Research Paper Volume 15, Issue 12 pp 5514—5534

Figure 5. hucMSCs treatment alleviated pulmonary fibrosis by preventing circANKRD42 reverse splicing biogenesis. (A) qRT-PCR result illustrated that hucMSCs treatment reduced the expression level of circANKRD42 compared with TGF-β1/BLM treatment in vivo and in vitro. (B) Rescue experiment of cell proliferation showed that hucMSCs treatment reduced the TGF-β1-induced cell proliferation. circANKRD42 overexpression increased the cell proliferation and reversed the downward trend caused by hucMSCs treatment. (C) Rescue experiment of cell scratch assay monitored with an IncuCyte S3 instrument validated that hucMSCs treatment blocked the cell migration. circANKRD42 overexpression promoted the cell migration and reversed the downward trend caused by hucMSCs treatment. (D) Rescue experiment showed that hucMSCs treatment reduced the expression of S100A4, FAP1, α-SMA, vimentin, and collagen I and III. circANKRD42 overexpression increased the expression of S100A4, FAP1, α-SMA, vimentin, and collagen I and III and reversed the downward trend caused by hucMSCs treatment. (E) Immunofluorescence images exhibited that hnRNP L was located in the nucleus and its expression decreased in the hucMSCs treatment group compared with that in the TGF-β1 group. (F) Example of hnRNP L immunofluorescence images depicted that hnRNP L expression was increased in patients with IPF compared with that in normal individuals. (G) Western blot analysis identified that hucMSCs treatment inhibited hnRNP L expression compared with that in the TGF-β1/BLM group in vivo and in vitro. (H) RIP uncovered that hnRNP L bound to pre-mRNA (ANKRD42) and hucMSCs treatment reduced the binding amount of hnRNP L with pre-mRNA (ANKRD42). (I) qRT-PCR was performed to prove that the inhibitory effect of hucMSCs on circANKRD42 depending on hnRNP L. (J) MiR-136-5p expression markedly decreased in the TGF-β1/BLM group and increased in the hucMSCs treatment group in vivo and in vitro. (K) RAP experiment showed that miR-136-5p was enriched in circANKRD42; this enrichment was intensified by TGF-β1 and decreased by hucMSCs treatment. Lac Z was the control. BP indicates blank plasmid, and RP indicates the recombinant plasmid of overexpressed circANKRD42. Each bar represents the mean ± SD; n = 6; ^*p < 0.05.