动物造模 z-bio 2023-01-17 357

　　Objective: To clarify the relationship between the effects of TOLL-like receptor 2 (TLR2) and microRNA144 (miR-144).

　　Methods: Different concentrations of miR-144 mimics (mimics) and inhibitors (inhibitor) were used to transiently transfect rat macrophage cell line NR8383 cells, and RT-qPCR was used to detect miR-144, TLR2 and its downstream molecule TNF-α Express. Using rat liver cDNA as a template, the target fragment (ie the 3'UTR region of TLR2 mRNA containing miR-144 wild and mutation binding site) was obtained by PCR; the pmirGLO reporter gene vector and miR-144 containing miR-144 were digested with SacⅠ and XbaⅠ The 3'UTR region of TLR2 mRNA in the wild and mutation binding sites, construct the dual luciferase reporter gene carrying the above fragments, and identify the constructed recombinant plasmid by PCR, double enzyme digestion, and DNA sequencing, namely pmir-TLR2-3' UTR and pmir-mutant-TLR2-3'UTR; and co-transfected with miR-144 mimics and dual luciferase reporter gene to clarify the targeting relationship between miR-144 and the 3'UTR region of TLR2 mRNA.

　　Results: After transiently transfecting 100 nmol/L miR-144 mimics, the expression of miR-144 in NR8383 cells was significantly increased, while the expression of TLR2 and its downstream molecule TNF-α decreased significantly; while the effect of 100 nmol/L miR-144 inhibitor was on the contrary. PCR and double enzyme digestion DNA sequencing results confirmed that pmir-TLR2-3’UTR and pmir-mutant-TLR2-3’UTR recombinant vector construct

　　was successfully established; after co-transfecting HEK 293T cells with 100 nmol/L miR-144 mimics, the empty vector and the above two constructs, the relative luciferase activity of the pmir-TLR2-3'UTR transfection group was significantly reduced.

　　Conclusion: miR-144 negatively regulates the expression of TLR2 and its downstream pro-inflammatory factors by targeting the 3'UTR region of TLR2 mRNA.