The Role Of TNF-α,MMP-2, MAPK And NF-κB In The Pathogenesis Of Human Leiomyoma

Objective:Human uterine leiomyoma, the most common benign tumour in female reproductive tract. But the mechanism underlying uterine leiomyoma is not known. Previous study have revealed that myometrium conversion into leiomyoma and an increase in its mass is accompanied by changes in the expression and contents TNF and TNF RI. Therefore, our objective is to investigate the mechanism and effect of TNF-α, MMP-2, MAPK and NF-κB in occurrence and development of UL and provide the basis of theory for instructing UL clinical treatment.Methods:(1)HL-and matched HM-were sampled for tissue homogenization and protein extraction,followed by western blot analysis the protein expression of TNF-a and MMP-2and real-time PCR analysis the expression of TNF-a mRNA and MMP-2mRNA;(2)SMCs were isolated from human leiomyoma and matched myometrium, also followed by western blot and real-time PCR analysis the expression of TNF-a and MMP-2;(3) HM-and HL-SMCs were treated with20ng/ml TNF-a, after0,5min,15min,30min,60min, the phosphorylation and expression of p44/42MAPK and IκBα were analysised by Western blot; HM-and HL-SMCs were treated with20ng/mlTNF-α, after0,2h,12h,24h,48h,Western blot detected the expression of MMP-2and real-time PCR detected the expression of MMP-2mRNA.(4) The phosphorylation and expression of p44/42MAPK were detected by Western blot after injection of p44/42MAPK inhibitor PD98059; The phosphorylation and expression of IκBα were detected after injection of a selective inhibitor ofNF-KB activity, Bay11-7082;We also detected the expression of MMP-2after inhibition of p44/42MAPK and NF-κB.Results:1.Western blots and real-time PCR showed that TNFa expression in leiomyoma tissue and cells was higher than that in the adjacent normal myometrial tissue and cells during proliferative phase, whereas the difference of TNFa expression between leiomyoma and myometrium was not obvious in secretory phase. TNF-α stimulated the expression of MMP-2protein and MMP-2mRNA in a time-dependent manner in proliferative phase HL-SMCs, but not in proliferative HM-SMCs.2.TNF-a induced a time-dependent activation of p44/42MAPK in proliferative phase HL-SMCs, but not in proliferative phase HM-SMCs,which was inhibited by p44/42MAPK inhibitor PD98059.3.TNF-a induced a time-dependent activation of NF-κB signaling pathways in proliferative phase HM-and HL-SMCs.Interestingly,inhibitor Bay11-7082only blocked TNF-induced IκBα activation in proliferative phase HL-SMCs, but not in proliferative phase HM-SMCs.4.PD98059and Bay11-7082blocked TNF-induced MMP-2expression in proliferative phase HL-SMCs, but not in proliferative phase HM-SMCs.Conclusion:The study indicted that TNF-a and MMP-2were increased in proliferative phase human leiomyoma compared with corresponding myometrium. TNF-a induced both p44/42MAPK and NF-κB signaling pathways activation time-dependently in in proliferative phase HL-SMCs, and interruption of TNF-induced p44/42MAPK activation and TNF-induced NF-κB activation resulted in decreased expression of MMP-2. These findings provide novel evidence to the pathogenesis of uterine leiomyoma.