miR-143 and miR-145 disrupt the cervical epithelial barrier through dysregulation of cell adhesion, apoptosis and proliferation

Abstract

Molecular mechanisms regulating preterm birth (PTB)-associated cervical remodeling remain unclear. Prior work demonstrated an altered miRNA profile, with significant increases in miR-143 and miR-145, in cervical cells of women destined to have a PTB. The study objective was to determine the effect of miR-143 and miR-145 on the cervical epithelial barrier and to elucidate the mechanisms by which these miRNAs modify cervical epithelial cell function. Ectocervical and endocervical cells transfected with miR-negative control, miR-143 or miR-145 were used in cell permeability and flow cytometry assays for apoptosis and proliferation. miR-143 and miR-145 target genes associated with cell adhesion, apoptosis and proliferation were measured. Epithelial cell permeability was increased in miR-143 and miR-145 transfected cervical epithelial cells. Cell adhesion genes, JAM-A and FSCN1, were downregulated with overexpression of miR-143 and miR-145. miR-143 and miR-145 transfection decreased cervical cell number by increasing apoptosis and decreasing cell proliferation through initiation of cell cycle arrest. Apoptosis genes, BCL2 and BIRC5, and proliferation genes, CDK1 and CCND2, were repressed by miR-143 and miR-145. These findings suggest that miR-143 and miR-145 play a significant role in cervical epithelial barrier breakdown through diverse mechanisms and could contribute to premature cervical remodeling associated with PTB.

Figure 1

Epithelial cell permeability is altered in ectocervical and endocervical cells transfected with miR-143 and miR-145 mimics. Epithelial cell permeability was significantly increased in ectocervical cells (a) and endocervical cells (b) overexpressing miR-143 and miR-145 when compared to miR-negative (miR-neg) control. Cell permeability is expressed as fluorescence OD measurements from a fluorescent plate reader and is indicative of the movement of FITC-dextran from the top to the bottom insert of a transwell chamber system. Values are mean ± SEM. *p < 0.001, **p < 0.0001.

Figure 2

miR-143 and miR-145 inhibit adhesion gene expression in ectocervical and endocervical cells. Ectocervical and endocervical cells were transfected with miR-negative control (miR-neg), miR-143 and miR-145 mimics and expression of downstream target genes were measured by QPCR and western blot. Junctional adhesion molecule-A (JAM-A), a member of the adherens junction complex, was significantly decreased by overexpression of miR-145 but not miR-143 in both ectocervical (a) and endocervical cells (b). JAM-A protein expression was reduced by miR-145 in ectocervical and endocervical cells (c). Fascin-1 (FSCN1), an actin bundling protein, was significantly repressed in both miR-143 and miR-145 transfected ectocervical (e) and endocervical (f) cells. FSCN1 protein expression was reduced by miR-143 and miR-145 in both cell types (g). 3′UTR luciferase reporter assays in HEK293T cells verified that JAM-A is a direct target of miR-145 (d) and FSCN1 is a direct target of both miR-143 and miR-145 (h). 3′UTR assay results are expressed as a ratio of Gaussia Luciferase (GLuc) activity over Secreted Alkaline Phosphatase (SEAP) which has been normalized to cells transfected with the plasmid alone. Values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

miR-145 inhibits JAM-A expression on the epithelial cell membrane of ectocervical and endocervical cells. Ectocervical and endocervical cells transfected to overexpress miR-143 and miR-145 were stained for JAM-A (red) and DAPI (cell nucleus, blue). JAM-A protein expression was reduced in both ectocervical and endocervical cells transfected with miR-145 but not miR-143 indicating that JAM-A is a direct target of miR-145.

Figure 4

Ectocervical and endocervical cell number is decreased by miR-143 and miR-145. Ectocervical and endocervical cells transfected to overexpress miR-143 and miR-145 were counted at 0, 72 and 144 hours after transfection. The total number of ectocervical (a) and endocervical (b) cells were significantly decreased in both miR-143 and miR-145 transfected cells when compared to the miR-negative transfected cells after 144 hours of growth. Values are mean ± SEM. *p < 0.05, **p < 0.001, ***p < 0.0001.

Figure 5

miR-143 and miR-145 increased ectocervical and endocervical cell apoptosis. Flow cytometry based annexin V/PI apoptosis assays were performed on ectocervical and endocervical cells transfected with miR-negative control (miR-neg), miR-143 or miR-145 at 0, 72 and 144 hours after transfection. Representative histograms showing the results of the apoptosis assay are depicted for each miRNA at 144 hours in ectocervical (a) and endocervical (c) cells. The results from four independent assays are shown in the bar graphs. In ectocervical cells, miR-145 significantly increased the percentage of apoptotic cells (b) while, in endocervical cells, both miR-143 and miR-145 increased the percentage of apoptotic cells (d) at 144 hours. Values are mean ± SEM *p < 0.05, **p < 0.001.

Figure 6

miR-143 and miR-145 repressed apoptosis gene expression. Ectocervical and endocervical cells were transfected with miR-negative control (miR-neg), miR-143 or miR-145 and expression of downstream target genes were measured by QPCR and western blot. BCL2, a negative regulator of apoptosis and a predicted target of miR-143, was significantly decreased with miR-143 but not miR-145 overexpression in ectocervical (a) and endocervical cells (b). Protein expression of BCL2 was reduced by miR-143 in transfected endocervical cells while BCL2 protein was too low to be detectable in ectocervical cells (c). 3′UTR luciferase assays in HEK293T cells confirmed that BCL2 is a direct target of miR-143 (d). BIRC5, a predicted target of miR-143 and miR-145, was significantly repressed by miR-145 in ectocervical (e) cells and by both miR-143 and miR-145 in endocervical cells (f). BIRC5 protein expression was reduced by miR-143 and miR-145 in endocervical cells while BIRC5 protein levels were decreased by miR-143 and unchanged by miR-145 in ectocervical cells (g). 3′UTR luciferase assays verified that BIRC5 is a target of miR-143 and miR-145 (h). 3′UTR assay results are expressed as a ratio of Gaussia Luciferase (GLuc) activity over Secreted Alkaline Phosphatase (SEAP) which has been normalized to cells transfected with the plasmid alone. Values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

miR-143 and miR-145 overexpression results in cell cycle arrest. Ectocervical and endocervical cells transfected with miR-negative control (miR-neg), miR-143 or miR-145 were used for cell cycle assays by flow cytometry. Representative cell cycle histograms show the percentage of ectocervical and endocervical cells in each phase of the cell cycle after miR-neg (a,g), miR-143 (b,h) and miR-145 (c,i) transfection. Bar graphs show the average (n = 4 independent experiments) percentage of cells in each phase of the cell cycle. miR-143 and miR-145 over expression resulted in cell cycle arrest at G0/G1 (d) in ectocervical cells (d,e,f). In endocervical cells (j,k,l), similarly, miR-143 transfection caused cell cycle arrest at G0/G1 (j), while, miR-145 transfection resulted in S-phase arrest (k). Values are mean ± SEM *p < 0.05, **p < 0.01.

Figure 8

miR-143 and miR-145 repress genes that regulate the cell cycle. Ectocervical and endocervical cells were transfected with miR-negative control (miR-neg), miR-143 or miR-145 and expression of downstream target genes were measured by QPCR and western blot. Cyclin dependent kinase 1(CDK1), was significantly decreased by both miR-143 and miR-145 in ectocervical cells (a). In endocervical cells, only miR-143 transfection resulted in a decrease in CDK1 expression (b). Protein expression of CDK1 was reduced by miR-143 and miR-145 in ectocervical cells and by miR-143 only in endocervical cells (c). 3′UTR luciferase assays confirmed that CDK1 is a direct target of miR-143 and miR-145 (d). Cyclin D2 (CCND2) was repressed by overexpression of both miR-143 and miR-145 in ectocervical (e) and endocervical (f) cells. CCND2 protein expression was reduced in miR-143 and miR-145 transfected ectocervical and endocervical cells (g). 3′UTR luciferase assays showed that CCND2 is a direct target of both miR-143 and miR-145 (h). 3′UTR assay results are expressed as a ratio of Gaussia Luciferase (GLuc) activity over Secreted Alkaline Phosphatase (SEAP) which has been normalized to cells transfected with the plasmid alone. Values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.