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

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.

Previous studies by our group and others suggest that compromise of the cervical epithelial barrier promotes cervical remodeling and contributes significantly to the pathogenesis of preterm birth [9][10][11] . Epithelial cells within the cervicovaginal space must be tightly regulated during pregnancy as they play an integral role in cervical remodeling and growth. Cervical epithelial cells line the cervical lumen creating a barrier to protect the cervical stroma from the invasion of microbes and to regulate paracellular transport through the apical junctional complex present on the epithelial cell membrane. The apical junctional complex regulates cell-cell adhesion, paracellular permeability, and cell polarity and is made up of both tight junction and adherens junction proteins 12 . Tight junctions, made up mostly of the claudin family of proteins 13 , and the adherens junctions, made up mostly of the cadherin family of proteins 14 , regulate the "tightness" of the epithelial cells to each other. Therefore, changes in the composition of the tight and/or adherens junctions can alter the cervical epithelial barrier significantly. In order to maintain the integrity of the cervical epithelial barrier during gestation, cervical epithelial cells also undergo a marked increase in growth and proliferation. Consequently, alterations in epithelial cell number can have a significant impact on barrier function.
While the mechanisms regulating cervical remodeling remain largely unknown, there are many factors that may have the ability to alter the cervical epithelial barrier and, hence, initiate cervical remodeling including alterations in inflammation and infection 9,15 , biomechanical properties of the cervix [16][17][18] , microRNAs (miRNAs) 19,20 and the cervicovaginal microbiome 21 and metabolome 22 . In a previous study, we investigated the expression of miRNAs in a cohort of women at high risk for preterm birth 20 . We showed the presence of an altered miRNA profile (99 differentially expressed miRNAs) in the cervicovaginal space weeks, if not months, prior to the initiation of spontaneous preterm birth. We identified two specific miRNAs, miR-143 and miR-145, that were significantly increased in the cervix of women destined to have a preterm birth. miRNAs are short, about 22 nucleotides in length, highly conserved single-stranded RNA molecules that play a critical role in post transcriptional gene regulation. One miRNA has the ability to interact with hundreds of messenger RNAs (mRNA) through matching of the miRNA seed sequence with the 3′ untranslated regions (3′UTR) of specific mRNA targets to negatively affect mRNA stability and translation. Therefore, miRNAs have the ability to regulate large gene networks and have been shown to play a significant role in almost every disease state including cancer and cardiovascular disease 23,24 among many others. The increased expression of miR-143 and miR-145 in cervical cells from women destined for a preterm birth suggested that these miRNAs have the ability to contribute significantly to cervical remodeling.
The objective of this study was to determine the effect of miR-143 and miR-145 on the integrity of the cervical epithelial barrier and to elucidate the mechanisms by which these miRNAs modify cervical epithelial cell function. For this study, we investigated the effects of miR-143 and miR-145 on cervical epithelial cell permeability to determine if these miRNAs have the ability to alter the cervical epithelial cell barrier. Additionally, we focused on the molecular mechanisms contributing to the breakdown of the cervical epithelial cell barrier by investigating the effects of increased miR-143 and miR-145 expression on cell adhesion and growth. Therefore, we hypothesize that increased expression of miR-143 and miR-145 disrupts the integrity of the cervical epithelial barrier through regulation of cell adhesion, apoptosis and cell proliferation which initiates premature cervical remodeling resulting in early delivery.

Results
miR-143 and miR-145 increase ectocervical and endocervical epithelial cell permeability. As we have previously identified miR-143 and miR-145 as being significantly increased in the cervicovaginal space in high risk women months prior to delivering preterm 20 , we wanted to determine if these specific miRNAs have an effect on cervical cell function. Therefore, we focused on epithelial cell permeability, as decreased epithelial tight junctions and increased water influx are primary events in cervical ripening and remodeling 25 . Ectocervical (Fig. 1a) and endocervical (Fig. 1b) cells transfected with miR-143 (n = 3, ecto: p = 0.0016, endo: p < 0.0001) and miR-145 (n = 3, ecto: p = 0.0003, endo: p = 0.0004) had a significant increase in epithelial cell permeability (when compared to those transfected with miR-negative control) as evidenced by a significant increase in the amount of 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.
FITC dextran present in the bottom well of the transwell inserts. These results suggest that increased expression of these miRNAs contribute to the breakdown of the cervical epithelial cell barrier.
Predicted gene targets of miR-143 and miR-145. In order to determine the specific genes that might be mechanistically involved in alterations of the cervical epithelial barrier, we focused on known or predicted downstream targets of miR-143 and miR-145 that regulate epithelial cell adhesion and cell number including apoptosis and cell proliferation. Using TargetScan 26 , an online software program that predicts biological targets of miRNAs by searching for the presence of 8mer, 7mer, and 6mer sites that match the seed region of a miRNA of interest, we identified several target genes known to be involved in epithelial tight junction formation and cell adhesion including junctional adhesion molecule-A (JAM-A, F11R) and Fascin1 (FSCN1). Additionally, we chose to focus on targets involved in inhibiting the intrinsic apoptosis pathway including B-cell lymphoma 2 (BCL2) and baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5, Survivin) as well as genes known to play a role in cell cycle progression such as cyclin dependent kinase 1 (CDK1) and cyclin D2 (CCND2). The seed region sequence for miR-143 and miR-145, their predicted genes of interest and their corresponding target sequences are shown in Tables 1 and 2, respectively. A full list of all miR-143 and miR-145 target genes investigated as part of this study and their expression in ectocervical and endocervical cells can be found in Supplementary Table S1. miR-143 and miR-145 decrease cell adhesion. JAM-A, a predicted target of miR-145, was significantly decreased in both ectocervical (Fig. 2a) and endocervical ( Fig. 2b) cells transfected with miR-145 (n = 3, ecto: p < 0.01, endo: p < 0.05) but not miR-143. JAM-A protein expression ( Fig. 2c) was similarly decreased in ectocervical and endocervical cells transfected with miR-145 but not miR-143. The JAM-A 3′UTR reporter assay (Fig. 2d), which shows repressed GLuc activity in the presence of a specific miRNA target, showed a significant decrease in GLuc expression in the presence of miR-145 when compared to the JAM-A plasmid alone (n = 9, p < 0.001). No changes in GLuc expression were seen in the presence of exogenous miR-neg control or miR-143 indicating that JAM-A is a direct target of miR-145. Staining of ectocervical and endocervical cells provide further evidence that transfected cells have the ability to form a monolayer and that miR-145 but not miR-143 represses JAM-A expression localized at the epithelial cell membrane (Fig. 3). Additional experiments showed a significant decrease in FSCN1 expression after transfection of ectocervical (Fig. 2e) and endocervical (Fig. 2f) cells with both miR-143(n = 3, ecto: p < 0.001, endo: p < 0.01) and miR-145 (n = 3, ecto: p < 0.001, endo: p < 0.001). FSCN1 is an actin bundling protein that regulates cytoskeletal structures for the maintenance of cell adhesion in epithelial cells and is a predicted target for both miR-143 and miR-145. FSCN1 protein expression was reduced after ectocervical and endocervical cell transfection with both miR-143 and miR-145 (Fig. 2g). The FSCN1 3′UTR assay (Fig. 2h) showed repressed GLuc activity in the presence of both miR-143 (n = 6, p < 0.01) and miR-145 (n = 6, p < 0.01) alone and in combination (n = 6, p < 0.01) but not with miR-negative control. These results confirmed that miR-143 and miR-145 specifically target FSCN1 in ectocervical and endocervical cells.    with miR-143 (ecto: p < 0.05, endo: p < 0.001) when compared to those transfected with miR-neg control. This effect was independent of the transfection reagent (Lipofectamine exposure) as we did not see any effect on cell number when transfecting the miR-negative control into these cells (compared to non-transfected cells, data not shown).

miR-143 and miR-145 promote apoptosis.
After observing a significant decrease in cell number after transfection with miR-143 and miR-145, we investigated the molecular mechanisms contributing to this effect. Preliminary experiments, using an ApoTox-Glo Triplex assay which assesses viability, cytotoxicity and caspase  miR-143 and miR-145 decrease negative regulators of apoptosis. BCL2, a predicted target of miR-143, was significantly decreased in both ectocervical (Fig. 6a) and endocervical (Fig. 6b) cells transfected with miR-143(n = 4, ecto: p < 0.05, endo: p < 0.01) but not miR-145. BCL2 protein expression was reduced in endocervical cells transfected with miR-143 and miR-145. However, in ectocervical cells, in agreement with very low mRNA levels, protein levels were too low to be detected by western blot (Fig. 6c). The BCL2 3′UTR reporter assay (Fig. 6d, n = 9) showed a significant decrease in GLuc expression in the presence of miR-143 (p < 0.001) when compared to the BCL2 plasmid alone. No changes in GLuc expression were seen in the presence of exogenous miR-neg control or miR-145 indicating that BCL2 is a direct target of miR-143. A significant decrease in BIRC5 expression was seen in ectocervical cells (Fig. 6e, n = 4) transfected with miR-145 (p < 0.01). Transfection with both miR-143 (p < 0.01) and miR-145 (p < 0.05) repressed BIRC5 in endocervical cells (Fig. 6f, n = 4). Protein expression of BIRC5 was decreased after transfection of endocervical cells with miR-143 and miR-145. However, in ectocervical cells, there was a reduction in BIRC5 protein expression in miR-143 transfected cells while BIRC5 was unchanged after transfection with miR-145 (Fig. 6g). Decreased BIRC5 3′UTR GLuc activity (Fig. 6h, n = 6) was seen in the presence of exogenous miR-143(p < 0.01), miR-145 (p < 0.001) and both miR-143 and miR-145 co-transfected together (p < 0.001) but not the miR-negative control indicating that BIRC5 is a direct target of both miRNAs.
miR-143 and miR-145 alter cell cycle progression. In addition to the role of miR-143 and miR-145 in promoting apoptosis, we also investigated if decreases in cell number could be due to alterations in cell proliferation and growth. Using flow cytometry, we performed standard cell cycle assays to assess if ectocervical and  endocervical cells transfected with miR-neg, miR-143 and miR-145 were progressing normally through the three phases of cell growth -G0/G1, synthesis (S-phase) and G2. Representative histograms demonstrating the cell cycle after transfection with miR-neg (Fig. 7a,g), miR-143 (Fig. 7b,h) and miR-145 (Fig. 7c,i) are shown for ectocervical and endocervical cells. Quantification of the percentage of cells in each of the three phases of the cell cycle show a significant increase in ectocervical cells (n = 3) in the G0/G1 phase (Fig. 7d, p = 0.0113) after transfection with miR-143 (p < 0.05) and miR-145 (p < 0.05) when compared with miR-neg. Consistent with this, there is a significant reduction in the percentage of cells in the S-phase (Fig. 7e, miR-143: p < 0.01, miR-145: p < 0.01) and G2 phase (Fig. 7f, miR-143: p < 0.01, miR-145: p < 0.01) indicating cell cycle arrest at G0/G1. A similar effect is seen in endocervical cells (n = 4) transfected with miR-143 at G0/G1 (Fig. 7j, p < 0.01), S-phase (Fig. 7k, p < 0.05) and G2 (Fig. 7l, p < 0.05) but not with miR-145. miR-145 transfection decreased the percentage of cells in G0/G1 (Fig. 7j, p < 0.05) and increased endocervical cells in S-phase (Fig. 7k, p < 0.01) followed by a reduction in cells in G2 (Fig. 7l, p < 0.05) suggesting that miR-145 causes cell cycle arrest in the S phase.

Discussion
This study provides novel insight into the molecular mechanisms contributing to cervical remodeling. Additionally, as we have shown in a previous study that miR-143 and miR-145 are significantly increased in the cervical vaginal space of women destined to have a premature delivery 20 , this study suggests that compromise of the cervical epithelial barrier is a possible mechanism for the pathogenesis of preterm birth. First, we have shown that overexpression of miR-143 and miR-145 results in a breakdown of the cervical epithelial barrier in both ectocervical and endocervical cells. Secondly, we have identified multiple molecular pathways and specific gene targets contributing to the increase in epithelial barrier breakdown. Increased expression of miR-143 and miR-145 alters both ectocervical and endocervical cell function by decreasing adherens junction proteins, reducing cell number, activating the intrinsic apoptosis pathway and initiating cell cycle arrest. Additionally, similar results in both ectocervical and endocervical cells indicate that miR-143 and miR-145 have global, and not region-specific, effects on the cervical epithelia. We acknowledge that the expression levels of miR-143 and miR-145 following transfection supersedes the physiological levels seen in our previous study 20 , however, understanding the limitations of in vitro work, we believe that our data support the biological plausibility of the role of these miRNAs in cervical epithelial biology. The results of this study support our hypothesis that the pathogenesis of preterm birth is initiated within the cervical vaginal space where alterations in the environment surrounding the cervix can alter cervical function leading to premature cervical remodeling. During pregnancy, the cervical epithelial cells must maintain a strong barrier in order to protect the upper reproductive tract and fetus from invading pathogens, regulate paracellular transport and maintain fluid balance. However, in the presence of an "unhealthy" cervicovaginal space, characterized by increased inflammation due to a bacterial infection or a dysbiotic microbiome, among other possibilities, the cervical epithelial barrier could become compromised. Once the epithelial barrier is disrupted, the cervical stroma is vulnerable to invasion by pathogenic bacteria, inflammatory mediators or water 25 which have all been previously associated with the initiation of cervical tissue remodeling. The concept of a compromised epithelial barrier resulting in a disease state is well described in the gut literature [27][28][29][30] . In the gastrointestinal tract, commensal bacteria and food-derived antigens directly interact with the gut epithelial barrier, which acts similarly in the cervix, to create a physical and immunological barrier against invading pathogens. Disruption of the gut epithelial barrier results in invasion by commensal and pathogenic bacteria causing recruitment of pro-inflammatory mucosal immune mediators. This detrimental inflammation ultimately leads to inflammatory bowel disease, Crone's disease and ulcerative colitis. In a previous study, we have shown that the presence of lipopolysaccharide (LPS), a gram negative inflammatory mediator, has the ability to breakdown the cervical epithelial barrier through increased cytokine expression and sECAD release 9 . The results from that study suggested that inflammatory agents, gaining access to the cervix through the vaginal canal, have the ability to compromise the epithelial barrier. Similarly, in this study, we showed that increased expression of miR-143 and miR-145 were able to disrupt the epithelial barrier. Interestingly, we have previously shown that ectocervical and endocervical cells exposed to LPS have increased expression of miR-143 and miR-145 suggesting that inflammation could be an upstream regulator of these miRNAs 20 . While some progress has been made in understanding the role of inflammation in cervical remodeling, the molecular mechanisms regulating this process remain largely unclear. As miR-143 and miR-145 are undoubtedly regulated by both inflammation-dependent and independent mechanisms, it is of interest to determine the molecular mechanisms altered by miR-143 and miR-145 that contribute to the breakdown of the cervical epithelial barrier.
Since cell to cell adhesion is regulated by the presence of both tight junctions and adherens junctions on the epithelial membrane, we first focused on investigating the proteins localized to these two junctional complexes. After doing a search of known or predicted target genes of miR-143 and miR-145 in TargetScan, two proteins in particular were of most interest including JAM-A and Fascin-1 (FSCN1). JAM-A, a predicted target of miR-145, is an integral part of the adherens junction complex, and has been shown to contribute significantly to epithelial cell barrier function 31,32 . FSCN1, a direct target of both miR-143 and miR-145, is an actin bundling protein that regulates cell adhesion and cellular interactions 33 . In this study, we found a significant reduction in both JAM-A and FSCN1 gene expression in the presence of miR-143 and miR-145 overexpression in ectocervical and endocervical cells suggesting that reductions in JAM-A and FSCN1 contribute to increased epithelial cell permeability. Although no previous studies have been done investigating the effects of miR-143 or miR-145 on the cervical epithelial barrier, miR-145 is known to target JAM-A in endometrial stromal cells 34 and breast cancer cells 35 where it inhibited cell proliferation, motility and invasion indicating that cervical cell function could be similarly affected. Similar studies investigating miR-145 on FSCN1 expression in several cancer cell lines (gastric, colorectal, non-small cell lung) have shown significant inhibition in cancer cell phenotypes including metastasis, invasion and proliferation [36][37][38] . As most research involving miR-143 and miR-145 has been done in the cancer field, no studies have investigated the effects of these miRNAs on cellular adhesion as it relates to cell barrier function. Therefore, this study is the first to associate miR-143 and miR-145 with deficits in the epithelial barrier.
Disruption of the cervical epithelial barrier is associated with alterations in both epithelial cell number and epithelial cell proliferation 39 . As previous studies have shown that miR-143 and miR-145 have significant effects on cancer cell growth 40 , we focused on cervical cell number and miRNA target genes known to affect cell apoptosis and proliferation. In the presence of overexpressed miR-143 and miR-145, we found decreased ectocervical and endocervical cell number as well as increased apoptosis. BCL2, a target of miR-143, and BIRC5, a target of miR-143 and miR-145, are both well studied inhibitors of the intrinsic apoptosis pathway. Increased miR-143 and miR-145 inhibited the expression of BCL2 and BIRC5, consequently, activating the apoptosis pathway. These results indicate that upregulated miR-143 and miR-145 expression within the cervical space could lead to increased apoptosis at the cervical epithelial barrier contributing to premature cervical remodeling. Previous studies have also found that BCL2 is a direct target of miR-143 resulting in apoptosis in the cervical cancer cell line, HeLa, among others 41,42 . Very little is known about the regulation and maintenance of the cervical epithelial barrier, however, in the gut, normal epithelial cell death occurs frequently in order to make room for new cells ensuring that the strongest epithelial cells are available to uphold the barrier. In disease states such as irritable bowel disease, significant pathological apoptosis is present 43,44 . While the exact role of epithelial cell death on barrier integrity remains unclear in both the gut and the cervix, the consequences of this are hypothesized to be 1) disruption of the barrier allowing bacteria to traverse into the stroma, 2) loss of the anti-microbial (and other) protective factors released by epithelial cells and 3) epithelial cell death itself leads to increased inflammation and inflammatory cytokines creating a negative cycle of epithelial barrier breakdown.
Secondary to apoptosis, we also investigated the effects of miR-143 and miR-145 on cell proliferation and the cell cycle as a potential mechanism contributing to cervical epithelial barrier breakdown. Ectocervical and endocervical cells transfected with miR-143 and miR-145 showed cell cycle arrest at either G0/G1 or S phase. Additionally, direct gene targets, CDK1 and CCND2 were repressed in the presence of miR-143 and miR-145. CDK1 is a cyclin dependent kinase known to play an integral role in cell cycle regulation especially in the progression from G2 to M-phase. CCND2, also known as the G1/S-specific cyclin D2, is responsible for regulating the G1 to S-phase transition through the cell cycle. Nothing has been reported in the current literature pertaining to CDK1 and CCND2 in cervical or gut epithelial barrier integrity, however, it has been well established that epithelial cell proliferation is necessary in order to maintain a strong and intact epithelial barrier. Therefore, it is easy to hypothesize that increased expression of miR-143 and miR-145 in the cervicovaginal space could significantly decrease epithelial cell proliferation contributing to the breakdown of the cervical epithelial barrier.
Due to the known effects of miR-143 and miR-145 on cell growth, proliferation and apoptosis, these miRNAs have been highly studied in the cancer field. Interestingly, in agreement with our results, miR-143 and miR-145 have been shown to be tumor suppressors/oncogenic factors in human cancers of epithelial origin including cervical, colon, gastric, breast and pancreatic carcinomas due to their effects on inhibiting cell growth and proliferation and activating apoptosis [45][46][47] . These findings have led to several research studies investigating the therapeutic potential for both miR-143 and miR-145 which have shown promise in their ability to stop tumor growth 48,49 . While overexpressing miR-143 and miR-145 has positive effects as a cancer therapy, in preterm birth, miR-143 and miR-145 would need to be inhibited in order to prevent premature breakdown of the cervical epithelial barrier and cervical remodeling. Nonetheless, these studies provide proof of principle that miR-143 and miR-145 could be potential biomarkers or therapy targets when administered directly into the cervical vaginal space.
When taken together, the results from this study show that increased expression of miR-143 and miR-145 in ectocervical and endocervical cells cause a breakdown in the epithelial barrier due to alterations in adherens junction proteins and a significant reduction in cell number due to cervical cell apoptosis and cell cycle arrest. As we know from our previous work that miR-143 and miR-145 expression is upregulated in cervical epithelial cells collected from women destined to have a preterm birth, we can conclude that it is biologically plausible that miR-143 and miR-145 contribute significantly to premature cervical remodeling due to a disruption in epithelial barrier integrity that ultimately leads to early delivery. This research study has identified multiple molecular pathways that are altered in the presence of increased miR-143 and miR-145. While miR-143 and miR-145 undoubtedly target hundreds of downstream genes and, consequently, have the widespread ability to effect many gene networks, it is clear that these miRNAs target pathways that contribute directly to cervical epithelial barrier integrity. Understanding the molecular pathways regulating cervical remodeling are critical to devising future therapies aimed at reducing the incidence of preterm birth.
Ectocervical and Endocervical Cell Transfection. Ectocervical cells were plated at 1.5 × 10 5 cells/well and endocervical cells were plated at 2 × 10 5 cells/well in 6-well plates in antibiotic-free K-SFM media. After 24 hrs, the cells were transfected with miRNA mimics (20 uM, final concentration of 40 nM). Hsa-miR-143-3p (miR-143), hsa-miR-145-5p (miR-145) and miR-negative (miR-neg, non-targeting control) miRNA mimics were purchased from Ambion (Applied Biosystems, Thermo-Fisher Scientific). Lipofectamine RNAiMAX (Invitrogen, Thermo-Fisher Scientific) was used for the transfection of the miRNA mimics according to the manufacturers' protocol. Cells were transfected for 48 to 72 hours and maintained under normal growth conditions. Efficiency of miR-143 and miR-145 transfection was verified by QPCR at the 72 hour time point (see Supplementary Fig. S1). Transfected cells were then used for cell permeability assays, immunohistochemistry, apoptosis or cell proliferation assays by flow cytometry or QPCR measurement of miRNAs or target genes.
Epithelial Cell Permeability Experiments. Endocervical and ectocervical cell permeability was determined using an In Vitro Vascular Permeability Assay (Millipore, Bedford, MA). Briefly, miR-neg, miR-143 and miR-145 transfected endocervical and ectocervical cells were plated at 1.0 × 10 6 cells/ml respectively into 24 well hanging cell culture inserts which contain 1 µm pores with a transparent polyethylene terephthlate (PET) membrane pre-coated with collagen. After 24, 48 and 72 hours of growth, an insert membrane from one representative well was stained and analyzed by brightfield imaging to assess monolayer integrity. At 72 hours, when a monolayer was completely formed, the media was removed and phenol red free K-SFM media (ScienCell hours prior to plating the ectocervical and endocervical cells. The cells were plated at 1.5 × 10 5 cells/ml onto the gelatin-coated slides. Ectocervical and endocervical cells were transfected with miR-neg, miR-143 and miR-145 for 48 hours. The cells were fixed with 10% buffered formalin for 30 minutes, washed with cold PBS and permeabilized using 0.25% triton-X for 10 minutes. The slides were blocked with 5% donkey serum for 30 minutes and incubated with primary antibody, human JAM-A polyclonal anti-goat (1:50, R&D systems, Minneapolis, MN) overnight at 4 °C. The next day, the slides were washed three times with cold PBS and incubated with secondary antibody, AlexaFluor 568 Donkey anti-goat (1:750, Life Technologies) for one hour at room temperature. After washing, the slides were stained with DAPI (1:500, Molecular Probes, Eugene, OR) for one hour. The slides were washed again, dehydrated and mounted with Krystalon (Harleco, Darmstadt, Germany). To validate the staining procedure, ectocervical and endocervical cells were stained for JAM-A in duplicate slides with each slide  Table 3. List of primary antibodies used for western blots.
containing a section incubated with and without (negative control) the primary antibody. Stained cells were photographed on a Zeiss LSM 710 confocal system set up on an AxioObserver inverted microscope.
Apoptosis Assay. Apoptosis in transfected ectocervical and endocervical cells was measured by flow cytometry using a FITC Annexin V/PI apoptosis detection kit (Invitrogen, Thermo-fisher Scientific) according to the manufacture's protocol. Ectocervical and endocervical cells transfected for 0, 72 and 144 hours were washed in cold sterile PBS, pelleted, counted (recorded for cell number determinations) and resuspended in 1X annexin-binding buffer at a concentration of 1 × 10 5 /100 ul. The cells were incubated for 15 minutes with FITC annexin V and propidium iodide (PI, 100 ug/ml) and then further diluted with 400 ul 1X annexin-binding buffer. The stained cells were then analyzed by a flow cytometer (Accuri C6, BD Biosciences, San Jose, CA) using a fluorescence emission at 530 nm (FL1) and >575 nm (FL3). Single color stains for FITC annexin and PI and unstained cells were included in all experiments as positive and negative controls.
After 24 hours, miTarget miRNA 3′UTR Target clones specific for BCL2, BIRC5, JAM-A, FSCN1, CDK1 or CCND2 inserted into a pEZX-MT05 vector (Genecopoeia, Rockville, MD) were transfected into the HEK293T cells using 3 ul of Lipofectamine 3000 (Invitrogen, Thermo-Fisher Scientific). The pEZX-MT05 vector contains a Gaussia luciferase (GLuc) reporter gene driven by an SV40 promoter and a secreted Alkaline Phosphatase (SEAP) reporter driven by a CMV promoter which serves as the internal control for transfection efficiency. Twenty four hours after transfection with the reporter plasmid, miR-neg, miR-143 or miR-145 were transfected into the cells using Lipofectamine RNAiMAX as described above. Twenty four hours later, media was collected for GLuc and SEAP activities using the Secrete-Pair Gaussia Luciferase Assay Kit (Genecopoeia). GLuc and SEAP activity was measured by a luminescent plate reader. EF1A-PG04 media (provided by Genecopoeia) was included in all GLuc measurements as a positive control for the assay. The results are expressed as a ratio of GLuc to SEAP and normalized to cells transfected with the target gene plasmid alone.