Cervical Epithelium Research Articles

Histone Lactylation-Driven GPD2 Mediates M2 Macrophage Polarization to Promote Malignant Transformation of Cervical Cancer Progression.

Cervical cancer (CC) is the most common cancer in women. This study aims to explore the molecular mechanism of lactate secreted by CC cells modulating macrophage polarization in CC via histone lactylation. Normal cervical epithelium (NCE), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), and cervical squamous cell carcinoma (CESC) were collected to assess H3K18la level and macrophage infiltration. Macrophages were incubated with SiHa cell-derived conditioned medium to detect M1 and M2 markers. NCE, HSIL, and CESC samples were used for ChIP-seq of H3K18la. Histone lactylation-dirven GPD2 was knocked down in macrophages. Compared to NCE, H3K18la level and M2 macrophage abundance were increased in LSIL, HSIL, and CESC. Lactate secreted by CC cells upregulated H3K18la and M2 markers but downregulated M1 markers in macrophages. ChIP-seq revealed that upregulated pathways in HSIL vs. NCE and CESC vs. HSIL were commonly enriched in lipid metabolism. Notably, lactate upregulated H3K18la-modified GPD2 expression in macrophages, and GPD2 knockdown reversed lactate induction to M2 macrophages. Collectively, lactate secreted by CC cells upregulates GPD2 via histone lactylation, thereby promoting M2 macrophage polarization in CC. This study provides new insights into the role of histone lactylation in macrophage polarization in the malignant transformation of CC.

Application of mass cytometry in multiparametric characterization of precancerous cervical lesions.

Cervical cancer (CC) is the fourth most common malignant tumor in women worldwide. Detecting different biomarkers together on single cells by novel method mass cytometry could contribute to more precise screening. Liquid-based cytology (LBC) cervical samples were collected (N = 53) from women categorized as normal and precancerous lesions. Human papillomavirus was genotyped by polymerase chain reaction, while simultaneous examination of the expression of 29 proteins was done by mass cytometry (CyTOF). Differences in cluster abundances were assessed with Spearman's rank correlation as well as high dimensional data analysis (t-SNE, FlowSOM). Cytokeratin (ITGA6, Ck5, Ck10/13, Ck14, Ck7) expression patterns allowed determining the presence of different cells in the cervical epithelium. FlowSOM analysis enabled to phenotype cervical cells in five different metaclusters and find new markers that could be important in CC screening. The markers Ck18, Ck18, and CD63 (Metacluster 3) showed significantly increasing associated with severity of the precancerous lesions (Spearman rank correlation rho 0.304, p = 0.0271), while CD71, KLF4, LRIG1, E-cadherin, Nanog and p53 (Metacluster 1) decreased with severity of the precancerous lesions (Spearman rank correlation rho -0.401, p = 0.0029). Other metaclusters did not show significant correlation, but metacluster 2 (Ck17, MCM, MMP7, CD29, E-cadherin, Nanog, p53) showed higher abundance in low- and high-grade intraepithelial lesion cases. CyTOF appears feasible and should be considered when examining novel biomarkers on cervical LBC samples. This study enabled us to characterize different cells in the cervical epithelium and find markers and populations that could distinguish precancerous lesions.

Adenoid basal carcinoma cervix – A rare epithelial neoplasm

ABSTRACT Adenoid basal carcinoma of the uterine cervix is a rare epithelial neoplasm. Adenoid basal carcinoma constitutes less than 1% of all cervical adenocarcinomas and has a favorable prognosis. Adenoid basal carcinoma is assumed to arise from a multipotential basal or reserve cell layer in the cervical epithelium. We report a case of adenoid basal carcinoma of the uterine cervix with complaints of bleeding per vagina in a 40-year-old female. The carbohydrate antigen 125 level was elevated (100 U/ml), and the MRI abdomen and pelvis revealed increased signal intensity in the anterior lip of the cervix. The local examination revealed thickening of the anterior lip of the cervix and induration in the left fornix. The patient was diagnosed with carcinoma cervix stage IB2 (FIGO Stage). The patient was operated by a radical robotic hysterectomy. Microscopic examination revealed small basaloid tumor cell nests with peripheral cell palisading and microcyst formation. The tumor cells showed p63 immunoreactivity in basaloid tumor cells and were negative for CD117 and S-100P. This tumor should be differentiated from adenoid basal cell hyperplasia, which has a favorable outcome, and adenoid cystic carcinoma, with a dismal course.

Prevalence, Clinical Features, and Management of Cervical Polyps: A One-Year Study at Rabia Balkhi Hospital in Afghanistan.

Background Cervical polyps are benign growths from the cervical epithelium, found in 2% to 5% of the population. They usually appear as small, stalk-attached lesions ranging from pea to cherry size. Contributing factors include cervical infections, congestion, and elevated estrogen levels. During pregnancy, these polyps may increase the risk of late abortion and preterm birth. While malignancy is rare, histopathological examination after removal is advised to rule out cancer. Materials and methods This current study, the first of its kind in Afghanistan, aimed to assess the scope of cervical polyps among Afghan women presenting to Rabia Balkhi National Hospital in 2018. We conducted a descriptive study of cervical polyps, exploring the prevalence, symptoms, complications, and procedures used to remove them. Data were collected from 3730 cases admitted to the hospital. Results Out of 3730 cases, 50 patients (1.34%) were diagnosed with cervical polyps. The polyps varied in size and were typically attached to the cervix by a slender stalk. Symptoms reported included inter-menstrual, post-coital, and post-menopausal bleeding. Histopathological examinations were performed following polyp removal to rule out malignancy. Conclusion This study provides a comprehensive overview of the prevalence and clinical presentation of cervical polyps among Afghan women. The findings highlight the importance of recognizing and managing cervical polyps to prevent potential complications, especially during pregnancy. Further research is needed to understand this population's epidemiology, pathogenesis, and optimal management strategies for cervical polyps.

Validation and functional follow-up of cervical cancer risk variants at the HLA locus.

Cervical cancer is the fourth most common cancer in females. Genome-wide association studies (GWASs) have proposed cervical cancer susceptibility variants at the HLA locus on chromosome 6p21. To corroborate these findings and investigate their functional impact in cervical tissues and cell lines, we genotyped nine variants from cervical cancer GWASs (rs17190106, rs535777, rs1056429, rs2763979, rs143954678, rs113937848, rs3117027, rs3130214, and rs9477610) in a German hospital-based series of 1122 invasive cervical cancers, 1408 dysplasias, and 1196 healthy controls. rs17190106, rs1056429 and rs143954678/rs113937848 associated with cervical malignancies overall, while rs17190106 and rs535777 associated specifically with invasive cancer (OR = 0.69, 95% CI = 0.55-0.86, p = 0.001) or adenocarcinomas (OR = 1.63, 95%CI = 1.17-2.27, p = 0.004), respectively. We tested these and one previously genotyped GWAS variant, rs9272117, for potential eQTL effects on 36 gene transcripts at the HLA locus in 280 cervical epithelial tissues. The strongest eQTL pairs were rs9272117 and HLA-DRB6 (p = 1.9x10E-5), rs1056429 and HLA-DRB5 (p = 2.5x10E-4), and rs535777 and HLA-DRB1 (p = 2.7x10E-4). We also identified transcripts that were specifically upregulated (DDX39B, HCP5, HLA-B, LTB, NFKBIL1) or downregulated (HLA-C, HLA-DPB2) in HPV+ or HPV16+ samples. In comparison, treating cervical epithelial cells with proinflammatory cytokine γ-IFN led to a dose-dependent induction of HCP5, HLA-B, HLA-C, HLA-DQB1, HLA-DRB1, HLA-DRB6, and repression of HSPA1L. Taken together, these results identify relevant genes from both the MHC class I and II regions that are inflammation-responsive in cervical epithelium and associate with HPV (HCP5, HLA-B, HLA-C) and/or with genomic cervical cancer risk variants (HLA-DRB1, HLA-DRB6). They may thus constitute important contributors to the immune escape of precancerous cells after HPV-infection.

LINE-1 ORF1p is a Promising Biomarker in Cervical Intraepithelial Neoplasia Degree Assessment.

Cervical intraepithelial neoplasia (CIN) represents a spectrum of preinvasive squamous lesions within the cervical epithelium, whose identification is a diagnostic challenge due to subtle histomorphological differences among its categories. This study explores ORF1p, a nucleic acid-binding protein derived from long interspersed nuclear element-1 (LINE-1), as a potential biomarker for enhancing CIN diagnosis. A comprehensive analysis of 143 cervical specimens, encompassing CIN I (n=20), CIN II (n=46), CIN III (n=14), invasive cancer (n=32), and nondysplastic cases (normal cervical epithelia (n=24) and atrophy (n=7) were conducted. ORF1p, Ki67, and p16 expressions were evaluated using immunohistochemistry. ORF1p immunopositivity was detected in the vast majority [110/112 (98.2%)] of dysplastic and neoplastic (CIN and invasive cancer) specimens, whereas 19/24 (79.2%) of normal cervical specimens lacked ORF1p expression. The observed pattern of ORF1p expression showed a progressively increasing extent and intensity with advancing CIN grades. CIN I exhibited mild ORF1p expression in the lower one or two-thirds of the cervical epithelium [14/16 (87.5%)], whereas CIN II demonstrated moderate to strong ORF1p expression spanning the lower two-thirds [29/46 (63.0%)]. Pronounced transepithelial ORF1p immunopositivity characterized CIN III cases [13/14 (92.8%)] and cervical cancer [30/32 (93.8%)]. These findings propose ORF1p as a valuable indicator even for detecting CIN I, effectively discerning them from normal cervical tissue (p < 0.0001). Our findings underscore the potential of ORF1p as an early diagnostic marker for cervical neoplasia.

Exploring molecular targets: herbal isolates in cervical cancer therapy

ObjectiveCervical cancer (CxCa) stands as a significant global health challenge, ranking fourth in cancer-related mortality among the female population. While chemotherapy regimens have demonstrated incremental progress in extending overall survival, the outlook for recurrent CxCa patients remains disheartening. An imperative necessity arises to delve into innovative therapeutic avenues, with molecular targeted therapy emerging as a promising candidate. Previous investigations have shed light on the therapeutic effectiveness of five distinct herbal compounds, epicatechin, curcumin, myricetin, jatrorrhizine, and arborinine, within the context of CxCa.MethodsA systems biology approach was employed to discern differentially expressed genes (DEGs) in CxCa tissues relative to healthy cervical epithelial tissues. A protein–protein interaction network (PPIN) was constructed, anchored in the genes related to CxCa. The central genes were discerned within the PPIN, and Kaplan–Meier survival curves explored their prognostic significance. An assessment of the binding affinity of the selected herbal compounds to the master regulator of prognostic markers in CxCa was conducted.ResultsA significant correlation between the overexpression of MYC, IL6, JUN, RRM2, and VEGFA and an adverse prognosis in CxCa was indicated. The regulation of these markers is notably influenced by the transcription factor CEBPD. Molecular docking analysis indicated that the binding affinity between myricetin and the CEBPD DNA binding site was robust.ConclusionThe findings presented herein have unveiled pivotal genes and pathways that play a central role in the malignant transformation of CxCa. CEBPD has emerged as a potential target for harnessing the therapeutic potential of myricetin in this context.

Identification and Functional Implications of the E5 Oncogene Polymorphisms of Human Papillomavirus Type 16.

The persistence of the human papillomavirus type 16 (HPV16) infection on the cervical epithelium contributes to the progression of cervical cancer. Studies have demonstrated that HPV16 genetic variants may be associated with different risks of developing cervical cancer. However, the E5 oncoprotein of HPV16, which is related to several cellular mechanisms in the initial phases of the infection and thus contributes to carcinogenesis, is still little studied. Here we investigate the HPV16 E5 oncogene variants to assess the effects of different mutations on the biological function of the E5 protein. We detected and analyzed the HPV16 E5 oncogene polymorphisms and their phylogenetic relationships. After that, we proposed a tertiary structure analysis of the protein variants, preferential codon usage, and functional activity of the HPV16 E5 protein. Intra-type variants were grouped in the lineages A and D using in silico analysis. The mutations in E5 were located in the T-cell epitopes region. We therefore analyzed the interference of the HPV16 E5 protein in the NF-kB pathway. Our results showed that the variants HPV16E5_49PE and HPV16E5_85PE did not increase the potential of the pathway activation capacity. This study provides additional knowledge about the mechanisms of dispersion of the HPV16 E5 variants, providing evidence that these variants may be relevant to the modulation of the NF-κB signaling pathway.

Cervical dysplasia, cervicitis and inflammatory bowel disease – is there a relationship? (Literature review)

The state of health of the female population of Ukraine is of particular concern due to the year-by-year increase in the frequency of precancerous and oncological diseases of the reproductive organs. Benign and precancerous pathologies of the cervix occupy an important place among gynecological diseases.The objective of this article is to review foreign literature and domestic data sources for assessment the risk of dysplasia and cervical cancer in patients with inflammatory bowel disease (IBD) and cervicitis.Early onset of sexual life and a large number of sexual partners are factors that increase the risk of contracting the human papillomavirus (HPV) and other sexually transmitted infections (STIs). Trauma to the cervical epithelium – mechanical one or caused by an STI – can cause HPV to enter the basal layer of the cervical epithelium. Cervical cancer (CC) is preceded by HPV lesions of the cervical epithelium, including low-grade and high-grade squamous intraepithelial lesions, which are usually diagnosed in women of reproductive age.Cervical intraepithelial neoplasia (CIN) is characterized by impaired maturation and differentiation of cells of the multilayered squamous epithelium and is the most frequent form of morphological precancer of the cervix. Up to 90% of CIN are localized at the border of single-layered cylindrical and multilayered flat epithelium (transformation zone). CIN of various degrees is a precancerous disease in which the index of malignancy (oncological transformation) can reach 50%.The clinical course of dysplastic changes in CIN is asymptomatic, and the patients have no specific complaints. During the infectious process in the vagina, CIN can be accompanied by an increase in the amount of vaginal discharges in the form of mucus or milky secretions. Therefore, in modern conditions timely diagnosis, effective treatment and prevention of precancerous processes, which include CIN of the cervix, is one of the important tasks of a gynecologist. Women of perimenopausal age, who are traditionally classified as a risk group, deserve special attention.Today, there is information in the literature that diseases of a multifactorial nature make up 90–95% of all diseases, which also includes CIN. Despite extensive research efforts and advances in screening and prevention strategies, CC remains an unsolved public health challenge, particularly in low- and middle-income countries. Every year in the world, more than half a million women are diagnosed with CC.The world scientific community pays great attention to the study of epidemiology, pathogenesis, treatment and development of methods of prevention of neoplastic processes in general and CC in particular. So far, this has not been thoroughly studied, and there are no large studies that take into account a variety of potential factors. Understanding the risk factors for the development of CC in patients with IBD and cervicitis may help develop screening and prevention strategies and ultimately improve treatment outcomes.

MicroRNA-875-5p inhibits the growth and metastasis of cervical cancer cells by promoting autophagy and apoptosis and inhibiting the epithelial-mesenchymal transition.

MicroRNA-875-5p (miR-875-5p) is a cancer-related microRNA. It has been demonstrated that miR-875-5p participates in the development of various types of cancer such as hepatocellular carcinoma, gastric carcinoma, prostate and bladder cancer. Previous research suggested that miR-875 is implicated in the development of cervical cancer cells. However, the exact role and function of miR-875-5p in cervical cancer remain unexplored. It is important to examine the role and function of miR-875-5p and the associated signaling pathway, as the findings may have diagnostic and therapeutic significance. Thus, in this study, we investigated the effect of miR-875-5p on the growth and metastasis of cervical cancer cells and the possible underlying mechanisms. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-875-5p in cervical cancer cells and normal cervical epithelium. After overexpression or co-expression of miR-875-5p in cells, the changes in cell function were analyzed. Western blot was used to detect the expression changes of epithelial-mesenchymal transition (EMT) -related proteins and autophagy-related proteins. Functional studies demonstrated that miR-875-5p overexpression significantly inhibited the proliferation, migration, invasion, and EMT, and promotes apoptosis and autophagy of cervical cancer cells., while miR-875-5p knockdown promoted the proliferation, migration, invasion, and EMT, and inhibited apoptosis and autophagy cervical cancer cells. Furthermore, Western blot results showed that overexpression of miR-875-5p downregulated the expressions of N-cadherin, Snail, Vimentin and microtubule-associated protein 1 light chain 3B I (LC3B I). Conversely, miR-875-5p upregulated the expression of E-cadherin. In conclusion, our findings suggest that miR-875-5p functions as a tumor inhibitor suppressing the growth and metastasis of cervical cancer. Overexpression of miR-875-5p inhibits malignant behavior and promotes autophagy and apoptosis in cervical cancer cells. These findings advance our understanding of the role and function of miR-875-5p in cervical cancer and could facilitate the development of early genetic markers or biomarkers and therapeutic targets for cervical cancer.

Laminin receptor 1 expression in premalignant and malignant squamous lesions of the cervix

ABSTRACT Laminin receptor 1 (LAMR) may have a role in the progression of premalignant squamous epithelial lesions to cervical cancer. Therefore, we aimed to investigate the expression of laminin receptor 1 (LAMR) in normal, premalignant, and malignant tissues of the uterine cervix. Paraffin blocks of 129 specimens with the diagnoses of normal cervical tissue (n = 33), cervical intraepithelial neoplasia (CIN) 1 (n = 30), CIN 2 (n = 14), CIN 3 (n = 28), and squamous cell carcinoma (n = 24) were immunohistochemically stained with LAMR antibody and its expression percentage, pattern, and intensity in these tissues were assessed. Compared to the other groups, the nonstaining with LAMR was highest in low grade squamous intraepithelial lesion (LSIL) (p < 0.0001). LAMR expression, which was positive in less than 50% of cells with weak staining, increased significantly between normal cervical epithelium and high-grade squamous intraepithelial lesion (HSIL) or invasive carcinoma, as well as between LSIL and HSIL (p < 0.0001). Between LSIL and invasive carcinoma, a significant increment was also observed for weak staining in less than 50% of cells (p < 0.001). LAMR expression, which was positive in more than 50% of cells with strong staining, was significantly higher in normal cervical tissue compared to the other groups (p < 0.0001). Disease progression related gradual increment of LAMR expression from normal cervical epithelium or LSIL towards HSIL or cervical cancer reveals that LAMR may play an important role in the transition from premalignant to malignant state in cervical lesions.

Laurencia johnstonii extract reverses early lesions in the K14E7HPV16 murine cervical carcinogenesis model.

Persistent infection with high-risk human papillomavirus (HR-HPV) is a well-established risk factor to the development of cervical intraepithelial neoplasia (CIN), a condition that can progress to cervical cancer (CC) a major health problem worldwide. Recently, there has been growing interest in exploring alternative therapies utilizing natural products, among which is the algae species Laurencia johnstonii Setchell & Gardner, 1924 (L. johnstonii), proposed for the management of precancerous lesions. The aim of this work was to determine the effect of an organic extract from L. johnstonii (ELj) in early cervical lesions (CIN 1). These CIN 1 lesions were generated in a murine model expressing the HR-HPV16 E7 oncoprotein (K14E7HPV transgenic mice) with a single exogenous hormonal stimulus using 17β-estradiol. The histopathological studies, the determination of cell proliferation and of the apoptotic levels in cervical tissue, showed that, seven doses of ELj (30 mg/kg weight per day diluted in a DMSO-saline solution [1:7]) lead to recovery the architecture of cervical epithelium. Accordingly, in the transgenic mice it was observed a statistically significant decrease of the PCNA expression levels, a marker of cell proliferation, and a statistically significant increase in the apoptosis levels using Caspase 3 as a marker. In addition, we determined the expression levels of the tumor suppressor miR-218 and the oncomiRNA miR-21. Interestingly, our results may suggest that ELj treatment tended to restore the normal expression of both miRNAs as compared with controls being more evident in the non-transgenic induced mice. Differences of p < 0.05 were considered statistically significant through the whole study. Based on these results, we propose that the use of ELj could be an alternative for the treatment of cervical early lesions.

Application of CytoPath®Easy Vials in Cervical Cancer Screening: Self-Sampling Approach.

CytoPath®Easy kit (DiaPath S.p.A.) offers a major advantage compared to other commercially available kits available for the screening of cervical cancer, as it does not require additional equipment for sample processing. Using this methodology, collected epithelial cells are immersed in a preservative liquid before setting as a thin layer on a slide via gravity sedimentation. To evaluate the suitability of the CytoPath®Easy kit for the processing of cervical samples, detection of pre-neoplastic lesions, and nucleic preservation and extraction for HR-HPV diagnosis. A total of 242 self-sampled cervical specimens were utilized, with 192 collected in CytoPath®Easy vials and 50 collected and processed using the ThinPrepTM for comparative analysis. The samples underwent processing, Papanicolaou staining, and microscopic evaluation for morphological parameters. The extracted nucleic acids were assessed for purity and integrity, and the detection of high-risk human papillomavirus (HR-HPV) was carried out using the Alinitym HR HPV system kit (Abbott Laboratórios Lda). Both methods demonstrated effective performance, enabling the morphological assessment of the cervical epithelium. Statistical analysis indicated that ThinPrepTM yielded significantly better results in terms of cellularity. Conversely, CytoPath®Easy exhibited superior performance in terms of the quantity of extracted DNA and its degree of purification. Concerning the time consumed during processing, both methods were comparable, with the CytoPath®Easy methodology standing out for its cost-effectiveness, as it does not necessitate additional instruments and consumables. The novel CytoPath®Easy methodology proves effective in preserving both nucleic acids and cell morphology characteristics, two crucial features for cervical cancer screening.

Abstract 7288: Evaluation of silica spin-column and bead formats for rapid promoter DNA methylation analysis by qMSP in clinical and point-of-care settings

Abstract Late-stage cancers often lack effective management and treatment options, emphasizing the importance of early diagnosis for better prognosis and lower mortality rates. Molecular markers, such as DNA methylation tests, have shown promise in predicting and detecting cancer at its earliest stages. The COVID-19 post-pandemic scenario is providing a one in one-hundred-year opportunity of utilizing the excess installed PCR capacity world-wide to develop novel Quantitative Methylation Specific PCR (qMSP) screening strategies for cervical cancer screening in low-resource settings. This study aimed to compare the use of commercial bisulfite conversion kits in cervical epithelium liquid cytology samples (liquid cytology) from cancer screening clinics in the United States. Commercially available rapid bisulfite conversion protocols using silica spin column and magnetic beads were compared with conventional DNA extraction followed by bisulfite conversion protocols to profile DNA methylation by qMSP in a panel of methylated genes we previously developed to distinguish normal and cervical cancer patient samples. The results revealed that small sample volumes are suitable for methylation studies. Comparisons among four spin-column kits showed no significant differences in β-actin Cycle Threshold (Cts) values between Abcam, Epigentek, QIAGEN and Zymo D5021 kits. Zymo The best performing spin-column kit was compared with manual and automated versions of magnetic bead kits using a 96-well plate format for higher throughput. Methylation profiling of three genes (β-actin ZNF516, and FKBP6) with manual and automated magnetic bead kits revealed that automation increases throughput without a reduction in amplification efficiency in open PCR systems. Cost analysis showed that the direct kits 96-well plate format were more affordable compared to the Gold Standard manual protocol that uses spin-columns. An optimized version of Zymo D5046 magnetic bead kit provides the fastest turnaround time with similar amplification efficiency, when compared to spin-column or other magnetic bead kits. The test cost per sample is $5.41 dollars to process 83 samples in a 96 well-plate format in less than 2 hours. Another significant finding was showing the feasibility of using as low as 12µL of liquid cytology samples for DNA methylation testing, proving that bodily fluids are a liquid biopsy alternative to plasma. Overall, this comprehensive comparison is useful in determining optimal options for efficient and cost-effective cancer DNA methylation diagnostic tests, particularly in low-resource settings. Citation Format: Fernando Zamuner, Ashley Ramos-Lopez, Amanda Garcia-Negron, Ana Purcell-Wiltz, Andrea Cortes-Ortiz, Aniris Roman, Keerthana Gosala, Eli Winkler, David Sidransky, Mariana Brait, Rafael E. Guerrero-Preston. Evaluation of silica spin-column and bead formats for rapid promoter DNA methylation analysis by qMSP in clinical and point-of-care settings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7288.

Frequent Inactivation of Secreted Frizzled-Related Protein 2 during the Development of Cervical Carcinoma: Identification of Susceptible Alleles and Clinical Implications

ABSTRACT Background: In this study, importance of SFRP2, wnt stem cell renewal pathway antagonist, in the development of cervical cancer (CACX) was evaluated Aims and Objectives: Alterations (expression/ methylation/ deletion) of SFRP2 were analysed in primary cervical lesions of different clinical stages followed by their correlation with different clinicopathological parameters. Then, susceptible allele(s) of SFRP2 was identified through case control study followed by and in vitro validation. Results: The mRNA expression of SFRP2 was gradually reduced with progression of CACX. In immunohistochemistry, SFRP2 membrane expression was mainly present in the spinous layers of normal cervical epithelium and its reduced protein expression in CACX samples showed concordance with mRNA expression. Frequent deletion/ methylation of SFRP2 were seen to be associated with development of cervical cancer. Methylation of SFRP2 was prevalently associated with early invasive lesions (stage I/II) while, deletion with late invasive lesions (stage III/IV). Overall alterations (deletion/ methylation) of SFRP2 were significantly increased from premalignant CIN to stage-I/II samples followed by comparable change to the next stage (stage III/IV) samples. Moreover, deletion and/or methylation of SFRP2 were associated with poor prognosis of the patients. In a case control study, out of its seven microsatellite alleles infrequent SFRP_CA15/16 alleles along with frequent SFRP_CA17 allelewere found to be associated with CACX development. Comparatively reduced expression (mRNA/ protein) of SFRP2 was seen in the tumor adjacent normal cervical epithelium having SFRP_CA15/16 alleles than the other alleles. This has been further validated in in vitro luciferase promoter activity assay where SFRP_CA16 repeat showed high reduced activity followed by SFRP_CA15 repeat than the other repeats. Conclusion: Thus, our data showed that presence of the infrequent susceptible alleles along with deletion/methylation might have synergistic effect on frequent inactivation of SFRP2 during development of CACX.

METTL3-mediated HSPA9 m6A modification promotes malignant transformation and inhibits cellular senescence by regulating exosomal mortalin protein in cervical cancer

The role of RNA methyltransferase 3 (METTL3) in tumor progression when tethered to aberrantly expressed oncogenes remains unknown. In especial, the correlation between cervical cancer (CCa)-derived exosomes and m6A methylation in malignant traits of cervical epithelium is currently elusive. Mortalin expression was found to be up-regulated in plasma exosomes isolated from CCa patients. Furthermore, mortalin gained increased mRNA stability and enhanced translation efficiency via the m6A methylation in the HSPA9 mRNA 3′UTR, which was catalysed by METTL3 in CCa cells. Exosomal mortalin overexpression significantly promoted the proliferation, migration and invasion of CCa both in vitro and in vivo. Additionally, exosome-encapsulated mortalin suppressed cellular senescence and facilitated malignant transformation by blocking nuclear transport of p53, thereby preventing the p53-Gadd45A interaction and resulting in inactivation of p53. Our studies demonstrated the significant role of METTL3 mediated exosomal mortalin in malignant transformation and cellular senescence suppression of CCa. Exosomal mortalin could clinically serve as a potential early-diagnosis biomarker and therapeutic target for CCa given its abundance and propensity to be found.

FOXM1 transcriptional regulation of RacGAP1 activates the PI3K/AKT signaling pathway to promote the proliferation, migration, and invasion of cervical cancer cells.

Cervical cancer (CC) is the most common gynecological tumor disease in women, which occurs at the junction of cervical squamous columnar epithelium. We investigated the effect and mechanism of transcription factor FOXM1 synergizing RacGAP1 in the proliferation, migration, and invasion of CC cells. Here, we analyzed the correlation between FOXM1 and RacGAP1 and the clinicopathological features of 68 CC patients. RT-qPCR was used to assess FOXM1 and RacGAP1 mRNA expression in CC tissues and cells. Cell proliferation was assessed by CCK-8 and EDU assays. Transwell assay was applied to test migration and invasion. Cell apoptosis was evaluated utilizing flow cytometry. ChIP and dual-luciferase reporter assays confirmed the interaction of FOXM1 and RacGAP1. Protein levels of FOXM1 and RacGAP1, as well as PI3K/AKT, were analyzed by Western blot. FOXM1 expression was correlated with FIGO stage and histological grade, and RacGAP1 expression was correlated with histological grade. FOXM1 and RacGAP1 levels were increased in CC tissues, and higher expressed in human CC cell lines than that in an immortalized HPV-negative skin keratinocyte line (HaCaT). Depleted RacGAP1 suppressed CC cell proliferation, migration and invasion, and promoted apoptosis. RacGAP1 was a target gene of FOXM1, and FOXM1 positively regulated RacGAP1 expression. FOXM1 had a synergistic effect with RacGAP1 to exert oncogenic function in CC by activating the PI3K/AKT signaling. FOXM1 cooperates with RacGAP1 to induce CC cell proliferation, migration and invasion, inhibit apoptosis, and regulate PI3K/AKT signaling to promote CC progression.

Оценка эффективности применения активированной глицирризиновой кислоты у пациенток с ВПЧ-ассоциированными поражениями шейки матки с учетом уровня половых стероидных и гонадотропных пептидных гормонов

Background: the persistence of human papillomavirus (HPV) infection with oncogenic protein expression causes changes in the stratified squamous epithelium, which is the immediate cause of cervical cancer manifestation. The use of the activated glycyrrhizic acid (GA) for HPV-associated cervical lesions is pathogenetically justified. During an experimental study on a culture of human cervical adenocarcinoma cells (HeLa line), it was shown that the antiproliferative effect of GA is enhanced by low concentrations of estradiol and weakened by testosterone. Aim: to evaluate the efficacy of the activated GA in female patients with HPV-associated cervical pathology with respect to the level of sex steroid and gonadotropin-releasing hormones Patients and Methods: a prospective study was conducted with the inclusion of 51 female patients with HPV-associated cervical pathology (ASCUS — atypical squamous epithelial cells of undetermined significance, LSIL — low-grade squamous intraepithelial lesion, HSIL— high-grade squamous intraepithelial lesion according to the Bethesda classification) and the absence of clinical and/or laboratory signs of hyperandrogenism. The following studies were performed: clinical examination, colposcopy, microscopic examination, cytology and immunocytochemistry, as well as hormonal status was evaluated. All female patients received Epigen Intim spray 0.1%, 3 times a day, for 30 days from the moment of inclusion in the study. If indicated, cervical conization was also performed. The treatment efficacy was evaluated 90±7 days after the therapy initiation based on the liquid-based HPV-PAP test results with the determination of p16/Ki67 oncogenic protein co-expression in the cervical epithelium. Changes in the colposcopy results were also evaluated. Results: it was found that the level of estradiol in patients with HSIL was lower versus the patients with ASCUS and LSIL (32 [32.0; 36.0] pg/ml versus 40 [34.5; 45.5] and 40 [33.0; 44.0] pg/ml, respectively, but the difference did not reach statistical significance (p=0.453). 90 days after the follow-up start, according to the cytology results of the cervical epithelium, 50 (98.0%) patients showed the absence of cervical intraepithelial lesions and the absence of p16/Ki67 oncogenic protein co-expression, 48 (94.1%) patients showed no high-risk (HR) HPV DNA, whereas HPV-associated colposcopy signs (anogenital warts, chronic cervicitis signs) persisted only in 6 (11.8%) patients. Conclusion: the use of activated GA in female patients of reproductive age with HPV-associated cervical lesions without clinical and/or laboratory signs of hyperandrogenism allowed to achieve high efficiency indicators: 98.5% — according to the cytology results, 83.3% — in reducing the HR-HPV viral load, 85.7% — in terms of p16/Ki67 oncogenic protein co-expression. KEYWORDS: human papillomavirus, squamous intraepithelial lesion of the cervix, oncogenic protein co-expression, sex steroid hormones, activated glycyrrhizic acid FOR CITATION: Mandrykina Zh.A., Dobrokhotova Yu.E., Ibragimova D.M., Kazieva M.D., Filimonova M.S., Shimanovsky N.L., Akhmetgaliev A.R. Assessment of the activated glycyrrhizic acid efficacy in female patients with HPV-associated cervical lesions with respect to the level of sex steroid and gonadotropin-releasing hormones. Russian Journal of Woman and Child Health. 2024;7(2):127–134 (in Russ.). DOI: 10.32364/2618-8430-2024-7-2-7.

24 Vaginal putrescine attenuates microbe-mediated inflammation in the cervical epithelium

24 Vaginal putrescine attenuates microbe-mediated inflammation in the cervical epithelium

Intra-horn insemination in the alpaca Vicugna pacos: Copulatory wounding and deep sperm deposition.

Alpacas (Vicugna pacos) are reported to be the rare mammal in which the penis enters the uterus in mating. To date, however, only circumstantial evidence supports this assertion. Using female alpacas culled for meat, we determined that the alpaca penis penetrates to the very tips of the uterine horns, abrading the tract and breaking fine blood vessels. All female alpacas sacrificed one hour or 24 hours after mating showed conspicuous bleeding in the epithelium of some region of their reproductive tract, including the hymen, cervix and the tips of each uterine horn, but typically not in the vagina. Unmated females showed no evidence of conspicuous bleeding. Histological examination of mated females revealed widespread abrasion of the cervical and endometrial epithelium, injuries absent in unmated females. Within one hour of mating, sperm were already present in the oviduct. The male alpaca's cartilaginous penis tip with a hardened urethral process is likely responsible for the copulatory abrasion. The entire female reproductive tract interacts with the penis, functioning like a vagina. Alpacas are induced ovulators, and wounding may hasten delivery of the seminal ovulation-inducing factor beta-NGF into the female's blood stream. There is no evidence of sexual conflict in copulation in alpaca, and thus wounding may also be one of a variety of mechanisms devised by mammals to induce a beneficial, short-term inflammatory response that stimulates blastocyst implantation, the uterine remodeling associated with placental development, and thus the success of early pregnancy.