动物造模 z-bio 2023-02-13 481

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

　　Methods: Rat macrophage cell line NR8383 cells were transiently transfected with different concentrations of miR-144 mimics and inhibitors, and RT-qPCR was used to detect the expression of miR-144, TLR2 and its downstream molecule TNF-α. Express. Using rat liver cDNA as a template, PCR was used to obtain the target fragment (that is, the 3'UTR region of TLR2 mRNA containing the wild and mutant binding sites of miR-144); the pmirGLO reporter gene vector and miR-144 containing miR-144 were double digested with SacⅠ and XbaⅠ The 3'UTR region of TLR2 mRNA in the wild and mutant binding sites was constructed to construct a dual-luciferase reporter gene carrying the above fragments, and the constructed recombinant plasmid, namely pmir-TLR2-3', was identified by PCR, double restriction digestion and DNA sequencing. 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 transient transfection of 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 was significantly decreased. on the contrary. The results of PCR and double-enzyme-digested DNA sequencing confirmed the structure of pmir-TLR2-3'UTR and pmir-mutant-TLR2-3'UTR recombinant vectors

　　After the HEK 293T cells were co-transfected 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.