Normal Cervical Epithelium Research Articles

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.

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.

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.

Human serum albumin-bound paclitaxel nanoparticle inhibits cervical carcinoma cell proliferation and oxidative damage through CYP3A4 and CYP2C8

BackgroundCervical cancer (CC) is currently the most common malignant tumour in the female reproductive tract, and paclitaxel (PTX) is a commonly used chemotherapeutic agent, but tumour cell resistance will seriously affect the therapeutic efficacy of PTX. Nanoparticle human serum albumin-bound paclitaxel (Nano-HSA-PTX) is a novel drug delivery modality that may have superior effects to PTX alone. ObjectiveTo clarify the effect of Nano-HSA-PTX on cervical carcinoma (CC) cells and the underlying mechanisms. MethodsAfter the preparation of Nano-HSA-PTX, its morphology was observed by electron transmission microscope (TEM), and its entrapment efficiency (EE%) and drug loading rate (DL%) were detected. Nano-HSA-PTX was compared with conventional PTX for drug metabolism. Additionally, CC HeLa and SiHa cells were purchased and divided into three groups to treat with Nano-HSA-PTX, PTX and normal saline, respectively. MTT, cell cloning, Transwell and cell scratch assays were carried out to determine cell proliferation, invasion and migration, flow cytometry and Western blotting were performed to detect apoptosis rate and apoptosis-related protein expression, and PCR was conducted to quantify oxidative damage indicators. Further, CYP3A4 and CYP2C8 expression patterns in CC cells (HeLa and SiHa) and human normal cervical epithelia (End1/E6E7) and the changes of their levels under the intervention of Nano-HSA-PTX were measured. Subsequently, C57BL/6mice were purchased for subcutaneous tumorigenesis experiment to observe the impact of Nano-HSA-PTX on tumor growth. ResultsUnder TEM, Nano-HSA-PTX was complete and arranged compactly, with a stable structure and markedly higher EE% and DL% than PTX (P < 0.05). Under Nano-HSA-PTX intervention, the proliferation, invasion, migration and oxidative damage of HeLa and SiHa were significantly decreased compared with the control and PTX groups, while the apoptosis was increased (P < 0.05). Besides, elevated CYP3A4 and CYP2C8 levels were observed in CC cells, which were inhibited by Nano-HSA-PTX and PTX (P < 0.05). Finally, tumorigenesis experiments in nude mice revealed that Nano-HSA-PTX could inhibit tumor growth. ConclusionCompared with PTX, Nano-HSA-PTX has a superior effect of inhibiting CC activity. And this mechanism of action was carried out by inhibiting the expression of CYP3A4 and CYP2C8.

Role of MLH1 and MSH2 deficiency in the development of tumorigenesis and chemo-tolerance of cervical Carcinoma: Clinical implications

Cervical cancer (CACX) is one of the top causes of cancer death in women globally. The involvement of several cellular pathways in carcinogenesis is still poorly understood. Here, we focused to evaluate the contributory role of Mismatch Repair (MMR) pathway genes-MLH1 and MSH2 in CACX and their association with chemo-tolerance of the disease. For this purpose, molecular profiles (expression/promoter methylation/deletion) of the genes were analysed in both normal cervical epithelium and tumour tissue, also validated in in-silico dataset as well. Later on, prognostic importance of the genes was identified through analysis of their methylation/expression status in plasma DNA of circulating tumour cells (CTCs) and cisplatin-tolerant CACX cell lines respectively. It was found that the expression profile of MLH1 and MSH2 genes was considerably reduced from undifferentiated basal-parabasal layers of normal cervical epithelium towards progression of the disease. Further analysis showed that frequent deletion [34–48%] and promoter methylation events [28–46%] of the genes were the plausible reasons for their reduced expression during tumorigenesis. Incidentally, the prevalence of MLH1 [32%] and MSH2 [27%] promoter methylation found in CTCs of plasma of the clinically advanced CACX patients implicated their prognostic importance of the disease. In addition, the patients having high alterations of those genes resulted in poor patient outcomes even after the therapy. In in-depth analysis of this result in cisplatin-tolerant CACX cell lines, we discovered that increased promoter methylation frequency of those genes at higher concentrations of cisplatin and gradual accumulation of the cells in the G2/M phase of the cell cycle were the rational causes for their reduced expression and MMR deficiency in the system. Hence, it is possible to conclude that the gradual down-regulation of MLH1 and MSH2 proteins may be a key event for MMR pathway inactivation in CACX. This might also be associated with chemo-tolerance and overall poor survival among the patients.

Potential diagnostic value of quantitative superb microvascular imaging in premalignant and malignant cervical lesions.

The purpose of this study was to assess the diagnostic efficacy of the vascular index (VI) on superb microvascular imaging (SMI) in distinguishing normal uterine cervical epithelium, high-grade cervical intraepithelial neoplasia (CIN), and cervical cancer. The retrospective study included women with pathology-confirmed CIN or cervical cancer, who underwent transvaginal ultrasound and SMI between April 2021 and October 2022. The SIM manifestations of normal cervix and cervical lesions were reviewed. SIM were measured and converted into vascular index (VI) which compared between cervical lesions and control groups. We have retrospectively compared ultrasound features of cervical lesions and characteristics of patients. Measurement reliability was evaluated by intra class correlation coefficient (ICC). A total of 235 consecutive females were enrolled, comprising 38 with high-grade CIN, 96 with cervical cancer, and 101 with a normal uterine cervix. The microvascular architecture exhibited significant variations between premalignant and malignant cervical lesions. Branch-like patterns were predominantly observed in high-grade CIN, while crab claw-like and fireball-like patterns were more commonly associated with cervical cancer. The median VI of cervical cancer (34.7 ± 10.3) was significantly higher than that of high-grade CIN (17.6 ± 4.2) (P < 0.001). Moreover, the VI values of cervical cancer differed significantly among different FIGO stages and pathological types (P < 0.001 and P = 0.003, respectively). The VI demonstrated superior diagnostic performance for cervical lesions compared to vascular patterns (AUC = 0.974 and 0.969, respectively). Using a cut-off value of 25.5, the VI yielded a sensitivity of 82.3% and a specificity of 99.3% for cervical lesion detection. The SMI parameter (VI) exhibited a significantly higher value in cervical cancer compared to high-grade CIN, with a high level of agreement among observers. These findings suggest that quantitative SMI holds promise as an imaging technique for the detection and characterization of cervical lesions.

Efficiency of CIN2+ Detection by Thyrotropin-Releasing Hormone (TRH) Site-Specific Methylation.

Cervical cancer screening typically involves a Pap smear combined with high-risk human papillomavirus (hr-HPV) detection. Women with hr-HPV positivity but normal cytology, as well as those with precancerous abnormal cytology, such as low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL), are referred for colposcopy and histology examination to identify abnormal lesions, such as cervical intraepithelial neoplasia (CIN) and cervical cancer. However, in order to enhance the accuracy of detection, bioinformatics analysis of a microarray database was performed, which identified cg01009664, a methylation marker of the thyrotropin-releasing hormone (TRH). Consequently, a real-time PCR assay was developed to distinguish CIN2+ (CIN2, CIN3, and cervical cancer) from CIN2- (CIN1 and normal cervical epithelia). The real-time PCR assay utilized specific primers targeting methylated cg01009664 sites, whereas an unmethylated reaction was used to check the DNA quality. A cut-off value for the methylated reaction of Ct < 33 was established, resulting in improved precision in identifying CIN2+. In the first cohort group, the assay demonstrated a sensitivity of 93.7% and a specificity of 98.6%. In the cytology samples identified as atypical squamous cells of undetermined significance (ASC-US) and LSIL, the sensitivity and specificity for detecting CIN2+ were 95.0% and 98.9%, respectively. However, when self-collected samples from women with confirmed histology were tested, the sensitivity for CIN2+ detection dropped to 49.15%, while maintaining a specificity of 100%. Notably, the use of clinician-collected samples increased the sensitivity of TRH methylation testing. TRH methylation analysis can effectively identify women who require referral for colposcopy examinations, aiding in the detection of CIN2+.

Spectrum, Clinicopathologic Profile, and p16 Expression Pattern of Nonmalignant Cervical Tissues in Enugu, South-East Nigeria

Abstract Background p16 is a marker for p16-induced transformation of high-risk human papilloma virus (hrHPV)-infected cervical epithelium. HPV is a known etiologic agent of cervical cancer. Persistent hrHPV infection of cervical epithelium causes transformation of the infected epithelial cells resulting in increased proliferative potential of the cells and subsequent progression to frank malignancy. Early detection of transformation in cervical cells is crucial in reducing morbidity and mortality associated with cervical cancer. Materials and Methods We aimed to study the clinicopathologic profile of nonmalignant cervical lesions and their p16 staining pattern. Histopathology requisition forms, blocks, and slides of cases containing cervical tissue with nonmalignant diagnosis received into the morbid anatomy department of University of Nigeria Teaching Hospital, Enugu, from 2009 to 2018 were studied. Fresh sections from the blocks were immunohistochemically stained with p16 and examined. Results One-hundred and ninety-one cases were studied. Majority of the cases are normal cervical epithelium. Chronic nonspecific cervicitis was the major non-neoplastic lesion present and accounted for 33.3% of the biopsy and the mean age was 50.5 years. Other lesions were nabothian cyst (8.4%), cervical polyp (10.5%), low-grade and high-grade squamous intraepithelial lesion (LSIL and HSIL; 6.3 and 2.6%, respectively). The mean age for LSIL was 40.3 years, while that of HSIL was 45.2years. Four LSIL, two HSIL, one polyp, one chronic nonspecific cervicitis, and one lobular endocervical hyperplasia stained positively with p16. Conclusion The most common benign lesion of the cervix is cervicitis. Chronic cervicitis is negative to p16 immunoreactivity. There are more low-grade cervical intraepithelial neoplasia (CIN) than high-grade CIN. The low-grade CIN overexpress p16 in one-third of cases. There are more cases of p16-negative high-grade CIN in this study.

Assessment and clinicopathological correlation of p16 expression in cervical squamous cell carcinoma of Indian population: Diagnostic implications.

Our aim was to assess the p16 expression in normal cervical epithelium and cervical lesions and how it correlated with HPV oncoprotein E7 and other etiological parameters of cervical cancer. For this purpose, we analyzed protein expression of p16 and E7 oncoprotein in total 20 normal cervical epithelium tissue (as control) and 62 cervical lesions. Next, the result was correlated with different clinico-pathological parameters. Out of 62 cases of cervical lesions, we found around 75%-100% of the cervical lesion samples exhibited E7 nuclear protein expression, whereas around 33.33%-75% samples were p16 positive. On the other hand, p16 expression showed strong association with E7 oncoprotein and other clinico-pathological parameters (like high parity, early age of sextual debut) in the same set of samples of our study. We concluded that overexpression of p16 is very practical and can be readily implemented in most diagnostic pathology laboratories.

The Effects of Dietary Nutrient Intake on Cervical Cancer: A Brief Review

AbstractCervical cancer (CC) results from a subsequent process, starting from the infection of normal cervical epithelium with oncogenic human papillomavirus and gradually progressing to cervical intraepithelial neoplasia (CIN), before finally developing into invasive cervical cancer (ICC). Over recent decades, dietary micronutrients have gained much attention due to their pivotal role in cancer prevention. We reviewed several relevant literature studies to investigate the protective roles of dietary nutrient intake in CC. Dietary intake of vitamin C, green–yellow vegetables, and provitamin A carotenoids that are rich sources of antioxidants may widely inhibit the process of CC development, whereas vitamins A and D might be more helpful in preventing the early events in the disease development. Vitamin E, lycopene, and folate are more effective for the treatment of high-grade CIN. Fruits exert their protective effects in the late stages of the cancer process, thus playing a vital role in ICC prevention. Polyphenols, flavonoids, and polyunsaturated fatty acids are more often used in cases of CC in combination with chemotherapy and radiotherapy. Thus, as a primary prevention strategy, the health benefits of various nutrients in CC must be clarified by vitro and in vivo approaches rather than epidemiological studies.

Elevated expression of endocan in the development of cervical squamous neoplasia of the uterus.

Accumulated evidence has shown that endocan, which was originally called endothelial cell-specific molecule-1, is an attractive prognostic factor in a variety of cancers. However, the relevance of endocan expression in human malignancies remains to be clarified. In the present study, the expression of endocan in cervical squamous neoplasia of the uterus, including low- and high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively), as well as in invasive squamous cell carcinoma was examined by immunohistochemistry. Endocan was not sufficiently expressed in the normal cervical epithelium. Endocan expression was present in LSIL cases but was limited to basal and parabasal areas of the cells. HSIL cases exhibited strong expression of endocan with widely distributed expression toward the epithelial surface. In contrast, further strong expression of endocan was not observed in patients with invasive carcinoma. This study is the first study showing increased expression of endocan in precancerous dysplastic lesions and malignancy of the cervix. The data suggest that a high expression level of endocan potentially contributes to the development of cervical squamous neoplasia of the uterus.

Mueller matrix imaging polarimeter at the wavelength of 265 nm.

Mueller matrix imaging polarimeters (MMIPs) have been developed in the wavelength region of >400n m with great potential in many fields yet leaving a void of instrumentation and application in the ultraviolet (UV) region. For the first time to our knowledge, an UV-MMIP is developed for high resolution, sensitivity, and accuracy at the wavelength of 265nm. A modified polarization state analyzer is designed and applied to suppress stray light for nice polarization images, and the errors of the measured Mueller matrices are calibrated to lower than 0.007 in pixel level. The finer performance of the UV-MMIP is demonstrated by the measurements of unstained cervical intraepithelial neoplasia (CIN) specimens. The contrasts of depolarization images obtained by the UV-MMIP are dramatically improved over those obtained by our previous VIS-MMIP at the wavelength of 650nm. A distinct evolution of depolarization in normal cervical epithelium tissue, CIN-I, CIN-II, and CIN-III specimens can be observed by the UV-MMIP with mean depolarization promotion by up to 20 times. This evolution could provide important evidence for CIN staging but can hardly be distinguished by the VIS-MMIP. The results prove that the UV-MMIP could be an effective tool in polarimetric applications with higher sensitivity.

Androgen promotes squamous differentiation of atypical cells in cervical intraepithelial neoplasia via an ELF3-dependent pathway.

Since the human papillomavirus vaccines do not eliminate preexisting infections, nonsurgical alternative approaches to cervical intraepithelial neoplasia (CIN) have been required. We previously reported that FOXP4 (forkhead box transcription factor P4) promoted proliferation and inhibited squamous differentiation of CIN1-derived W12 cells. Since it was reported that FOXP expressions were regulated by the androgen/androgen receptor (AR) complex and AR was expressed on the CIN lesions, in this study we examined the effects of androgen on CIN progression. Since AR expression was negative in W12 cells and HaCaT cells, a human male skin-derived keratinocyte cell line, we transfected AR to these cell lines and investigated the effects of dihydrotestosterone (DHT) on their proliferation and squamous differentiation. We also examined the immunohistochemical expression of AR in CIN lesions. DHT reduced the intranuclear expression of FOXP4, attenuating cell proliferation and promoting squamous differentiation in AR-transfected W12 cells. Si-RNA treatments showed that DHT induced the expression of squamous differentiation-related genes in AR-transfected W12 cells via an ELF3-dependent pathway. DHT also reduced FOXP4 expression in AR-transfected HaCaT cells. An immunohistochemical study showed that AR was expressed in the basal to parabasal layers of the normal cervical epithelium. In CIN1 and 2 lesions, AR was detected in atypical squamous cells, whereas AR expression had almost disappeared in the CIN3 lesion and was not detected in SCC, suggesting that androgens do not act to promote squamous differentiation in the late stages of CIN. Androgen is a novel factor that regulates squamous differentiation in the early stage of CIN, providing a new strategy for nonsurgical and hormone-induced differentiation therapy against CIN1 and CIN2.

Evaluation of Sirtuin1 Overexpression by Immunohistochemistry in Cervical Intraepithelial Lesions and Invasive Squamous Cell Carcinoma.

Cervical cancer is one of the most common genital cancers in the woman with approximately half a million new cases per year. Development of invasive squamous cell carcinoma (SCC) is the result of persistent and frequent human papilloma virus infection in premalignant lesions of cervix. Thereby identification of biomarkers that could predict progression of dysplastic mucosa to invasive cancer is of great clinical significance. Overexpression of SIRT1 has been reported to induce tumorogenesis in several organs. We evaluated SIRT1 expression in normal squamous epithelium of cervix, low-grade and high-grade cervical intraepithelial lesions and invasive SCC. A total of 104 cases were selected including 34 low-grade cervical intraepithelial lesions (CINs), 37 high-grade CINs, and 35 cases of invasive SCC. The normal cervical epithelium showed negative or weak SIRT1 positivity only in basal layers. SIRT1 cytoplasmic expression was found in 13 of 34 (38.2%) of low-grade CINs, 31 of 37 (83.8%) of high-grade CINs and all 35 (100%) cases of invasive SCC. Expression between 2 groups of CIN was statistically significant ( P =0.001). Thus, SIRT1 appears to be a promising biomarker for predicting the progression of CIN to invasive SCC.

MiR-95-3p acts as a prognostic marker and promotes cervical cancer progression by targeting VCAM1.

Cervical cancer patients have a high risk of metastasis and a poor prognosis with shorter disease-free survival. Thus, novel biomarkers and feasible therapies urgently need to be discovered. Previous studies have shown that miR-95-3p plays crucial roles in several cancer types. However, the roles of miR-95-3p in cervical cancer remain unknown. The micro ribonucleic acid (miRNA) expression data and clinical characteristics of cervical cancer samples were downloaded from The Cancer Genome Atlas (TCGA) database. Univariate and multivariate Cox regression analyses were conducted to identify the prognostic-related miRNAs. The potential target genes of miR-95-3p were predicted by the TargetScan database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to explore the target gene of miR-95-3p. The effects of miR-95-3p inhibition and overexpression on cell proliferation were inspected by cell counting kit-8 (CCK-8) assays and cell colony formation assays. Wound-healing assays and transwell assays were also used to examine cell migration ability in HeLa and SiHa cells. MiR-95-3p was the only miRNA significantly associated with the poor prognosis of cervical squamous cell carcinoma. A further analysis suggested that vascular cell adhesion molecule 1 (VCAM1) is a target gene of miR-95-3p in cervical cancer, and miR-95-3p promotes the malignant behavior of cervical cancer cells by inhibiting the expression of VCAM1. The CCK-8 and cell colony assays showed that miR-95-3p downregulation significantly suppressed cell proliferation in the HeLa and SiHa cells. The transwell and wound-healing assays showed that miR-95-3p inhibition suppressed cell migration in the HeLa and SiHa cells. Further the Western blot analysis and the quantitative real-time-polymerase chain reaction (qRT-PCR) showed that the knockdown of miR-95-3p in HeLa cells resulted in increased VCAM1 expression. And VCAM1 was highly expressed in the paired adjacent normal cervical epithelium tissue samples, but lowly expressed in the cervical tumor tissue samples. Our study was the first to show that miR-95-3p could serve as a prognostic biomarker of cervical cancer. Mechanistically, we discovered that miR-95-3p inhibited the expression of the cell adhesion molecule VCAM1 and thus promoted further tumor progression.

LINE-1 and Alu Methylation in hrHPV-Associated Precancerous Cervical Samples.

This study was conducted to determine global DNA methylation patterns in cervical cells cytologically identified as atypical squamous cells of unknown significance (ASCUS) with a normal, LSIL, or HSIL histopathological result. Methylation patterns of long interspersed nuclear elements (LINE-1) and short interspersed element (Alu) sequences were assessed using the combined bisulfite restriction analysis (COBRA) method in cervical samples with cytology-diagnosed cervical lesions. In cervical precancerous lesions with hrHPV positive, the percentage of overall (mC) and mCmC LINE-1 methylation levels showed a stepwise increase from hrHPV positive normal to HSIL with significant differences (p<0.001). However, both methylation levels were significantly higher in hrHPV negative normal than in hrHPV positive normal (p<0.001). The overall (mC) Alu methylation in hrHPV positive LSIL and HSIL was lower than in hrHPV positive normal, with a significant difference (p<0.05). Remarkably, the percentage of uCmC and mCuC of LINE-1 and Alu in three different hrHPV positive cervical lesions showed a stepwise decrease from hrHPV positive normal, LSIL and HSIL, respectively. Furthermore, receiver operating characteristic (ROC) curve analyses revealed that the LINE-1 mC and mCmC patterns have high sensitivity and specificity for distinguishing HSIL from normal/LSIL in hrHPV positive cases at the appropriate cutoff levels. We have demonstrated the LINE-1 and Alu methylation data in normal and premalignant cervical epithelia. LINE-1 hypomethylation was found in hrHPV positive normal cells, with lower methylation levels associated with cancer features. In cytologically diagnosed Atypical Squamous Cells of Unknown Significance (ASCUS), the levels of mC and the mCmC pattern could be utilized in concert with hrHPV detection to classify the ASCUS sample prior to colposcopy.

SOX2 expression in the pathogenesis of premalignant lesions of the uterine cervix: its histo-topographical distribution distinguishes between low- and high-grade CIN

SOX2 expression in high-grade cervical intraepithelial neoplasia (CIN3) and cervical squamous cell carcinoma is increased compared to that in the normal cervical epithelium. However, data on the expression and histological distribution of SOX2 in squamous epithelium during progression of CIN are largely lacking. We studied SOX2 expression throughout the epithelium in 53 cases of CIN1, 2, and 3. In general, SOX2 expression increased and expanded from basal/parabasal to the intermediate/superficial compartment during early stages of progression of CIN. An unexpected, specific expression pattern was found in areas classified as CIN2 and CIN3. This pattern was characterized by the absence or low expression of SOX2 in the basal/parabasal compartment and variable levels in the intermediate and superficial compartments. It was significantly associated with CIN3 (p = 0.009), not found in CIN1 and only seen in part of the CIN2 lesions. When the different patterns were correlated with the genetic make-up and presence of HPV, the CIN3-related pattern contained HPV-positive cells in the basal/parabasal cell compartment that were disomic. This is in contrast to the areas exhibiting the CIN1 and CIN2 related patterns, which frequently exhibited aneusomic cells. Based on their SOX2 localisation pattern, CIN1 and CIN2 could be delineated from CIN3. These data shed new light on the pathogenesis and dynamics of progression in premalignant cervical lesions, as well as on the target cells in the epithelium for HPV infection.

Significance of Altered Fatty Acid Transporter Expressions in Uterine Cervical Cancer and Its Precursor Lesions.

High-risk human papilloma virus (HR HPV) infection is a major factor leading to the development of uterine cervical cancer. Data suggest that alterations in lipid metabolism are related to the pathogenesis of cervical cancer. Specifically, the uptake of exogenous fatty acids and their intracellular storage in lipid droplets enables cancer cells to survive and adapt to the changing tumor environment. We compared the immunohistochemical expression of fatty acid transport protein 4 (FATP4), and cluster of differentiation 36/fatty acid translocase (CD36/FAT) in normal cervical epithelium, low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC) tissues of the uterine cervix. We also investigated the clinicopathological implications of these fatty acid transporters in SCC. Compared with that in normal cervical tissues, the expression of FATP4 was lower in LSIL (p=0.002), HSIL (p=0.006), and SCC (p=0.001). In contrast, CD36 expression was higher in SCC tissues than in normal cervical tissues (p<0.001). In normal cervical tissues, HR HPV-infected lesions exhibited a decrease in FATP4 (p<0.001) and an increase of CD36 (p=0.134), compared to those that were not infected with HR HPV. High CD36 expression was associated with a shorter recurrence-free survival (p<0.001). However, high FATP4 levels showed no significant correlation with the clinicopathological parameters of SCC. Altered expression levels of FATP4 and CD36 are unique features that might be related to HR HPV infection and promote tumorigenesis and progression of cervical cancer.

PiRNA-14633 promotes cervical cancer cell malignancy in a METTL14-dependent m6A RNA methylation manner

BackgroundCervical cancer (CC) is one of the most common gynecological tumors that threatens women's health and lives. Aberrant expression of PIWI-interacting RNA (piRNA) is closely related with a range of cancers and can serve as a tumor promoter or suppressor in proliferation, migration and invasion. In this study, the aim was not only to discover differential expression of piRNA in CC tissue (CC cells) and normal cervical tissue (normal cervical epithelium cells), but also to investigate the biological function and action mechanism of piRNA in CC.MethodsThe DESeq2 approach was used to estimate fold change in piRNA between CC tissue and normal cervical tissue. The relative expressions of piRNAs (piRNA-20657, piRNA-20497, piRNA-14633 and piRNA-13350) and RNA m6A methyltransferases/demethylases were detected using RT-qPCR. After intervention with piRNA-14633 and METTL14 expression, the viability of CaSki cells and SiHa cells was detected by CCK8. CC cell proliferation was detected by colony formation assay. Apoptosis rate and cell cycle were detected by flow cytometry. Transwell assay was performed to detect cell migration and invasion. EpiQuik m6A RNA Methylation Quantification Kit was used to evaluate m6A RNA methylation levels. Expression of methyltransferase-like protein 14 (METTL14), PIWIL-proteins and CYP1B1 were detected by RT-qPCR and western blot. The effect of piRNA-14633 on METTL14 was evaluated by a dual-luciferase reporter assay. The in vivo effects of piRNA-14633 on CC was assessed by nude mice experiments.ResultspiRNA-14633 showed high expression in CC tissues and cells, piRNA-14633 mimic (piRNA-14633 overexpression) promoted viability, proliferation, migration and invasion of CaSki cells and SiHa cells. Besides, piRNA-14633 mimic increased m6A RNA methylation levels and METTL14 mRNA stability. Results of dual luciferase reporter assays indicated that METTL14 was a directed target gene of piRNA-14633. Knockdown of METTL14 with siRNA attenuated proliferation, migration and invasion of CC cells. piRNA-14633 increased CYP1B1 expression, while silencing of METTL14 impaired its expression. The effect of piRNA overexpression on METTL14 expression has concentration-dependent characteristics. Results from in vivo experiment indicated that piRNA-14633 promoted cervical tumor growth.ConclusionpiRNA-14633 promotes proliferation, migration and invasion of CC cells by METTL14/CYP1B1 signaling axis, highlighting the important role of piRNA-14633 in CC.