19 and increased Muc5ac in murine asthma model and 16-HBE cells treated with TNFα. q-PCR analysis of Muc5ac, Muc5b (A) and lncRNA H19 (B), in lung tissues of murine asthma group and control group. q-PCR analysis of LncRNA Muc5ac (C) and lncRNA H19 (D) in 16-HBE cells treated with TNFα for 24 hours. The study was repeated for three times. *P<0.05; **P<0.01; ***P<0.001.
Figure 3 LncRNA H19 positively regulated Muc5ac mRNA expression in 16-HBE cells. (A) Fluorescent visions of 16-HBE cells transfected with plasmids phblv-vc (left) and phblv-H19 (right), respectively. (B) Verification of lncRNA H19 overexpression by q-PCR. (C) Comparison of knockdown efficacy of four siRNAs targeting lncRNA H19 after transfection for 24 hours by q-PCR. (D) q-PCR analysis of Muc5ac in 16-HBE cells after overexpression and knockdown of lncRNA H19 for 24 hours. q-PCR analysis of lncRNA H19 (E and G) and Muc5ac (F and H) in 16-HBE cells transfected respectively with plasmids phblv-vc and phblv-H19 (E and F), or si-NC and si-H19 (G and H) for 24 hours and subsequently with TNFα treatment for 24 hours. The study was repeated for three times. *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001.
Figure 4 LncRNA H19 regulated Muc5ac mRNA expression via PI3K/Akt/NF-κB in 16-HBE cells. (A) After knock down of lncRNA H19 in 16-HBE cells for 24 hours, p-Akt, p-p65 and GAPDH were assayed by Western Blotting. Expressions of p-Akt and p-p65 were normalized to GAPDH. (B) Western Blotting analysis of p-Akt and p-p65 in 16-HBE cells treated with TNFα for different time. After overexpression of lncRNA H19 for 24 hours (right), 16-HBE cells was advancely treated with inhibitors of PI3K for 40 minutes and then with (D) or without (C) TNFα for 24 hours. Muc5ac was assayed by q-PCR. The study was repeated for three times. *P<0.05; **P<0.01; ***P<0.001; ns, not significant.
Figure 5 miR-675 processed from lncRNA H19 had no regulatory effects on Muc5ac mRNA expression in 16-HBE cells. (A) miRNA-675 encoded by exon 1 of lncRNA H19 gene. (B) q-PCR analysis of miR-675 in 16-HBE cells after overexpression of lncRNA H19 for 24 hours. After overexpression (left) and knockdown (right) of miR-675-3p in 16-HBE cells for 24 hours, miR-675-3p (C) and Muc5ac expressions (D) were assayed by q-PCR. The study was repeated for three times. **P<0.01; ***P<0.001; ****P<0.0001, ns, not significant.
