UTERINE FIBROID-CAUSING MUTAGENESIS INDUCED BY DEVELOPMENTAL EXPOSURE TO ENDOCRINE DISRUPTING CHEMICALS IS MEDIATED VIA ABROGATION OF NUCLEOTIDE EXCISION REPAIR PATHWAY IN MYOMETRIAL STEM CELLS THROUGH UPREGULATION OF TGF-β1

Abstract

The cause of uterine fibroid (UFs) is largely unknown, but it is well established that one of the major risk factors for the development of these tumors is early environmental exposure to endocrine-disrupting chemicals (EDCs). It has been shown that UFs originate from abnormal stem cells (SCs) in the myometrium (MM) that acquire a driver mutation in pivotal genes such as Tsc-2 (Eker rat) or Med12 (human), and there is evidence that DNA repair capacity could be involved in the emergence of such mutations. Furthermore, the pleiotropic cytokine TGF-β1 has been related to UF development. Recent studies have suggested a link between TGF-β1 and the DNA damage response with implications for tumor initiation and growth. In this context, this work aimed to evaluate the possible role of TGF-β1 and the nucleotide excision repair (NER) on the tumorigenesis process on EDC-exposed MMSCs. Laboratory research studies using an Eker rat fibroid model MMSCs. Female Eker rats were received subcutaneous injections of 10 μg of Diethylstilbestrol (DES, endocrine-disrupting chemical) per rat per day or 50 μl of sesame seed oil (vehicle, VEH) on days 10, 11, and 12 after birth. MMSCs were isolated from 5 months adult MM tissue (N=5 for each group) using Stro-1 and CD44 surface markers. Whole-genome RNA-sequencing was performed in VEH- and DES-MMSCs to determine global gene expression profiles. Latent-TGFβ-binding protein 1 (Ltbp-1), Tgf-β1, Xpc, Ddb1, and Ddb2 (three proteins involved in DNA damage recognition on NER pathway) mRNA levels in VEH- and DES-MMSCs were measured using qRT-PCR. Thrombospondin 1 (TSP-1), LTBP1, TGF-β1, XPC, and DDB1 protein expressions were evaluated by Western Blot (WB). Two-tailed unpaired Student t-test was used to assess any statistically significant differences (P-value<0.05). The RNA-seq data analysis demonstrated that the expression of 19 genes belonging to TGF-β1 signaling were altered in DES-MMSCs compared to VEH-MMSCs. Moreover, 6 genes involved in the NER pathway showed changes in RNA expression between DES-MMSCs. The mRNA levels of Ltbp-1 and Tgf-β1 in MMSCs showed an increase when animals were exposed neonatally to DES. Regarding NER related genes, Xpc, and Ddb2 were higher in DES-MMSCs than VEH-MMSC, while Dbb1 mRNA levels showed the opposite results (p<0.05). The protein levels of TSP-1, LTBP1, and TGF-β1 were increased in DES-MMSCs compared to VEH-MMSCs (p<0.05). We did not find differences in XPC protein levels (p>0.05). However, DDB1 protein levels were upregulated in DES-MMSCs in comparison to VEH-MMSCs. Our results showed that early-life exposure to DES provokes changes in TGF-β1 and NER pathways on MMSCs, therefore it could increase the predisposition to develop UFs later in life. Further studies are needed to determine the relation between TGF-β1 and NER pathways on DES-exposed MMSCs and parallel studies in the human uterus.