ENHANCING MIR-183-5P ABUNDANCE IN RODENT MAMMARY EPITHELIAL CELLS: EFFECT ON DIFFERENTIATION

Abstract

Breast cancer is the most common type of cancer occurring in women worldwide. Many cellular mediators contribute to breast cancer initiation and progression, among those are microRNAs. microRNAs (miRNAs) are a class of single stranded RNA molecules that control important cell functions including differentiation, cell growth, apoptosis, and migration through post- transcriptional gene silencing. Panels of miRNAs are dysregulated in multiple cancer types including breast cancer. In breast cancer, miR-183-5p is among the overexpressed miRNAs in both ductal carcinomas in situ (DCIS) and lobular neoplasia subtypes. Previous reports correlated miR-183-5p overexpression to loss of epithelial cell polarity and enhanced migration and proliferation of breast epithelial cells of ductular origin. Despite being identified as overexpressed in lobular carcinoma, the mechanisms by which miR-183-5p mediate lobular cancer initiation remain uninvestigated. Knowing that differentiation and cancer initiation are fundamentally opposite processes, and that the common cellular factors contribute to both processes, we aimed to determine the effect of miR-183-5p on tumor initiation in a model that specifically recapitulates mammary epithelial differentiation in vitro. To investigate the role of miR-183-5p on loss of differentiation and tumor initiation in lobular mouse mammary epithelial SCp2 cells, we sought to trigger miR-183-5p abundance in SCp2 cells and monitor its effect on cell-cell/ cell-ECM-mediated differentiation through the expression of the differentiation marker β-casein.

Virally infecting SCp2 epithelial and SCg6 myoepithelia-like cells with miR-183-5p expression vector under the control of CMV promoter resulted in low transduction efficiency and diminished expression of miR-183-5p. Two alternative approaches were therefore, adopted to trigger miR-183-5p abundance in SCp2 cells. The first was treating SCp2 cells with conditioned media derived from miR-183-5p-infected S1 epithelial cells. The results suggested that miR-183-5p is elevated in SCp2 cells treated with conditioned media deriving from S1-miR-183-5p infected cells. However, inhibitors-possibly sodium selenite- within the conditioned media resulted in the abolishment of β-casein expression independent from miR-183-5p. An alternative solution was treating SCp2 with exosome extracts isolated from the conditioned media.

The exosomal extracts deriving from miR-183-5p-S1 conditioned media had the highest miR-183-5p levels compared to controls. Moreover, a higher level of miR-183-5p was detected in SCp2 cells after treatment with the aforementioned exosome extracts. Further experiments are needed to assess the effect of exosome treatment on differentiation and β-casein expression. The third approach aimed to increase miR-183-5p in SCp2 by treating the cells with the potential carcinogen Glyphosate. Preliminary results suggest that Glyphosate treatment (10-11M) increases in miR-183-5p expression in SCp2 cells. On the other hand, β-casein expression was diminished in SCp2 after being treated with 10-11 M glyphosates.

In conclusion, we suggest that human CMV promoter efficiency is reduced in mouse derived SCp2 cells. Moreover, miR-183-5p could be released into S1-culture conditioned media and transferred to target SCp2 cells through exosomes. Finally,

Glyphosate treatment induced the overexpression of miR-183-5p and culminated with the downregulation of β-casein expression and the loss of normal mammary epithelial differentiation in SCp2 cells despite being cultured in differentiation permissive conditions. Overall, to increase miR-183-5p in SCp2 and SCg6 cells we suggest virally infecting SCp2 and SCg6 with a viral vector suitable for mouse mammary epithelial cells. Alternatively, exosomes could also be purified from pre-tumorigenic Cx43 knockout cells expressing high miR-183-5p levels. We also propose that glyphosate treatment could be downregulating β-Casein expression and imposing other tumor-initiating, or cytotoxic effect on SCp2.