Influence Of Silencing NMBR And NF-κB By RNA Interference On IL-6 Expression In Primary Uterine Smooth Muscle Cells Of Mice

Object:To study the influence of silencing NMBR and NF-κB by specific siRNA on expression of IL-6 in primary uterine smooth muscle cells of mice.Methods:1. Primary cells culture and identify were executed using the mice's uterine smooth muscle cells which were intrapartum or after 2 hours of postpartum. NMB concentrations were difined as 10-10M,10-8M,10-6M,10-4M.Three groups were set up as follow:(1)control group without any interference factor;(2)NMB group;(3) siRNA+NMB group.2. NMBR siRNA and NF-κB p65 siRNA were transfected respectively into uterine smooth muscle cells.The transfection efficiency was detected.Then we added NMB of different concentrations as above.3. Detect the binding activity of NF-κB—65 DNA in uterine smooth muscle cells before and after siRNA tranfection by ELISA assay. The mRNA and protein expression of IL-6 were detected by real-time PCR and ELISA at the same time.Results:1. The influence of NMBR siRNA on the binding activity of NF-κB—p65 DNA and expression of IL-6 in uterine smooth muscle.The result was as follow:The values in NMB group(10-4,10-6,10-8) are higher than that in control group (P<0.05). Data showed the obvious correlation between the variation of the binding activity of NF-κB p65 DNA,expression of IL-6 and concentration of NMB. With the increase of NMB concentration, the binding activity of NF-κB—p65 DNA and the expression of IL-6 increased correspondingly. There is significant difference between the subgroups in NMB groups(P<0.05). The values in NMB+NMBR siRNA group are lower than those in NMB group. But there is no significant difference between the subgroups in NMB+NMBR siRNA group(P>0.05).2. The influence of NF-κB p65 siRNA on the expression of IL-6 in uterine smooth muscle. The result was as follow:The values in NMB group(10-4,10-6,10-8) are higher than that in control group (P<0.05). Data showed the obvious correlation between the mRNA expression of IL-6 and concentration of NMB.With the increase of NMB concentration, the mRNA expression of IL-6 increased correspondingly. There is significant difference between the subgroups in NMB groups(P<0.05). The values in NMB+ p65 siRNA group are lower than those in NMB group(P<0.05). But there is no significant difference between the subgroups in NMB+NMBR siRNA group(P>0.05).3. The correlation analysis between binding activity of NF-κB—p65 DNA and IL-6 by blocking of NMBR siRNA.There is a significant positive correlation between the expression of IL-6 mRNA and p65 (r=0.952, P<0.01)4. The comparison of the influence on the expression of IL-6 mRNA by blocking NMBR and NF-κB.There is no significant difference between NMBR siRNA group and NF-κB p65 siRNA group(P>0.05).Conclusion:1. The expression of IL-6 in primary uterine smooth muscle cells can be down regulated via silencing of NMBR and NF-κB by specific siRNA.2. NMBR may regulates the expression of IL-6 in uterine smooth muscle cells of mouse by NF-κB pathway and then affect the activity of uterine smooth muscle cells of mouse.