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. 2020 Jun 25;11(6):480. doi: 10.1038/s41419-020-2685-8

Fig. 6. MiR-200b in HBM-exo targets Hmgb3 to attenuate the inflammatory injury of IECs.

Fig. 6

a TargetScan, miRWalk, and miRDB database predicted the microRNAs targeting the 3′ UTR of Hmgb3. b QRT-PCR was used to detect the relative expression levels of miR-200b, miR-17, miR-139, miR-429, miR-191a, and miR-214a in TNF-α-exo, BM-exo, and HBM-exo groups (normalized by U6, fold change to TNF-α-exo, n = 5). c QRT-PCR was used to detect the relative expression of miR-200b in the Mock, TNF-α, TNF-α-exo, BM-exo, and HBM-exo groups (fold change relative to the Mock group, n = 4). d, e The plasmids HMGB3-WT (wild-type), HMGB3-MUT (mutant-type), positive control (Positive Con.) and negative control (Negative Con.) were constructed. Dual luciferase reporter assays were used to verify the targeted binding relationship between miR-200b and the 3′ UTR region of Hmgb3 in HEK293T cells and IEC-6s (n = 3). f, g The protective effect of overexpression of miR-200b on inflammatory injury of IEC-6s. Transfection of miR-200b mimic and Negative Con., western blotting to verify the Mock group, Control group under TNF-α damage, and the Negative Con group. The relative expression levels of HMGB3, phosphorylated (p-) JNK, and other proteins in IEC-6s of the control and miR-200b mimic groups (n = 3); h, i Interference with miR-200b expression to block the protective effect of HBM-exo on inflammatory IEC-6s. Transfection of an miR-200b inhibitor and Negative Con. After treatment, the relative expression levels of HMGB3 and p-JNK were detected in the model of IEC-6s protected by HBM-exo (n = 3). j The relative expression levels of miR-200b in BMMSCs, HO-1/BMMSCs, TNF-α + BMMSCs, TNF-α + HO-1/BMMSCs cells, and exocrine bodies were detected (fold change to BMMSCs, n = 5). *P < 0.05, # undetected. BMMSCs bone marrow mesenchymal stem cells; BM-exo BMMSCs co-culture system exosomes; Exo exosomes; HMGB3 high mobility group box 3; HO-1 heme oxygenase-1; HBM-exo HO-1/BMMSCs co-culture system exosomes; IECs intestinal epithelial cells; JNK C-Jun NH2-terminal kinase; qRT-PCR quantitative real-time reverse transcription polymerase chain reaction; TNF-α tumor necrosis factor alpha; UTR untranslated region.