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MiR-183-5p and miR-492 over-expression trigger loss of polarity and nuclear circularity and enhanced invasion and proliferation in nontumorigenic breast epithelium

ABSTRACT: microRNAs (miRNAs) are stable and abundant in body fluids and exhibit unique dysregulation signatures in cancers, making them attractive novel noninvasive cancer biomarkers. In an HMT-3522 S1 (S1) breast epithelial risk-progression 3D culture model, non-tumorigenic S1 cells form a fully polarized epithelium. Their pretumorigenic counterparts, silenced for gap junction and tumor suppressor Cx43, Cx43-KO-S1, lose epithelial polarity, multilayer, exhibit proliferative and invasion potential, and mimic tumor-initiated in vivo mammary epithelial morphology. To characterize the miRNA profile specific to Cx43 loss in the breast epithelia, miRNA-sequencing was performed and revealed 65 differentially expressed miRNAs in Cx43-KO-S1 as compared to S1 cells. A comparative analysis was conducted between the 65 differentially expressed miRNAs and 15 tumor-associated miRNAs from a young Lebanese patient validation cohort. miR-183-5p, downstream of Cx43 loss and commonly upregulated in the patient cohort and the breast epithelia and miR-492, not attributed to Cx43 loss, and only up-regulated in the young Lebanese patients were chosen for stable over-expression in the nontumorigenic S1 cells. Ectopic over-expression of miR-183 and miR-492 in S1 cells, through pLenti-III-miR-GPF tagged vectors, resulted in the formation of larger acini devoid of lumen assembly with cell multilayering, disrupted epithelial polarity and decreased nuclear circularity in 3D cultures, as well as in enhanced proliferation and invasion capacity and altered apical localization of Cx43 and apico-lateral localization of ß-catenin and Scrib. miR-183-5p and miR-492 over-expression in nontumorigenic S1 cells thus recapitulate pretumorigenic phenotypes similar to those reported upon Cx43 loss and act as oncomiRs and prognostic biomarkers.

ORGANISM(S): Homo sapiens

PROVIDER: GSE196062 | GEO | 2022/12/28

REPOSITORIES: GEO

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Project description:We tested the hypothesis that a panel of placental mammal-specific miRNAs and their targets play important to establish receptivity to implantation and their dysregulated expression may be a feature in women with early pregnancy loss. Relative expression levels of miR-340-5p, −542-3p, and −671-5p all increased following treatment of Ishikawa cells with progesterone (10 μg/ml) for 24 hrs (p < 0.05). RNA sequencing of these P4-treated cells identified co-ordinate changes to 6,367 transcripts of which 1713 were predicted targets of miR-340-5p, 670 of miR-542-3p, and 618 of miR-671-5p. Quantitative proteomic analysis of Ishikawa cells transfected with mimic or inhibitor (48 hrs: n=3 biological replicates) for each of the P4-regulated miRNAs was carried out to identify targets of these miRNAs. Excluding off target effects, mir-340-5p mimic altered 1,369 proteins while inhibition changed expression of 376 proteins (p < 0.05) of which, 72 were common to both treatments. A total of 280 proteins were identified between predicted (mirDB) and confirmed (in vitro) targets. In total, 171 proteins predicted to be targets by mirDB were altered in vitro by treatment with miR-340-5p mimic or inhibitor and were also altered by treatment of endometrial epithelial cells with P4. In vitro targets of miR-542-3p identified 1,378 proteins altered by mimic while inhibition altered 975 a core of 200 proteins were changed by both. 100 protein targets were predicted and only 46 proteins were P4 regulated. miR-671-mimic altered 1,252 proteins with inhibition changing 492 proteins of which 97 were common to both, 95 were miDB predicted targets and 46 were also P4-regulated. All miRNAs were detected in endometrial biopsies taken from patients during the luteal phase of their cycle, irrespective of prior or future pregnancy outcomes Expression of mir-340-5p showed an overall increase in patients who had previously suffered a miscarriage and had a subsequent miscarriage, as compared to those who had infertility or previous miscarriage and subsequently went on to have a life birth outcome. The regulation of these miRNAs and their protein targets regulate the function of transport and secretion, and adhesion of the endometrial epithelia required for successful implantation in humans. Dysfunction of these miRNAs (and therefore the targets they regulate) may contribute to endometrial-derived recurrent pregnancy loss in women.

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MiR-183-5p and miR-492 over-expression trigger loss of polarity and nuclear circularity and enhanced invasion and proliferation in nontumorigenic breast epithelium

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