miR-339-3p regulated acute pancreatitis induced by caerulein through targeting TNF receptor-associated factor 3 in AR42J cells

Figures

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  Figure 1. Caerulein induced inflammation and apoptosis in AR42J cells. (a–c) ELISA was used to detect the contents of TNF-α (a), IL-6 (b), and IL-1β (c) in AR42J cells induced by caerulein. (d) Flow cytometry was applied to detect cell apoptosis in AR42J cells induced by caerulein. (e) Western blot was applied to measure the protein expression of Bcl-1, C-caspase 3/caspase 3, Bcl-2/Bax, and Bax in AR42J cells induced by caerulein. Data are represented as mean ± SD of at least triple experiments. *P < 0.05.

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  Figure 2. miR-339-3p expression was inhibited while TRAF3 expression was increased in AR42J cells induced by caerulein. (a) qRT-PCR was applied to determine the expression of miR-339-3p under different caerulein concentrations (0, 25, 50, and 100 nM). (b) qRT-PCR was applied to determine the expression of miR-339-3p under 100 nM caerulein concentration at different times (0, 2, 4, 6, and 8 h). (c) Western blot was used to measure the protein expression of TRAF3 under different caerulein concentrations (0, 25, 50, and 100 nM). (d) Western blot was used to measure the protein expression of TRAF3 under 100 nM caerulein concentration at different times (0, 2, 4, 6, and 8 h). Data are represented as mean ± SD of at least triple experiments. *P < 0.05.

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  Figure 3. Promotion of miR-339-3p weakened the effect of caerulein on cell inflammation and apoptosis in AR42J cells. (a) The expression of miR-339-3p was detected in 100 nM caerulein, 100 nM caerulein + NC, and 100 nM caerulein + miR-339-3p groups using qRT-PCR. (b–d) ELISA was used to detect the contents of TNF-α (b), IL-6 (c), and IL-1β (d) in 100 nM caerulein, 100 nM caerulein + NC, and 100 nM caerulein + miR-339-3p groups. (e) Flow cytometry was applied to detect cell apoptosis in 100 nM caerulein, 100 nM caerulein + NC, and 100 nM caerulein + miR-339-3p groups. (f) Western blot was applied to measure the protein expression of Bcl-1, C-caspase 3/caspase 3, Bcl-2/Bax, and Bax in 100 nM caerulein, 100 nM caerulein + NC, and 100 nM caerulein + miR-339-3p groups. 100 nM caerulein + NC were used as a negative control. Data are represented as mean ± SD of at least triple experiments. *P < 0.05.

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  Figure 4. TRAF3 is a target mRNA of miR-339-3p. (a) The binding sites between TRAF3 and miR-339-3p were predicted by TargetScanHuman. (b) Luciferase activities of TRAF3-wt and TRAF3-mut were detected in NC and miR-339-3p groups using the luciferase reporter assay. NC was used as a negative control. (c) The enrichment of TRAF3 was measured in NC and miR-19b-3p groups using the RIP assay. (d) Western blot was used to detect the protein expression of TRAF3 in NC, miR-339-3p, anti-NC, and anti-miR-339-3p groups. NC or anti-NC were used as a negative control. Data are represented as mean ± SD of at least triple experiments. *P < 0.05.

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  Figure 5. Promotion of TRAF3 could reverse the effects of miR-339-3p on cell inflammation and apoptosis in AR42J cells induced by caerulein. (a) Western blot was used to detect the protein expression of TRAF3 in 100 nM caerulein + miR-339-3p, 100 nM caerulein + NC, 100 nM caerulein + miR-339-3p + vector, and 100 nM caerulein + miR-339-3p + TRAF3 groups. (b–d) ELISA was used to detect the levels of TNF-α (b), IL-6 (c), and IL-1β (d) in 100 nM caerulein + miR-339-3p, 100 nM caerulein + NC, 100 nM caerulein + miR-339-3p + vector, and 100 nM caerulein + miR-339-3p + TRAF3 groups. (e) Flow cytometry was applied to detect cell apoptosis in 100 nM caerulein + miR-339-3p, 100 nM caerulein + NC, 100 nM caerulein + miR-339-3p + vector, and 100 nM caerulein + miR-339-3p + TRAF3 groups. (f) Western blot was applied to measure the protein expression of Bcl-1, C-caspase 3/caspase 3, Bcl-2/Bax, and Bax in 100 nM caerulein + miR-339-3p, 100 nM caerulein + NC, 100 nM caerulein + miR-339-3p + vector, and 100 nM caerulein + miR-339-3p + TRAF3 groups. 100 nM caerulein + NC or 100 nM caerulein + miR-339-3p + vector were used as a negative control. Data are represented as mean ± SD of at least triple…

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  Figure 6. miR-399-3p/TRAF3 axis regulated AP induced by caerulein through the p38 pathway. (a) Western blot was used to detect the protein expression of p-p38 and p38 in 100 nM caerulein + miR-339-3p, 100 nM caerulein + NC, 100 nM caerulein + miR-339-3p + vector, and 100 nM caerulein + miR-339-3p + TRAF3 groups. 100 nM caerulein + NC or 100 nM caerulein + miR-339-3p + vector was used as a negative control. (b) Western blot was used to detect the protein expression of p-p38 and p38 in control, 100 nM caerulein + DMSO, 100 nM caerulein + SB203580, 100 nM caerulein + miR-339-3p + SB203580, and 100 nM caerulein + miR-339-3p + TRAF3 + SB203580 groups. 100 nM caerulein + DMSO or 100 nM caerulein + miR-339-3p + SB203580 were used as a negative control. Data are represented as mean ± SD of at least triple experiments. *P < 0.05.