Figure 6

Identification of target-regulating mechanism in miR-344a-5p, map3k11, and lnc-PCF. (a) Map3k11 3′-UTR luciferase activities assay. Two types of 3′ UTR (WT: 5′-GAGCCUGU-3′; MT: 5′-CGCGAACA-3′) were cloned for the miR-344a-5p seed sequence (5′-CUCGGACU-3′) and tested for their luciferase activity, as regulated by miR-344a-5p. The luciferase activity of the WT 3′-UTR–map3k11 was significantly decreased in cells transfected with miR-344a-5p mimics, whereas miR-344a-5p mimics could not inhibit the luciferase activities of the MU 3′UTR–map3k11 (1–4). Lnc-PCF could protect map3k11 by competitive binding to miR-344a-5p (5–10). WT indicates wild type, MT indicates mutant type, NC indicates negative control, and BP indicates blank plasmid. (b) Western blot analysis showed that map3k11 protein levels decreased after miR-344a-5p mimic (MI) transfection. Protein samples were obtained from RLE-6TN cells after being transfected with miR-344a-5p mimic for 24 h and then exposed to TGF-β1 for 72 h. (c) Western blot analysis showed that map3k11 protein levels increased after miR-344a-5p inhibitor (IN) transfection. Protein samples were obtained from RLE-6TN cells after being transfected with miR-344a-5p inhibitor for 24 h and then exposed to TGF-β1 for 72 h. (d) Knockin lnc-PCF could promote map3k11expression. (e) miR-344a-5p mimic (MI) could block map3k11 expression increased by overexpression of lnc-PCF. After transfection with RP/BP for 24 h, cells were then cotransfected with miR-344a-5p mimic (MI) for 48 h. Mut indicates mutation binding site with miR-344a-5p in lnc-PCF. (f) Knockin lnc-PCF could increase the map3k11 expression, which was blocked with miR-344a-5p mimic (MI). After transfection with miR-344a-5p mimic (MI) for 24 h, cells were then cotransfected with RP/BP for 48 h. (g) miR-344a-5p inhibitor (IN) could increase the map3k11 expression by lnc-PCF smart silencer. Data are shown as mean±S.D. *P< 0.05, **P<0.01, ***P<0.01