Fig. 4
MIR181A1HG in CRC cell-derived EVs functions as a ceRNA by sponging miR373-3p to activate HSCs via the TGFβRII/Smad2/3 pathway. (a ) Sequences of the predicted binding sites between MIR181A1HG and miR373-3p. (b) MS2-RIP assays followed by qPCR assays were used to measure miR373-3p endogenously associated with MIR181A1HG. LX2 cell lysates were incubated with biotin-labeled MIR181A1HG and miR373-3p, and qPCR analyses of the expression of miR373-3p (c) and MIR181A1HG (d) in the products of pull-down by biotin were performed. Luciferase reporter gene assays were performed to determine the effects of the miR373-3p mimic (e) and anti-miR373-3p (f) constructs on the luciferase activity of the wild-type and mutant MIR181A1HG in LX2 cells. g. AGO2-RIP assays followed by qPCR assays were used to evaluate the expression levels of MIR181A1HG in LX2 cells transfected with miR373-3p overexpression or knockdown constructs. h. qPCR analysis of the expression levels of miR373-3p in LX2 cells transfected with wild-type or mutant MIR181A1HG overexpression or knockdown constructs. i. Sequences of the predicted binding sites between miR373-3p and the 3′UTR of wild-type (Wt)/mutant (Mut) TGFβRII. j. Luciferase reporter gene assays were performed to determine the effects of the wild-type/mutant-type miR373-3p mimic and anti-miR373-3p on the luciferase activity of wild-type and mutant TGFβRII in LX2 cells. qPCR (k) and western blot (l) analyses of the expression levels of TGFβRII in LX2 cells transfected with the miR373-3p overexpression or knockdown construct. m. MIR181A1HG and TGFβRII share the same miRNA binding site. LX2 cells were first transfected with wild-type and mutant MIR181A1HG overexpression and knockdown constructs and with miR373-3p mimics and anti-miR373-3p constructs. The luciferase activities of wild-type and mutant TGFβRII and the expression levels of TGFβRII were subsequently determined by luciferase reporter gene assays (n), qPCR (o), and western blot assays (p). LX2 cells were first cocultured with EVs derived from HT29 and RKO cells that had been pretransfected with MIR181A1HG-overexpressing or MIR181A1HG-knockdown constructs. Then, LX2 cells were transfected with miR373-3p mimics or anti-miR373-3p. q. Western blot analysis of the expression levels of TGFβRII, Smad2/3 and phosphorylated Smad2/3 in the above LX2 cells. r. s. Exogenous TGFβRII or TA-02, a potent and selective inhibitor of TGFβRII, was added to the above LX2 cells, and the expression levels of TGFβRII, Smad2/3 and phosphorylated Smad2/3 were subsequently examined by western blot assays. (nsP>0.05, *P < 0.05, **P < 0.01)