Endocervical Cells Research Articles

Interpretation of Endocervical Cells With Gastric-Type Mucin on Pap Smears.

Early detection of endocervical adenocarcinoma is especially important for cancers that are human papillomavirus (HPV) negative. We investigated the clinicopathologic significance of yellow gastric-type mucin observed on Papanicolaou smears. We described "atypical endocervical cells with gastric-type mucin" (AEC-GAM) when yellow mucin was observed in endocervical cells. We retrieved AEC-GAM samples from 58,752 cervical smears performed at Yamanashi University Hospital during our study period and reviewed clinical, cytologic, and pathologic features. We detected AEC-GAM in 172 (0.29 %) smears from 65 patients, and 41 of these 65 patients were histologically diagnosed with lobular endocervical glandular hyperplasia (LEGH) (43%) or pyloric gland metaplasia (20%). The prevalence of adenocarcinoma was 25% (7/28) in LEGH cases and 11% (7/65) in AEC-GAM smears. Yellow mucin is a diagnostic clue for endocervical glandular lesions with gastric differentiation. We recommend describing AEC-GAM on cytologic reports to improve cytologic screening for HPV-negative cervical cancers.

Endocervical and Neutrophil Lipoxygenases Coordinate Neutrophil Transepithelial Migration to Neisseria gonorrhoeae.

Infection with Neisseria gonorrhoeae (GC) is characterized by robust neutrophil influx that is insufficient to clear the bacteria. Sustained neutrophilic inflammation contributes to serious clinical sequelae that particularly affect women, including pelvic inflammatory disease and infertility. We established a 3-component system using GC, End1 polarized human endocervical cells, and primary human neutrophils to investigate neutrophil transepithelial migration following infection. Neutrophil migration across endocervical monolayers increased with the infectious dose and required GC-epithelial cell contact. Epithelial protein kinase C, cytosolic phospholipase A2, 12R-lipoxygenase (LOX), and eLOX3 hepoxilin synthase were required for neutrophil transmigration to GC, and migration was abrogated by blocking the MRP2 efflux pump and by adding recombinant soluble epoxide hydrolase. These results are all consistent with epithelial cell production of the neutrophil chemoattractant hepoxilin A3 (HXA3). Neutrophil transmigration was also accompanied by increasing apical concentrations of leukotriene B4 (LTB4). Neutrophil 5-lipoxygenase and active BLT1 receptor were required for apical LTB4 and neutrophil migration. Our data support a model in which GC-endocervical cell contact infection stimulates HXA3 production, driving neutrophil migration that is amplified by neutrophil-derived LTB4. Therapeutic targeting of these pathways could limit inflammation and deleterious clinical sequelae in women with gonorrhea.

EGFR-mediated matrix metalloproteinase-7 up-regulation promotes epithelial-mesenchymal transition via ERK1-AP1 axis during ovarian endometriosis progression.

Endometriosis, characterized by extrauterine development of endometrial glands and stroma, is associated with increased risk of ovarian cancer development. In the present study, we investigated the role of matrix metalloproteinase-7 (MMP-7) on epithelial-mesenchymal transition (EMT) during ovarian endometriosis ( N = 40) progression. We found that the expressions of EMT markers such as vimentin, slug, and N-cadherin were significantly elevated in late stages of ovarian endometriosis compared with those found in early stages. In addition, the activity and expression of ectopic MMP-7 were significantly higher in the late stages of endometriosis. In vitro studies revealed that increased expression of MMP-7 as well as epidermal growth factor (EGF), which was significantly elevated in severe stages of ovarian endometriosis, induced EMT in endocervical epithelial cells (End1/E6E7). Silencing the MMP-7 transcripts using small interfering RNA attenuated EMT responses, whereas treatment with recombinant active MMP-7 promoted EMT by cleaving E-cadherin. In addition, EGF receptor (EGFR) inhibitor treatments regressed endometriotic lesions and decreased MMP-7 activities in a mouse model of endometriosis. Chromatin immunoprecipitation assay identified EGFR-mediated ERK1 and activator protein 1 signaling for the transcriptional activation of MMP-7 in End1/E6E7 epithelial cells.-Chatterjee, K., Jana, S., DasMahapatra, P., Swarnakar, S. EGFR-mediated matrix metalloproteinase-7 up-regulation promotes epithelial-mesenchymal transition via ERK1-AP1 axis during ovarian endometriosis progression.

MAPK and NF-κB signalling pathways regulate the expression of miRNA, let-7f in human endocervical epithelial cells.

MicroRNAs (miRNAs) mediate post-transcriptional gene suppression and are a critical component of the complex regulatory networks in epithelial immune responses. Transcription of miRNA genes in epithelial cells can be elaborately controlled through Toll-like receptors (TLRs), and associated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, leading to nuclear transcription factor associated-transactivation and transrepression of miRNAs. MiRNA, let-7f is involved in the regulation of innate immune responses post TLR3 stimulation in human endocervical cells (End1/E6E7) and decreased let-7f is associated with poor immune activation. Thus, expression of let-7f is under strict control. However, the mechanism by which let-7f is regulated in these cells is not known. Therefore, in the present study, we have investigated the role of MAPK and NF-κB in the transcription of let-7f. We report that signalling of TLR3, results in activation of multiple signalling pathways including MAPK/ERK, JNK, p38, and NF-κB. Of these MAPK/ p38 and JNK directly influence the expression of let-7f in End1/E6E7 cells. Inhibition of ERK and NF-κB up regulates the expression of let-7f and its transcription factor, C/EBPβ. In conclusion, we have identified a system through which TLR3 mediated immune response is regulated by C/EBPβ and let-7f through the temporal activation of MAPK and NF-κB in human endocervical cells.

Effects of Nordy on the proliferation, differentiation, and apoptosis of HPV16 subgene-immortalized human endocervical cells*

Abstract Objective The aim of this study was to investigate the effects of Nordy on the proliferation, differentiation, and apoptosis of HPV16 subgene-immortalized human endocervical cells (H8 cells). Methods After treatment with Nordy, H8 cell proliferation was evaluated using the MTT assay. The effects of Nordy on the cell cycle and apoptosis of H8 cells were analyzed by flow cytometry (FCM) and the Annexin V-FITC method. H8 cell MCM5 expression was detected by immunocytochemistry. Morphological changes were observed by light and electron microscopy. Telomerase activity was evaluated by TRAP-ELISA. Results We found that 10 µmol/L-100 µmol/L Nordy significantly inhibited H8 cell proliferation. After treatment with Nordy, H8 cells were blocked in the G0/G1 phase, and the rate of cell apoptosis increased significantly. Cells differentiated toward innocuousness, and MCM5 expression and telomerase activity notably decreased. Conclusion Nordy was observed to inhibit proliferation and promote apoptosis in H8 cells. Nordy also induced H8 cell differentiation; this effect may have been achieved by blocking the cell cycle and decreasing telomerase activity.

Rehydration of Air-Dried Smears with Normal Saline: An Alternative for Conventional Wet Fixation Method in Cervical Cytological Study.

Context:Papanicolaou (Pap) smear is an effective exfoliative cytological investigation done for early recognition of cervical cancer. It also plays role in diagnosis of inflammatory lesions of cervix.Aims:The aim of this study is (1) to compare the cytomorphological features in conventional Pap smear (C-PAPS) and rehydrated air-dried Pap smear (RADPS) and (2) to evaluate the efficacy of RADPS in cytodiagnosis of cervical lesion by comparing with cytomorphological features of conventional wet-fixed Pap smear.Subjects and Methods:Paired cervical smears were prepared for 247 patients. One was labeled as C-PAPS and another was labeled as RADPS. Comparison of both smears was done for various cytomorphological parameters.Results:Out of 247 smears, 2.4% RADPS and 7.3% C-PAPS were reported as unsatisfactory. Red blood cell (RBC) background was present in 2% of RADPS and 42% of C-PAPS. Cytolysis and air-drying artifact were observed more in C-PAPS amounting to 2% and 4% in RADPS and 11% and 15% in C-PAPS. Cytoplasmic staining (97% RADPS vs. 94% C-PAPS) was superior in RADPS. Cell border, nuclear border, and chromatin of squamous and endocervical cells were better appreciated on RADPS compared to C-PAPS, and also statistically significant difference was observed.Conclusion:Rehydrated air-dried technique can be satisfactory alternative for conventional wet fixation method which can be followed routinely or in conjugation with C-PAPS, especially in cervical screening programs.

Key molecules in the GABA signalling pathway are present in mouse and human cervical tissue.

Cervical mucus modulates fertility by cyclical changes of its biophysical and functional properties. Based on an analogy with bronchial goblet cells we set out to investigate the possible role of the gamma-aminobutyric acid (GABA) signalling pathway in the mediation of oestrogen-induced mucus secretion from endocervical secretory cells. The aim of the study was to examine the existence of GABAA receptor (GABAAR), glutamic acid decarboxylase 65/67 (GAD65/67) and vesicular GABA transporter (VGAT) in human and mouse cervical tissue. The mouse cervical tissue expressed GabaAR mRNA transcripts throughout the oestrous cycle. GABAAR-positive immunolabelling was present in the superficial layer of the mouse cervico-vaginal epithelium in pro-oestrus. Human cervical tissue showed the presence of GABAAR, GAD67 and VGAT mRNA transcripts and clear immunofluorescent signals of all three molecules were detected in the endocervical secretory epithelium. The results of this study confirmed that elements of the GABA signalling pathway are present in the secretory epithelium of mouse and human cervical tissue and that GABA signalling pathway could be considered a possible mediator in oestrogen regulation of mucus secretion in the endocervical glands.

Utility of p16 Immunohistochemistry in Evaluating Negative Cervical Biopsies Following High-risk Pap Test Results.

The Lower Anogenital Squamous Terminology (LAST) Standardization Project for human papilloma virus (HPV)-associated lesions specifically recommends the use of p16 immunohistochemistry (IHC) as an adjunct to morphologic assessment of cervical biopsies interpreted as negative or low-grade squamous intraepithelial lesion (LSIL) from patients with prior high-risk Pap test results (high-grade squamous intraepithelial lesion [HSIL], atypical squamous cells cannot exclude HSIL, atypical glandular cells [AGC], or HPV16 atypical squamous cells of undetermined significance [ASC-US]). The impetus for this recommendation is to increase detection of missed high-grade disease. However, the quality of evidence supporting this recommendation was lower than that for the other LAST recommendations addressing improved consistency in the diagnosis of HSIL with the use of p16. A database search spanning 10 years identified 341 cases (encompassing 736 discrete biopsy specimens) interpreted as negative for dysplasia from 330 patients with a prior high-risk Pap result (atypical squamous cells cannot exclude HSIL, HSIL, atypical glandular cells, not otherwise specified [AGC-NOS], atypical endocervical cells--NOS [AEC-NOS], and AEC-favor neoplastic). p16 IHC was performed and detected missed abnormalities in 11/341 (3.2%) cases. The abnormalities corresponded to missed foci of HSIL (cervical intraepithelial neoplasia [CIN] 2) (n=1), SIL-indeterminate grade (n=7), atypical squamous metaplasia (n=2), and LSIL [CIN1]) (n=1). Subsequent histologic follow-up identified HSIL or greater in 6/8 (75%) p16 cases versus 20/79 (25.3%) p16 cases (P=0.0079). p16 IHC performed on biopsies interpreted as negative from patients with prior high-risk Pap test results increased the detection rate of missed SIL. A p16 result also significantly increased the likelihood of HSIL on subsequent biopsy. Although further studies are required to determine what percentage of missed HSIL justifies the additional cost, improved detection of HSIL in high-risk patients may lead to fewer diagnostic procedures and fewer patients lost to follow-up.

Host Defense Peptide Expression in Human Cervical Cells and Regulation by 1,25-Dihydroxyvitamin D3 in the Presence of Cytokines and Bacterial Endotoxin.

Host defense peptides (HDPs) in the pregnant female reproductive tract provide protection against infection. The relationship between HDPs and infection/inflammation is poorly understood. Therefore, we investigated the regulation of HDPs by 1α, 25-dihydroxyvitamin D3 (1,25-(OH)2) in the presence/absence of infectious/inflammatory agents. Endocervical epithelial cells (END1/E6E7, n = 6) were exposed to 1,25-(OH)2, calcipotriol, interleukin 1β (IL-1β), granulate-macrophage colony-stimulating factor (GM-GSF), and lipopolysaccharide (LPS). Elafin, human beta defensin (hBD2), cathelicidin, secretory leucocyte protease inhibitor, interleukin 8, 1,25-(OH)2 receptor, and toll-like receptor 4 (TLR4) expression was determined using quantitative polymerase chain reaction and/or enzyme-linked immunosorbent assay. Host defense peptide gene and protein expression was assessed in cervicovaginal cells/fluid, respectively, from first trimester pregnant women (n = 8-12). Interleukin 1β induced elafin and hBD2. The 1,25-(OH)2 induced cathelicidin expression in the presence of IL-1β and LPS. The 1,25-(OH)2 also attenuated IL-1β-induced IL-8 expression and LPS enhancement of TLR4. Host defense peptides and TLR4 profiles in cervicovaginal cells and fluid samples from pregnant women were similar to END1/E6E7 cells. In conclusion, HDPs are differentially regulated in END1/E6E7 cells. The 1,25-(OH)2 induction of cathelicidin and suppression of IL-8 highlights a mechanism by which 1,25-(OH)2 supplementation could enhance the pregnant innate immune defenses.

Chlamydia trachomatis antimicrobial susceptibility in colorectal and endocervical cells.

Rectal Chlamydia trachomatis infections represent one of the most common sexually transmitted infections in the MSM population. Although current treatment guidelines suggest the use of either azithromycin or doxycycline, several clinical studies reported on azithromycin treatment failures in the case of rectal C. trachomatis localizations. In this context, the biological reasons behind the lack of azithromycin efficacy for C. trachomatis infections at the rectal level are still poorly understood. To evaluate the in vitro antimicrobial susceptibility of several C. trachomatis strains in two different cell lines, mimicking the urogenital localization and the rectal site of infection. The susceptibility to macrolides (i.e. azithromycin and erythromycin), doxycycline and levofloxacin was assessed for 20 C. trachomatis strains, belonging to the most frequently reported genovars (D, E, F and G), both in human endocervical cells (HeLa cells) and in colorectal cells (Caco-2 cells). Moreover, a correlation between MIC values and C. trachomatis bacterial load was investigated in both cell lines. For all the C. trachomatis strains, regardless of the genovar, macrolides showed higher MIC and MBC values (2-fold dilutions) in Caco-2 cells compared with HeLa cells, whereas for doxycycline and levofloxacin, no significant differences were found between the two cell lines. Moreover, azithromycin MICs were significantly higher with increasing levels of C. trachomatis elementary bodies on Caco-2 cells. The higher azithromycin MICs observed in colorectal cells, together with the positive correlation between MICs and C. trachomatis loads found, could explain azithromycin treatment failure for C. trachomatis infections at the rectal site.

Transcription factor CCAAT/enhancer-binding protein-β upregulates microRNA, let-7f-1 in human endocervical cells.

In endocervical epithelial cells (End1/E6E7), miRNA let-7f plays an important role in the control of innate immunity. The underlying molecular mechanism for let-7f regulation in these cells remains largely unclear. let-7f was knocked down in End1/E6E7 cells using siRNA, and differential gene expression was analyzed by microarray. Differentially expressed genes were validated by qPCR and Western blot. Expression of let-7f was studied by qPCR after inhibition of C/EBPβ with betulinic acid (BA) and pCMVβ plasmid and after overexpression of C/EBPβ with pCMVβ+ plasmid. ChIP assay was performed to confirm binding of C/EBPβ to let-7f promoter. Levels of Lin28A/B were checked by qPCR after similar treatment. let-7f knockdown (KD) affects the expression of many transcription factors (eg, C/EBPβ) which are important regulators of immune responses. We observed let-7f-1 promoter to contain 6 C/EBPβ binding sites. KD of C/EBPβ led to decreased let-7f expression while overexpression of C/EBPβ increased its expression. Treatment of End1/E6E7 cells with TLR-3 ligand, poly(I:C) increased binding of C/EBPβ at binding sites 3, 5, and 6. Expression of Lin28A/B also changed upon inhibition and overexpression of C/EBPβ. Its expression is opposite to that of let-7f in End1/E6E7 cells. let-7f-1 is a direct target of transcription factor, C/EBPβ in End1/E6E7 cells.

Cytologic parameters predicting neoplasia in Papanicolaou smears with atypical glandular cells and histologic follow-up: a single-institution experience

Studies investigating histologic follow-up of The 2001 Bethesda System diagnosis of atypical glandular cells (AGC) have focused on various screening methods, patient populations, and Papanicolaou preparations. Our aim was to report the histologic follow-up of AGC diagnoses from ThinPrep slides and evaluate specific cytologic features predicting benign or malignant follow-up results. A retrospective search identified liquid-based cervical cytology results interpreted as AGC. AGC diagnoses were stratified into four groups: atypical endometrial cells (AGC-EM); atypical endocervical cells (AGC-EC); AGC, favor neoplastic (AGC-FN); and AGC not otherwise specified (AGC-NOS). Evidence of disease was based on histologic follow-up (biopsy or resection specimen) with a diagnosis of cancer, complex endometrial hyperplasia, or high-grade squamous dysplasia. Available slides were blindly reviewed for specific cytologic features. Statistical analysis compared cytologic factors that would predict benign or malignant follow-up. We interpreted 264 samples as AGC from 2005 through 2009. Of the 246 (93.2%) with follow-up histologic material, 60 (24.4%) were AGC-EM; 36 (14.6%) were AGC-EC; 28 (11.4%) were AGC-FN; and 122 (49.6%) were AGC-NOS. Neoplasia was diagnosed in 80 (32.5%). Neoplastic cases showed significantly increased numbers of single cells, cells in 3-dimensional clusters, engulfed neutrophils, nuclear enlargement, increased nuclear-to-cytoplasmic ratio, irregular nuclear borders, reniform nuclei, loss of polarity, and macronucleoli. Cytologic parameters can be used to predict benign from neoplastic glandular lesions. Biopsy follow-up is necessary to correlate cytologic findings when AGC is diagnosed on a Papanicolaou smear.

Clinicopathological significance of atypical glandular cells on cervicovaginal Pap smears

To evaluate the histologic correlation and clinical significance of atypical glandular cells (AGC) detected on Pap tests. Pap tests interpreted as AGC were retrieved from an institutional cytopathology database. The AGC subcategories according to the Bethesda system 2001 include atypical glandular cells, not otherwise specified (AGC-NOS), atypical endometrial cells (AEM), and atypical endocervical cells (AEC). Clinicopathologic variables were collected from medical records retrospectively, and histologic follow-up diagnoses were correlated with AGC subcategories. From January 2009 to December 2014, 741 (0.17%) of 435778 cervical smears were interpreted as AGC. Of 713 cases included in the study analysis, the distribution of AGC subcategories was as follows: AGC 15.3%, AEM 59.6%, and AEC 25.1%. Of 508 cases with histologic follow-up results, 33.9% of cases were diagnosed to have clinically significant lesions, which include cervical squamous or glandular neoplasia (1.4% and 10.0%), endometrial neoplasia (21.5%), and other metastatic carcinoma (1.0%). Endometrial neoplasia was the most common significant pathology in women aged ≥40 years (28.4%), whereas cervical glandular neoplasia was the most commonly diagnosed lesion in women aged <40 years (14.5%, P < 0.001). When considering past histories which could affect the Pap results, IUD use (10.3%) and pregnancy (7.3%) were most frequently documented. Although IUD users showed low prevalence of significant pathology (2.6%), AGC cases related with pregnancy preceded significant pathologic lesions in 14.3%. The relatively high incidence of significant neoplasia linked to AGC emphasizes the importance of adherence to the AGC management guidelines which recommend early and intensive investigation using multiple testing modalities.

High Maternal Serum Interleukin-8 and Endocervical Neutrophil Cell Count as Risk Factors for Preterm Labor

High Maternal Serum Interleukin-8 and Endocervical Neutrophil Cell Count as Risk Factors for Preterm Labor

Abstract 5752: Susceptibility of cells from cervical transformation zone to HPV-16 induced immortalization and dysplastic differentiation

Abstract Persistent infection of high-risk human papillomavirus (HPV) is a major risk factor for cervical cancer. Greater than 90% of these cancers originate in the cervical transformation zone (CTZ), a narrow band of squamous metaplastic epithelium between ectocervix and endocervix. It is unclear why the CTZ has high susceptibility to malignant transformation and few studies have specifically examined cells from this region. We hypothesized cells cultured from human CTZ are uniquely susceptible to cellular immortalization and dysplastic differentiation, two alterations that contribute to malignant development. Primary epithelial cells were cultured from each region of human cervix (ectocervix, endocervix and CTZ) and characterized for expression of differentiation markers to assess their origin. We examined susceptibility of cells from each region to immortalization after transfection with the complete HPV-16 genome or infection of HPV-16 E6/E7 retroviruses. In addition, we evaluated the extent of dysplastic epithelial differentiation in organotypic cultures of HPV16-immortalized cell lines derived from each region of cervix. Our results showed that cells from the CTZ express specific keratin markers (keratin 14 and 18) that confirm their origin. Primary cell cultures from CTZ and ectocervix are more susceptible to immortalization by the HPV16 complete genome or E6/E7 retroviruses. In contrast, preliminary results indicate that HPV-immortalized CTZ and endocervical cells are more susceptible to dysplastic differentiation when maintained in organotypic culture. Our results may help to identify important signal pathways or biomarkers that are unique to cells from CTZ and that can be targeted for improved screening, prevention or therapy. Citation Format: Han Deng, Eric Hillpot, Philomina Yeboah, Sumona Mondal, Craig D. Woodworth. Susceptibility of cells from cervical transformation zone to HPV-16 induced immortalization and dysplastic differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5752. doi:10.1158/1538-7445.AM2017-5752

Abstract 1292: Germline genetic, molecular and environmental factors modulate APOBEC mutagenesis in human tumors

Abstract Background: Activity of APOBEC3 enzymes has been identified as a main source of somatic mutations of a specific type (C to T or G substitutions in the TCA or TCT motifs). This mutation pattern, known as the APOBEC signature, has been described in 16 of 30 tumor types explored by TCGA, with bladder cancer being one of top cancers enriched with APOBEC-signature mutations. Recently, we demonstrated that a risk allele of a bladder cancer GWAS SNP rs1014971 was significantly associated with increased APOBEC3B (A3B) mRNA expression and APOBEC mutagenesis in bladder tumors. Here, we aimed to further explore factors that can modulate APOBEC mutagenesis in human tumors. Methods: Isoform-specific expression analysis of all APOBEC3 genes (A3A, A3B, A3C, A3D, A3G, A3F, and A3H) was performed in TCGA datasets for seven tumor types enriched for APOBEC mutations (&amp;gt; 25% of the samples) - bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical and endocervical cancers (CESC), head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and ovarian serous cystadenocarcinoma (OV). Expression and APOBEC mutagenesis pattern were analyzed by multivariate linear regression in relation to rs1014971 genotypes and adjusting for age, sex, race, DNA CpG methylation, copy number alterations (CNA), and human papilloma virus (HPV) infection status (where relevant). Survival analysis was performed using multivariate Cox regression models that included age, sex, tumor stage, rs1014971, and load of APOBEC mutations or mRNA expression of APOBEC3 splicing forms. Results: Analysis of APOBEC mutagenesis identified significant interplay between SNP rs1014971, expression of specific APOBEC3 splicing isoforms, and environmental factors (such as HPV infection). For example, SNP was important in BLCA and sub-types of HNSC, while HPV infection status was the main factor associated with APOBEC mutagenesis in CESC and HNSC. Importantly, variable contribution of mutagenic and non-mutagenic isoforms of A3A and A3B, which are generated by alternative splicing, emerged as significant factors contributing to APOBEC mutagenesis across human tumors. Survival of the cancer patients was also differentially influenced by these factors. Conclusions: These findings suggest that APOBEC mutagenesis pattern is tissue-specifically affected by germline genetic, molecular and environmental factors. Deeper understanding of these factors and their interplay may pave the way for preventive and treatment measures of human cancers towards precision medicine. Citation Format: A. Rouf Banday, Ariunaa Bayanjargal, Krizia-Ivana Udquim, Olusegun O Onabajo, Ludmila Prokunina-Olsson. Germline genetic, molecular and environmental factors modulate APOBEC mutagenesis in human tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1292. doi:10.1158/1538-7445.AM2017-1292

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.

MiR-205 serves as a prognostic factor and suppresses proliferation and invasion by targeting insulin-like growth factor receptor 1 in human cervical cancer.

MicroRNAs are a kind of small and non-coding RNAs, which have been demonstrated to play an important role in the progression of human cervical cancer. Here, we found that the expression of miR-205 was low in cervical cancer cell lines and tissues, compared with matched non-tumor tissues and human endocervical epithelial cells. Also, miR-205 was inversely correlated with histological differentiation, metastasis, International Federation of Gynecology and Obstetrics stage, and the expression of insulin-like growth factor receptor 1 messenger RNA and protein. Besides, miR-205 or insulin-like growth factor receptor 1 expression is an independent prognostic factor. Mechanically, ectopic expression of miR-205 decreased proliferation, colony formation, and some proliferation/apoptosis-related proteins in cervical cancer cells. Ectopic expression of miR-205 caused G1 arrest. Luciferase reporter assays confirmed that binding of miR-205 to the 3' untranslated region of insulin-like growth factor receptor 1 may potentially decrease the expression of insulin-like growth factor receptor 1. Notably, insulin-like growth factor receptor 1 overexpression attenuated the inhibitory effects of miR-205 on cell proliferation and invasion, while small interfering RNA-insulin-like growth factor receptor 1 enhanced the inhibitory effects of miR-205 on cell proliferation and invasion. In conclusion, our findings suggested that miR-205 serves as a prognostic factor and suppresses proliferation and invasion by targeting insulin-like growth factor receptor 1 in human cervical cancer. Thus, miR-205/insulin-like growth factor receptor 1 pathway may be of great benefit to cervical cancer patients.

Is nitric oxide an essential mediator in cervical inflammation and preterm birth?

Objective: Cervical ripening is an obligatory step in the process of preterm birth. We hypothesize an inflammatory challenge to the cervix, which leads to an increase in nitric oxide production, disrupting the cervical epithelial barrier leading to preterm birth.Study design: For this study, three experiments were performed: (i) Using a mouse model, pregnant mice were treated with an intrauterine injection of saline or lipopolysaccharide (LPS). Mice were sacrificed and cervices were collected for molecular analysis. (ii) Immortalized ectocervical and endocervical cells were treated with either LPS or the nitric oxide donor sodium nitroprusside (SNP). Media and RNA was collected for analysis. (iii) The integrity of the epithelial cell barrier was evaluated using an in vitro permeability assay.Results: The expression of inducible nitric oxide synthase (iNOS) was increased in our mouse model with LPS (p < .005). In vitro, LPS did not increase nitrate or nitrite concentrations or mRNA expression of iNOS. Permeability increased in the presence of LPS (p < .01), but was unchanged after treatment with SNP.Conclusions: These studies show that LPS increases the expression of the iNOS in an animal model of preterm birth, but the nitric oxide metabolites nitrate and nitrite do not initiate the pro-inflammatory LPS-induced breakdown of the cervical epithelial barrier.

Neisseria gonorrhoeae infects the human endocervix by activating non-muscle myosin II-mediated epithelial exfoliation.

Colonization and disruption of the epithelium is a major infection mechanism of mucosal pathogens. The epithelium counteracts infection by exfoliating damaged cells while maintaining the mucosal barrier function. The sexually transmitted bacterium Neisseria gonorrhoeae (GC) infects the female reproductive tract primarily from the endocervix, causing gonorrhea. However, the mechanism by which GC overcome the mucosal barrier remains elusive. Using a new human tissue model, we demonstrate that GC can penetrate into the human endocervix by inducing the exfoliation of columnar epithelial cells. We found that GC colonization causes endocervical epithelial cells to shed. The shedding results from the disassembly of the apical junctions that seal the epithelial barrier. Apical junction disruption and epithelial exfoliation increase GC penetration into the endocervical epithelium without reducing bacterial adherence to and invasion into epithelial cells. Both epithelial exfoliation and junction disruption require the activation and accumulation of non-muscle myosin II (NMII) at the apical surface and GC adherent sites. GC inoculation activates NMII by elevating the levels of the cytoplasmic Ca2+ and NMII regulatory light chain phosphorylation. Piliation of GC promotes, but the expression of a GC opacity-associated protein variant, OpaH that binds to the host surface proteins CEACAMs, inhibits GC-induced NMII activation and reorganization and Ca2+ flux. The inhibitory effects of OpaH lead to reductions in junction disruption, epithelial exfoliation, and GC penetration. Therefore, GC phase variation can modulate infection in the human endocervix by manipulating the activity of NMII and epithelial exfoliation.