The Study Of MIR-125b Targeting DNMT3b Regulating P53DNA Methylation Involving In The Vascular Smooth Muscle Cells Proliferation Induced By Homocysteine

Objective To investigate the effects of Hcy on the status of methylation of p53, andclarify whether DNMT3b regulates p53methylation; To observe the effects of Hcy on theexpression of miR-125b in vascular smooth muscle cells, and to determine whether there existthe targeting and regulation relationships between miR-125b and DNMT3b, and clarify themechanism of miR-125b targeting DNMT3b thereby regulating the methylation of p53involving in VSMCs proliferation induced by Hcy；To look for the therapeutic targets ofVSMCs proliferation induced by Hcy.Methods Normal primary cultured human umbilical vein VSMCs were randomlydivided into control group, Hcy stimulation group (50,100,200,500μM) and folic acidintervention group (Hcy l00μM+folic acid30μM) total6groups; The methylation changesof p53gene promoter detect by nested touchdown methylation specific PCR PCR (ntMS-PCR)in VSMCs treated by Hcy; DNMT3b RNAi plasmids were transfected into VSMCs, and p53methylation changes were analyzed by ntMS-PCR; The target relationship between miR-125band DNMT3b was analyzed by bioinformatics; The expression of miR-125b assayed byqRT-PCR detected in VSMCs treated by Hcy; the pGL3luciferase reporter gene recombinantplasmid carrying DNMT3b mRNA3’UTR was constructed and co-transfected into VSMCswith miR-125b pEZX-miR-125b and miR-125b inhibitor, and the luciferase activities wereanalyzed by dual-Luciferase Reporter Assay System; and then DNMT3b mRNA and protein levels measured by qRT-PCR and Western blot; and then MTT cell proliferation rates wereassayed after pEZX-miR-125b and miR-125b inhibitor transfected into VSMCs.Results1. After p53promoter methylation changes were measured in VSMCs treated by Hcy, theresults showed a hypermethylation in p53promoter, but not dose-dependent, the most obviousdifference showed in100μM Hcy group compared with the control group, and these werestatistically significant (P<0.01); After downregulating DNMT3b expression by DNMT3bRNAi, p53methylation levels decreased significantly compared with the control group (P<0.01).2. The result of bioinformatic analysis showed that found a site action7nt of the miR-125b inNMT3b mRNA1000-1006bp sequence; the free energy of the secondary structure is-26.7kcal/mol.3. After assayed by qRT-PCR, the resluts showed that miR-125b expression in each group ofVSMCs decreased, but not dose-dependent, the most obvious difference showed in100μMHcy group compared with the control group, and these were statistically significant (P<0.01);After intervention by folic acid, miR-125b expression increased compared with100μM group,the difference was statistically significant (P<0.05).4. After miR-125b overexpression vector (pEZX-miR-125b) and inhibitor (miR-125binhibitor) respectively co-transfected with pGL3-DNMT3b into VSMCs, the luciferaseactivities were analyzed by dual-Luciferase Reporter Assay System. The result showed thatpEZX-miR-125b significantly inhibited luciferase activity compared with the empty plasmidgroup (P<0.01); after adding Hcy, luciferase activity increased (P<0.01); while the oppositeresults were found after miR-125b inhibitor added (P<0.01, P<0.05).5. After pEZX-miR-125b and miR-125b inhibitor transfected into VSMCs, DNMT3b mRNAand protein levels were measured by qRT-PCR and Western blot; the results showed thatcompared with the empty plasmid control group, pEZX-miR-125b group can significantly reduce DNMT3b mRNA and protein levels (P<0.01); after adding Hcy, this inhibitionattenuated (upregulated)(P<0.05); while the opposite results were found after miR-125binhibitor added (P<0.01, P<0.05).6. After pEZX-miR-125b and miR-125b inhibitor transfected into VSMCs, MTT assayshowed that, pEZX-miR-125b can inhibit the proliferation of VSMCs group compared withthe control group, and the difference was statistically significant (P<0.01), after adding Hcy,this inhibition is weakened; while the opposite results were found after miR-125b inhibitoradded (P<0.01).Conclusions1. Hcy increased p53promoter methylation and consequencely affected the expression of p53via upregulating DNMT3b, which may be an important mechanism of VSMCs proliferationinduced by Hcy.2. Hcy affects the expression of miR-125b in VSMCs, and miR-125b inhibit gene expressionof DNMT3b by site-specificly targeting on its mRNA3’ untranslated region, which maythereby involved in the biological processes of VSMCs proliferation induced by Hcy.3. Folic acid play a certain antagonism in the process of VSMCs proliferation induced by Hcy.