Fig 2: miR-4293 function was attenuated by blocking WFDC21P.The above-mentioned data showed that miR-4293 transfection elevated miR-4293 levels, which could be attenuated by ASO. pcDNA-WFDC21P transfection increased WFDC21P. Si-WFDC treatment reduced WFDC21P level. a MTT assay of A549 at 24 h post-transfection of si-WFDC + scramble, miR-4293, miR-4293+si-WFDC, and scramble. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; ANOVA test. b FACS analysis of A549 cell apoptosis at 24 h post-transfection of si-WFDC + scramble, miR-4293, miR-4293+si-WFDC, and scramble. c Western blot detection p-STAT3, Bcl-2, BAX, cleaved-caspase 3, 8, and 9 in si-WFDC + scramble-, miR-4293-, miR-4293+si-WFDC- or scramble-treated cells. d, e RTCA station analysis of migration for A549 with transcription of si-WFDC + scramble-, miR-4293-, miR-4293+si-WFDC- or scramble. f MTT of A549 at 24 h post-transfection of pcDNA+ASO-4293, pcDNA-WFDC21P + scramble, pcDNA-WFDC21P + ASO-4293, and pcDNA+ scramble. Data are expressed as mean ± SD for triplicate experiments. (* P < 0.05). g FACS analysis of A549 cell apoptosis at 24 h post-transfection of pcDNA+ASO-4293, pcDNA-WFDC21P + scramble, pcDNA-WFDC21P + ASO-4293, and pcDNA+ scramble. h Western blot detection of STAT3 phosphorylation and apoptosis-related factors (Bcl-2, Bax and Caspase 3) in pcDNA+ASO-4293, pcDNA-WFDC21P + scramble, pcDNA-WFDC21P + ASO-4293, and pcDNA+ scramble-treated A549 cells. i–l RTCA station analysis of migration for A549. i, j pcDNA-WFDC21P vs pc-DNA; (k) and (l), pcDNA-WFDC21P + scramble vs. pcDNA-WFDC21P + ASO-4293. Data are expressed as mean ± SD for triplicate experiments. *p < 0.05, ***p < 0.001; Student’s t-test.

Fig 3: DHA inhibited the JAK2/STAT3 pathway in pneumonia tissue. (A) Tgfb1, (B) Jak2, and (C) Stat3 mRNA levels in the lungs were determined using qRT-PCR (n = 6), data are expressed as the fold change in mRNA expression normalized to Gapdh expression, with respect to the control group. Representative images of (D) TGF-ß1 and (E) JAK2 protein expression in lung tissue detected using IHC. Mean IOD of (F) TGFß1 and (G) JAK2 protein by IHC staining. Data are expressed as the means ± standard error. *P < 0.05, **P < 0.001. Abbreviations: BLM: bleomycin; DHA-1: dihydroartemisinin 25 mg/kg/d; DHA-2: dihydroartemisinin 50 mg/kg/d; DHA-3: dihydroartemisinin 100 mg/kg/d; IHC: immunohistochemistry; IOD: integrated optical density; NS: not statistically significant.

Fig 4: DCP is targeted by WFDC21P, which is elevated by miR-4293.a TargetScan analysis predicted that the DCP2 mRNA-3'-UTR is targeted by miR-4293. We synthesized a mutant miR-4293 (Mu-4293) and cloned DCP2 mRNA-3’-UTR and mu-DCP2–3’-UTR sequences. b Analysis of miR-4293 regulating luciferase expression of reporter plasmids containing DC2–3’UTR (right panel) or mu-DCP2–3’UTR (left panel). Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; Student’s t-test. c Analysis of ASO-4293 regulating luciferase expression of reporter plasmids containing DC2–3’UTR (right panel) or mu-DCP2–3’UTR (left panel). Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; Student’s t-test. d Western blot detection of DCP2 in miR-4293-, ASO-4293-, or scramble-treated A549 cells. e Western blot detection of DCP2 in siRNA-DCP2–1-, siRNA-DCP2–2-, or scramble-treated A549 cells and H129 cells. f qRT-PCR analysis of WFDC21P level in pcDNA-WFDC21P + scramble-, si-DCP2-, si-DCP2 + pcDNA-WFDC21P-, or scramble-treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; ANOVA test. g Western blot detection of STAT3 phosphorylation in pcDNA-WFDC21P + scramble-, si-DCP2-, si-DCP2 + pcDNA-WFDC21P-, or scramble-treated A549 cells. h qRT-PCR analysis of DCP2 level in pcDNA or pcDNA-DCP2 -treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; Student’s t-test. i qRT-PCR analysis of WFDC21P level in 0 h, 4 h, 8 h, 12 h, 24 h, 48 h after transfection of pcDNA-DCP2. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; ANOVA test. j RIP assay of the interaction between WFDC21P and DCP2. Upper panel, qRT-PCR analysis of WFDC21P pull-down by IgG or DCP2; lower panel, electrophoresis for WFDC21P pull-down by IgG or DCP2 after PCR amplification. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; Student’s t-test. k qRT-PCR analysis of WFDC21P levels in miR-4293-, ASO-4293, Mu-4293 or scramble-treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; ANOVA test. l qRT-PCR analysis of WFDC21P levels in miR-4293+pcDNA-WFDC21P-, or scramble+ pcDNA-WFDC21P-treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; Student’s t-test. m Western blot detection of STAT3 phosphorylation in pcDNA-WFDC21P + scramble-, miR-4293-, miR-4293+pcDNA-WFDC21P-, or scramble-treated A549 cells. n Western blot detection of STAT3 phosphorylation in AG490 + pcDNA-, pcDNA-WFDC21P-, AG490 + pcDNA-WFDC21P-, or pcDNA treated A549 cells. o qRT-PCR analysis of miR-4293 level in AG490 + pcDNA-, pcDNA-WFDC21P-, AG490 + pcDNA-WFDC21P-, or pcDNA treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01, *P < 0.05; ANOVA test.

Fig 5: miR-4293 regulates lung cancer cell proliferation, migration, and apoptosis.a qRT-PCR analysis of miR-4293 levels in NSCLC tissues and corresponding para-carcinoma tissues (n = 16). Data are expressed as median (interquartile range). **P < 0.01; Mann–Whitney U test. b qRT-PCR analysis of miR-4293 levels in A549, H1299, H1975, and HBE cells. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; ANOVA test. c qRT-PCR analysis of miR-4293 level in A549 cells with transfection of miR-4293 mimic, ASO-4293, mu-4293 (mutant), or scrambled. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; ANOVA test. d MTT assay of A549 at 24 h post-transfection of miR-4293, ASO-4293, mu-4293 or scrambled. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01, *p < 0.05; ANOVA test. e FACS analysis of A549 cell apoptosis at 24 h post-transfection of miR-4293, ASO-4293, mu-4293, or scrambled. f Western blot detection of STAT3 and apoptosis-related factors (Bcl-2, Bax, BIRC3, and Caspase 3,8,9) in miR-4293-, ASO-4293-, or scramble-treated A549 cells. g, h Transwell migration assay of A549 at 24 h post-transfection of miR-4293, ASO-4293, mu-4293, or scrambled. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; ANOVA test. i, j Colony formation assays of A549 at 24 h post-transfection of miR-4293, ASO-4293, mu-4293, or scrambled. Data are expressed as mean ± SD for triplicate experiments. **p < 0.01; ANOVA test.