Uterine leiomyoma (fibroids) develop during reproductive years in over 70% of women; however, their etiology remains unknown. Our group has identified two miRNAs, miR-200c and miR-29c, which are critical to regulation of genes functionally associated with cell cycle, cellular transformation, inflammation and extracellular matrix accumulation and thus of great significance to fibroid pathogenesis. Our recent findings using next generation sequencing provided a comprehensive profile of non-coding RNAs including long non- coding RNAs (lncRNAs) as well as novel groups of small non-coding RNAs (sncRNAs). Our preliminary findings here have identified aberrant expression of linc-MD1 and miR-135 family in fibroids and thus the impetus for this proposal. Using qRT-PCR we detected markedly reduced levels of linc-MD1 while significantly higher expression of miR-135 family in leiomyomas. The sequences of the linc-MD1 and our preliminary data suggest that linc-MD1 can act as miRNA sponge for miR-135 family (miR-135a/-135b) in leiomyoma smooth muscle cells. Therefore, based on this preliminary data we hypothesize that leiomyoma as compared to myometrium displays reduced expression of linc-MD1 which through a miRNA-guided mechanism involving miR-135 regulates the expression of specific target genes functionally associated with Wnt/?-catenin signaling pathway which is known to be central to leiomyoma pathogenesis. To test our core hypothesis we propose 2 specific aims.
In Aim 1 we will determine the interaction between linc-MD1 and miR-135 family by RNA immunoprecipitation and RNA pull-down assay. We will over or under express linc-MD1 in LSMC or MSMC spheroid cells and determine its effect on miR-135 family and its downstream target genes namely GSK3? and APC which are known to regulate ?-Catenin degradation. In these experiments ?-Catenin, its phosphorylated form and its nuclear localization will be determined in response to overexpression and knockdown studies in vitro. To establish the clinical relevance and therapeutic potential of linc-MD1 in Aim 2 we will determine the effect of linc-MD1 overexpression in leiomyoma cells on fibroid progression in a leiomyoma animal model. This translational proposal addresses a significant gap in knowledge on the role of a novel lncRNA-miRNA network in the pathogenesis of fibroids which is a priority area of investigation for NIH, and could potentially be targeted for therapeutic purposes for fibroids.
Uterine leiomyoma (fibroids) are benign gynecologic tumors that develop during the reproductive age and cause abnormal uterine bleeding, pain and pressure symptoms. The factors that initiate leiomyoma?s development are unknown. In this proposal we have proposed a new mechanism involving long non coding RNA that can explain how fibroids develop and grow and have proposed new treatments based on our proposed model.
