Expression of Pregnancy Specific ß-1 Glycoprotein 1 in Cervical Cancer Cells.

BACKGROUND: Cervical Cancer (CC) is a worldwide public health concern associated with genetic alterations, among these the gain of the 19q chromosome harboring the Pregnancy Specific Glycoproteins (PSG) gene family. These proteins play a critical role in pregnancy, with participation in immunotolerance, angiogenesis, and invasion processes, which are also observed in carcinogenesis. The aim of this study was to determine the molecular alterations of PSG1 and its relationship with CC. METHODS: PSG1 Copy Number Variation (CNV) was evaluated in 31 CC and eight normal cervical tissues by qPCR. PSG1 expression was correlated with HPV detection and IL-10 and TGF-ß expression in CC samples. Finally, PSG1 protein expression was evaluated by immunofluorescence in CC cell lines, by immunohistochemistry in a tissue microarray, and by immunoblotting in the sera of women with normal cervix, pre-invasive lesions, and CC. RESULTS: PSG1 showed a gain of 25.6% in CNV and gene expression in CC. There was a lack of PSG1 expression in normal cervical epithelium and positive immunostaining in 57% of CC tissues, while all CC cell lines expressed PSG1. Finally, PSG1 was immunodetected in 90% of pre-invasive lesions and in all CC serum samples, but not in healthy women. PSG1 expression correlates with the expression of IL-10 and TGF-ß in CC tissues, but not with the presence of HPV. CONCLUSION: These data show evidence of the differential expression of PSG1 in CC that could explain its participation in tumor-biology and immunotolerance mechanisms. Further, its immunodetection could provide early detection of this cancer.

Interferon epsilon mRNA expression could represent a potential molecular marker in cervical cancer.

The effects of the immune system response in the malignant transformation process have been described. Molecules such as interferons are involved in such process. Interferons are small single-chained glycoproteins, involved in the first line of defense against pathogens such as viruses, bacteria, and parasites. Interferon epsilon (IFNε) is located in the 9p21.3 cytogenetic region, transcribes into a single exon mRNA. Contrary to other family members, IFNε exerts low antiviral activity. In the present work molecular alterations such as copy number variation (CNV) and expression were analyzed by available microarrays and fifty-nine cervical tissues ranging from normal to cancer and three cell lines were assessed for IFNε expression by RT-PCR, immunohistochemistry, and immunocytofluorescence. No significant CNV alterations were observed. Positive immunosignal was primarily present in the proliferative basal strata cells in the normal tissue, whereas in cervical cancer, all epithelial transformed cells were positive. The cell lines analyzed were HPV16, -18, and negative, all three cell-lines were positive for cytoplasmic protein presence. Interestingly, at the mRNA level, increased band intensity was observed, as the lesions were higher, and IFNε up-regulation in CC (P=0.0001) is reported here. Our results suggest that up-regulation is present as an independent event from single or multiple HPV infection (P=0.90). In conclusion, we suggest that IFNε mRNA up-regulation could represent a potential molecular marker in CC. Expression of IFNε might not be related to HPV infection or CNV, which could have an important role in cellular homeostasis and could influence immune related events in cervical carcinogenesis.

Krüppel-like factor 5 as potential molecular marker in cervical cancer and the KLF family profile expression.

Cervical cancer (CC) as other cancer types, presents molecular deregulations, such as the alterations of transcription factors. Krüppel-like factors (KLF) are a family of transcriptional regulators. They are involved in diverse cellular processes, such as proliferation, apoptosis, and angiogenesis among others. Here, we analyzed the expression of all 17 KLF members at messenger RNA (mRNA) level, and protein expression of the two most commonly altered KLF5 and KLF6 in cervical tissues. Fifty-nine cervical tissues ranging from normal tissue to CC were evaluated for KLF1-17 mRNA expression by end-point RT-PCR and KLF5 by qRT-PCR. For KLF5 and KLF6 protein analysis, a tissue microarray was constructed containing the 59 cases and subjected for immunohistochemistry assay and KLF6 IVS1-27G>A single nucleotide polymorphism by direct DNA sequencing. KLF2-16 expressions were present in normal tissue, whereas all 17 were present in Low-Grade Squamous Intraepithelial Lesion, High-Grade-SIL and CC, unrelated to presence of human papillomavirus (HPV). KLF5 mRNA expression gradually increased throughout the subgroups and overexpressed in CC (p=0.01). KLF5 and KLF6 proteins were immunodetected in all samples. For the KLF6 SNP analysis, 80% of the CC population analyzed presented GG genotype and the remaining 20% presented GA genotype (p=0.491). Our present data show that KLFs expression could not be related to HPV presence, at least at transcriptional level, and KLF5 mRNA overexpression could represent a potential molecular marker for CC; KLF6 SNP has no relation to increased risk of CC.

Heterogeneity of microRNAs expression in cervical cancer cells: over-expression of miR-196a.

In recent years, the study of microRNAs associated with neoplastic processes has increased. Patterns of microRNA expression in different cell lines and different kinds of tumors have been identified; however, little is known about the alterations in regulatory pathways and genes involved in aberrant set of microRNAs. The identification of these altered microRNAs in several cervical cancer cells and potentially deregulated pathways involved constitute the principal goals of the present study. In the present work, the expression profiles of cellular microRNAs in Cervical Cancer tissues and cell lines were explored using microRNA microarray, Affymetrix. The most over-expressed was miR-196a, which was evaluated by real time PCR, and HOXC8 protein as potential target by immunohistochemistry assay. One hundred and twenty three human microRNAs differentially expressed in the cell tumor, 64 (52%) over-expressed and 59 (48%) under-expressed were observed. Among the microRNAs over-expressed, we focused on miR-196a; at present this microRNA is poorly studied in CC. The expression of this microRNA was evaluated by qRT-PCR, and HOXC8 by immunohistochemistry assay. There is not a specific microRNA expression profile in the CC cells, neither a microRNA related to HPV presence. Furthermore, the miR-196a was over-expressed, while an absence of HOXC8 expression was observed. We suggest that miR-196a could be played as oncomiR in CC.

Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions.

Trichomonosis, a chronic sexually transmitted disease, remains a public health problem affecting yearly over 170 million people worldwide. This disease is caused by Trichomonas vaginalis, a protozoan flagellate rich in cysteine proteinases (CPs). Although CPs are involved in trichomonal cytopathogenicity, only few of them have been defined as virulence factors. In this study, we characterize a T. vaginalis 39-kDa proteinase (CP39) found in vaginal secretions from patients with trichomonosis. The CP39 proteinase bound to HeLa epithelial cells, vaginal epithelial cells (VECs), and human prostatic cancer cells (DU-145). CP39 did not bind to a human colon cancer (CaCo) cell line, suggesting tissue-specific binding. CP39 was found in six fresh trichomonad isolates tested. In two-dimensional gels, CP39 appeared as a single spot with a pI 4.5. CP39 is inhibited by E-64, stable at 50 degrees C, and active in a wide pH range (3.6-9.0), with an optimum pH at 7.0. In addition, CP39 degraded collagens I, III, IV, and V, human fibronectin, human hemoglobin, and human immunoglobulins A and G. Indirect immunofluorescence detected CP39 on the parasite surface with specific polyclonal antibody to purified CP39. Finally, CP39 was found to be immunogenic, as evidenced by detection on immunoblots with serum of patients with trichomonosis, but not control individuals. These data suggest that CP39 may play a role during trichomonal infection.