MicroRNAs (miRNAs) play important roles in regulation of gene expression during cervical carcinogenesis. We investigated expression profiles of miRNAs in cervical cancer and its precursor lesions by utilizing cervical mucus. Cervical mucus was collected from 230 patients with a normal cervix, cervical intraepithelial neoplasia (CIN), squamous cell carcinoma (SCC), or adenocarcinoma (AD). The levels of miRNA in the mucus were quantified by miRNA array and real-time reverse-transcriptase polymerase chain reaction (RT-PCR). The performance for detecting diseases was statistically analysed. The expression of miRNAs was further validated in the surgical tissues of enrolled patients. Four miRNAs (miR-126-3p, -20b-5p, -451a, and -144-3p) were significantly up-regulated in SCC and AD compared with normal, and their expression levels correlated with disease severity and high-risk human papillomavirus infection. Receiver operating characteristic curve analyses revealed that the area under the curve values for miR-126-3p, -20b-5p, -451a, and -144-3p were 0.89, 0.90, 0.94, and 0.93, respectively, for SCC plus AD compared with normal, showing high accuracy of cancer detection. Real-time RT-PCR analyses confirmed the expression of these four miRNAs in frozen tissues from cervical cancer. miR-126-3p, -20b-5p, -451a, and -144-3p in cervical mucus are promising biomarkers for cervical cancer and high-grade CINs.

Human papillomavirus (HPV) infection can persist in the cervical epithelium without provoking a strong host immune response, leading to the development of cervical cancer. Cytokines, which mediate innate and adaptive immune activities, are secreted in the cervical mucus; however, there is currently no appropriate method for assessing cytokine levels in mucus specimens. Here, we employed multiplexed bead-based immunoassays to examine cytokine levels in cervical mucus using both weighted-volume and total protein concentration methods to adjust for different specimen volumes in individual patients. Out of 18 cytokines initially examined in the primary cohort patient group (n = 28), 14 were detected in more than 10% of the samples. Of these 14 cytokines, expression levels of interferon (IFN)-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), RANTES, and eotaxin were significantly increased with the disease severity in the secondary cohort patient group (n = 235). We also examined associations between cytokine levels and clinical parameters, such as cytology and HPV genotype. Of the 14 cytokines, granulocyte colony-stimulating factor (G-CSF) was downregulated in HPV-positive specimens. Examination of co-expression patterns of cytokines in relation to HPV infection status revealed that several pairs of cytokines were simultaneously upregulated in HPV-positive cases, including INF-γ and interleukin (IL)-17A, GM-CSF and monocyte chemoattractant protein-1 (MCP-1), GM-CSF and RANTES, IL-17A and RANTES, and MCP-1 and eotaxin. Interestingly, upregulation of GM-CSF and RANTES might reflect a shift in immuno-regulatory cytokines in HPV-positive specimens, potentially associated with more severe cervical neoplasia.

Persistent HPV infection associated with immune modulation may result in high-grade squamous intraepithelial lesions (CIN)2/3. Currently, there is little information on the cervicovaginal microbiome, local cytokine levels and HPV infection related to CIN. Follow-up of patients after local surgery provides an opportunity to monitor changes in the cervicovaginal environment. Accordingly, we undertook this longitudinal retrospective study to determine associations between HPV genotypes, cervicovaginal microbiome and local cytokine profiles in 41 Japanese patients with CIN. Cervicovaginal microbiota were identified using universal 16S rRNA gene (rDNA) bacterial primers for the V3/4 region by PCR of genomic DNA, followed by MiSeq sequencing. We found that Atopobium vaginae was significantly decreased (p < 0.047), whereas A. ureaplasma (p < 0.022) increased after surgery. Cytokine levels in cervical mucus were measured by multiplexed bead-based immunoassays, revealing that IL-1β (p < 0.006), TNF-α (p < 0.004), MIP-1α (p < 0.045) and eotaxin (p < 0.003) were significantly decreased after surgery. Notably, the level of eotaxin decreased in parallel with HPV clearance after surgery (p < 0.028). Thus, local surgery affected the cervicovaginal microbiome, status of HPV infection and immune response. Changes to the cervicovaginal microbiota and cervical cytokine profile following surgery for cervical intraepithelial neoplasia may be important for understanding the pathogenesis of CIN in future.

Nectins are cell adhesion molecules that play a pivotal role in adherens junctions and tight junctions. Our previous study using whole-genome oligonucleotide microarrays revealed that nectin-4 was upregulated in pre-eclamptic placentas. We investigated the role of nectin-4 in the etiology of pre-eclampsia. We investigated the expression of nectin-4 using real-time RT–PCR, western blot and immunostaining. Additionally, we performed matrigel invasion assay and cytotoxicity assay using cells overexpressing the nectin-4. NECTIN4 transcripts were elevated in pre-eclamptic placentas relative to uncomplicated pregnancies. Nectin-4 protein levels in pre-eclamptic placentas were higher on a semi-quantitative western blot. Nectin-4 was localized at the apical cell membrane in syncytiotrophoblast cells and not at the adherens junctions. Nectin-4 was also detected in cytotrophoblasts and a subset of cells in the decidua. Nectin-4 overexpressing trophoblast cells migrated normally in the matrix. However, Natural killer (NK) cells showed a strong cytotoxic effect against nectin-4 overexpressing trophoblast cells. No causative genetic variation was evident in the NECTIN4 gene from a pre-eclamptic placenta. There are as yet unknown factors that induce nectin-4 overexpression in trophoblast cells that may contribute to abnormal placentation via an aberrant immune response and the onset of a pre-eclamptic pregnancy.

A fluid-based Papanicolaou test has been established to improve sample collection and preparation. This study was the first large-scale investigation in Japan to examine the feasibility of using fluid-based Papanicolaou specimens to detect human papillomavirus (HPV) using Hybrid Capture II and polymerase chain reaction (PCR). Three thousand patients who visited Keio University Hospital between October 2000 and February 2001 were enrolled in the study. The results of the fluid-based Papanicolaou tests corresponded well with those of conventional Papanicolaou smears (96.8% concordance). The sensitivities of cervical neoplasia detection using the fluid-based Papanicolaou test (73.9%) and Hybrid Capture II (76.3%, P=0.55) were not significantly different. Among the cervical intraepithelial neoplasia 3 and squamous cell carcinoma specimens, HPV 16 and HPV 52 were predominantly detected using the PCR method. Although some DNA samples extracted from the fluid-based specimens were degradaded, PCR and direct sequencing could be performed without difficulty even after 1 year of specimen storage. We conclude that fluid-based Papanicolaou specimens can be applied to investigate HPV infection.

Nishio S, et al. J Gynecol Oncol. 2013 Jul;24(3):215-221. http://dx.doi.org/10.3802/jgo.2013.24.3.215