Abstract
We have previously reported that leiomyoma expressed lower levels of miR-200c and elevated IL8 as compared to paired myometrium. Here we addressed the regulatory functions of miR-200c on the expression of inflammatory mediators and cellular viability using leiomyomas and paired myometrium and their isolated primary smooth muscle cells. Our results indicated that gain-of function or knockdown of miR-200c in leiomyoma smooth muscle cells (LSMC) regulated IL8 mRNA and protein expression through direct targeting of IKBKB and alteration of NF-kB activity. Additionally, leiomyoma expressed higher levels of phosphorylated IKBKB with no significant difference in the level of IKBKB mRNA and protein as compared to matched myometrium. Gain-of function of miR-200c in LSMC resulted in decreased IkBαphosphorylation and p65 nuclear translocation, which led to decreased p65 transcriptional activity of IL8 promoter, and increased caspase 3/7 activity which was not reversible following IL8 restoration. Collectively, our results suggest that NF-κB signaling pathway is a target of miR-200c regulatory function, and low level of miR-200c expression in leiomyoma by transcriptional regulation of inflammatory mediators such as IL8, in part account for development of leiomyomas.
Highlights
Uterine leiomyoma are benign gynecologic tumors that develop during the reproductive age and symptomatic tumors account for 1/3 of all hysterectomies performed in the United States
Since phosphorylation of IkBa by IkB kinases [IKKa (IKBKA) or IKKb (IKBKB)], and rapid proteasome-dependent degradation, results in Nuclear factor-kB (NF-kB) dissociation and nuclear translocation, where NF-kB binds to consensus motif of specific target genes and regulates their expression, we examined the expression of IkBa and assessed the level of phosphorylated IkBa at serine 32/36 following gain- and loss-of function of miR-200c
We demonstrated that in leiomyoma smooth muscle cells (LSMC) miR-200c regulates IL8 expression which occurred indirectly through downregulation of NF-kB signaling pathway by targeting IKBKB 39UTR
Summary
Uterine leiomyoma (fibroids) are benign gynecologic tumors that develop during the reproductive age and symptomatic tumors account for 1/3 of all hysterectomies performed in the United States. Leiomyomas are composed of cells with aberrant proliferation and exhibit elevated expression of network of genes with pro-inflammatory and pro-fibrotic activities which play a central role in their growth and associated symptoms [1,2,3]. Accumulated evidence suggests that microRNAs (miRNA), a member of non-protein coding small RNA, functions as key regulator of protein coding genes expression [4,5], and their aberrant expression has been associated with a wide range of disorders, including inflammatory and fibrotic disorders [6,7]. Nuclear factor-kB (NF-kB) is an established key transcriptional regulator of many genes functionally associated with inflammation, fibrosis and tumorigenesis [8,9,10]. The expression and nuclear localization of NF-kB p65 has been demonstrated in myometrium during parturition leading to regulation of several pro-inflammatory cytokines, including IL8 which in myometrial smooth muscle cells (MSMC) promotes premature labor [12]
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