The Prediction And Identification Of PDE4D Gene-related MiRNA And Its Mechanism Of Action By Targeting Regulation Of Atopic Dermatitis

Objective: This study used bioinformatics software to perform gene loc us analysis and exogenous cell experiments to verify the targeted regula tion of PDE4 D and related miRNAs,and to detect PDE4D-related miR NAs,inflammatory factors TNF-?,IL-1?,IL-6and PDE4 subtypes in A D The expression in skin lesions determines its possible molecular mec hanism role in AD and provides a certain basis for exploring the target ed therapy research of AD.Methods:(1)The biological information predicts the miRNAs that inter act with the 3?UTR of the PDE4 D gene,and selects the miRNAs with higher scores.Skin lesions and normal skin tissues from AD patients w ere collected,and total RNA from skin lesions and normal skin tissues was extracted by Trizol method.The real-time fluorescence quantitative PCR was used to determine miRNAs,PDE4 D,inflammatory factors TN F-?,IL-1?,IL-6 and Expression of other PDE4 isoforms.(2)Construct 3'UTR of PDE4 D gene and miRNAs for primer design,c onstruct a vector,detect the double luciferase activity after cell transfect ion,and then transfect miRNAs mimics and inhibitors in Ha Cat cells,r espectively.The expression levels of miRNAs,PDE4 D and inflammator y factors TNF-?,IL-1? and IL-6 were measured.Result:(1)collect skin tissues from 30 AD patients and 25 normal con trols.QPCR was used to detect the expression of four PDE4 subtypesPDE4 A,PDE4B,PDE4 C,and PDE4 D in the skin lesions.The expressi on was high in AD group,and the expression of PDE4 C was the lowe st among the four subtypes.The expression levels of PDE4 A and PDE4 C in the AD group were slightly higher than those in the normal cont rol group,the difference was not statistically significant(P> 0.05);the expression levels of PDE4 B and PDE4 D in the AD group were signific antly higher than those in the normal control group,the difference wass tatistically significant(P<0.05).(2)The miRNAs that interact with the 3?UTR of the PDE4 D gene were initially predicted through the analysis o f biological information,and miR-199b-5p with higher score was selecte d as the follow-up research object.q PCR was used to detect the express ion of miR-199b-5p,PDE4 D,TNF-?,IL-1? and IL-6 in all skin tissues.The results showed that miR-199b-5p expression was reduced in the A D group(P <0.05),PDE4 D,IL-1?,and IL-6 were significantly increase d in the AD group(P<0.05),and TNF-? was slightly expressed in the AD group Increased(P>0.05);the expression of miR-199b-5p in AD lesi ons was weakly correlated with the expression of PDE4 D,IL-1?,and I L-6(P<0.05);There was no significant correlation between miR-199b-5p expression and TNF-? expression(P>0.05);PDE4D expression wasp ositively correlated with IL-1? and IL-6 expression(P<0.05);no correlat ion with TNF-? expression(P>0.05).(3)After miR-199b-5p was transfect ed with PDE4 D gene 3'UTR mutant and wild-type vectors,the detectio n of dual luciferase activity revealed that the relative luciferase activity of the PDE4D-WT + miR-199b-5p group was significantly reduced,in dicating that miR-199b-5p can target the 3'UTR of the PDE4 D gene.(4)Levels of PDE4 D and inflammatory factors TNF-?,IL-1?,and IL-6 after 5groups of samples including miR-199b-5p mimic,inhibitor and negativecontrol were co-transfected into Ha Ca T cells The results showed that miR-199b-5p inhibited the expression of IL-1? and IL-6,but failed to inhibit the expression of PDE4 D and TNF-?.Conclusion:(1)Different subtypes of PDE4 are differentially expressed in AD,which provides some theoretical guidance for the design of high-efficiency inhibitors in the future.(2)miR-199b-5p,PDE4 D,TNF-?,IL-1?,and IL-6 participate in the regulation of AD expression.The effect of miR-199b-5p on the pathogenesis and progression of AD is related to inflammatory factors,and The mechanism of action of miR-199b-5p and inflammatory factors in AD may be related to oxidative stress and lipid metabolism.(3)The cellular level results of miR-199b-5p and PDE4 D are inconsistent with the results of skin lesions.Whether miR-199b-5p inhibits the expression of PDE4 D is not yet known,and its mechanism of action needs further study.