Functional Study Of MiR-6857-3p Regulating Sp110 Gene Of Human Macrophages

Objective: 1.To predict micro RNA(mi RNA)targeting Sp110 through bioinformatics websites.2.To further screen mi RNA by detecting mi RNA background level in THP-1 cells.3.To verify the targeting relationship between mi R-6857-3p and Sp110 with dual-luciferase experiment.4.To study the regulatory effect of mi R-6857-3p on Sp110 with real-time fluorescent quantitative PCR(q PCR)technology.5.To study the effect of mi R-6857-3p on apoptosis of Bacillus Calmette Guerin vaccine(BCG)-infected macrophages.Methods: 1.miRNAs that might have a targeting relationship with Sp110(miR-6857-3p,miR-19a-5p,mi R-4772-3p,mi R-4659-5p and mi R-5696)were predicted with bioinformatics websites(Target Scan,mi RDB and mi RTar Base).2.Total RNA was extracted from human macrophages(THP-1 cells),mi RNA was reverse transcribed to c DNA,q PCR technology was used to detect the background expression level of5 previously predicted mi RNAs,the relative expression was calculated by 2-?ct method,3 mi RNAs with the highest expression(mi R-6857-3p,mi R-4659-5p,mi R-4772-3p)were screened for dual-luciferase experiment.3.Binding sequence of mi R-6857-3p,mi R-4659-5p,mi R-4772-3p and Sp110 3'UTR was obtained from database,the sequence was cloned into the downstream of firefly fluorescent protein 3'UTR in psi CHECK-2 plasmid and named Sp110 wild-type plasmid(psi CHECK-2-Sp110-3'UTR).Meanwhile,mi RNA over-expression plasmids(mi R-6857-3p mimic,mi R-4772-3p mimic,mi R-4659-5p mimic and mi RNA mimic nc)were constructed.Different mi RNA over-expression plasmids and Sp110 wild-type plasmids were co-transfected into 293 T cells.Experiment was divided into 4 groups,and fluorescence activity was measured 48 h after transfection.In addition,the binding sequence of mi R-6857-3p and Sp1103'UTR was mutated,and the mutated sequence was cloned into reporter vector in the same way,and named Sp110 mutant plasmid(psi CHECK-2-Sp110-3'UTR-6857mut).mi R-6857-3p mimic and mi RNA mimic nc were co-transfected with Sp110 mutant plasmid into 293 T cells,and fluorescence activity was measured48 h after transfection.4.The differentiation of THP-1 cells into macrophages was promoted with phorbol myristate acetate(PMA),differentiated THP-1 cells were transfected by mi R-6857-3p mimics and inhibitors,and the expression of mi R-6857-3p in THP-1 cells was detected by q PCR technology after 24 h.RNA extraction was performed on THP-1 cells that were successfully transfected,and then reverse transcribed into c DNA,and then q PCR technology was used to detect the expression of Sp110 m RNA.The relative expression was calculated using 2-?ct method to compare differences.5.The differentiated THP-1cells were transfected with mi R-6857-3p mimic and mi R-6857-3p inhibitor.24 h after transfection,BCG was used to infect cells at a multiplicity of infection(MOI)of 10: 1,it was recorded as 0h after 6h of infection.Excess bacteria were removed by water,and flow cytometry was adopted to detect macrophage apoptosis at 24 h.Result: 1.miRNAs targeting Sp110 that were screened out from bioinformatics websites were mi R-6857-3p,mi R-19a-5p,mi R-4772-3p,mi R-4659-5p and mi R-5696.2.Expression detection of 5mi RNAs showed that the expression of mi R-6857-3p was the highest,followed by mi R-4659-5p,mi R-4772-3p,mi R-19a-5p and mi R-5696.3.Dual-luciferase experiment results showed that compared with control group,the luciferase activity carrying mi R-6857-3p mimic and Sp110 wild-type plasmids significantly decreased(P < 0.001),and luciferase activity of mi R-4659-5p and mi R-4772-3p groups also decreased(P < 0.05).Luciferase activity of mi R-6857-3p mimic rebounded after Sp110 3'UTR mutation.4.After transfection of mi R-6857-3p mimic in THP-1 cells,the expression of mi R-6857-3p increased.After transfection of mi R-6857-3p inhibitor in THP-1 cells,the expression of mi R-6857-3p decreased.q PCR results showed that Sp110 m RNA level in mi R-6857-3p mimic group decreased significantly(P < 0.001),and Sp110 m RNA level in mi R-6857-3p inhibitor group increased significantly(P < 0.001).5.Apoptosis detection by flow cytometry showed that mi R-6857-3p mimic group had less apoptosis than control group(P < 0.05),while mi R-6857-3p inhibitor group had more apoptosis than control group(P < 0.01).Conclusion: 1.mi R-6857-3p and Sp110 had a targeting relationship.2.mi R-6857-3p negatively regulated the expression of Sp110 m RNA.3.mi R-6857-3p inhibited apoptosis of BCG-infected macrophages.