Normal Cervical Tissues Research Articles

The profile analysis of circular RNAs in cervical cancer.

Cervical cancer (CC) is the third most common cancer among women and has a high mortality rate at the advanced stage. The mechanisms underlying the development and progression of CC are still elusive. Circular RNAs (circRNAs) play an important role in various physiological and pathological processes. The aim of this study was to identify the circRNAs significantly associated with cervical squamous cell carcinoma (CSCC), in order to discover novel diagnostic markers and elucidate their mechanistic basis.The circRNA expression profiles of CSCC and paired para-cancerous cervical tissues was downloaded from the Gene Expression Omnibus. Bioinformatics analysis were used to screen for the differentially expressed circRNAs (DECRs). The expression levels of hsa_circ_0000745, hsa_circ_0084927, hsa_circ_0002762, hsa_circ_0075341, hsa_circ_0007905, hsa_circ_0031027, hsa_circ_0065898, hsa_circ_0070190, and hsa_circ_0078383 were verified in CC and normal cervical tissues by quantitative real-time PCR.A total of 197 DECRs were identified between the CSCC and normal tissues, including 87 upregulated and 110 downregulated circRNAs. In addition, 37 miRNAs were predicted for the upregulated circRNAs and 39 for the downregulated circRNAs. Functional analysis showed that the DECRs were associated with positive regulation of substrate adhesion-dependent cell spreading, metabolism, positive regulation of GTPase activity, protein regulation, and intercellular adhesion. The MAPK signaling pathway that plays a significant role in the progression of CC, was also enriched. Consistent with the in-silico analysis, hsa_circ_0000745, hsa_circ_0084927, hsa_circ_0002762, hsa_circ_0007905 were upregulated and hsa_circ_0078383 was downregulated in CC tissues (P < .001), whereas hsa_circ_0075341 (P < .001) and hsa_circ_0031027 (P = .001) showed opposite trends.We identified novel diagnostic and therapeutic biomarkers of CSCC along with the mechanistic basis.

Downregulation of GTSE1 leads to the inhibition of proliferation, migration, and Warburg effect in cervical cancer by blocking LHDA expression.

G2 and S phase-expressed-1 (GTSE1) has been identified to play a vital role in several kinds of cancers, but its role in cervical cancer development remains unknown. Herein, we aimed to reveal the role and underlying mechanism of GTSE1 in cervical cancer cell growth, migration, and aerobic glycolysis. GTSE1 expression levels in cervical cancer tissues and normal cervical tissues were determined by real time PCR and immunohistochemistry. Human short hairpin RNA was used to downregulate GTSE1 level in cervical cancer cells SiHa and HeLa cells. Colony formation, cell counting kit-8, and wound-healing assays were used for cell function evaluation. Lactate production, lactate dehydrogenase activity, and glucose concentration were tested to assess the Warburg effect. GTSE1 expressions at both mRNA and protein levels were significantly elevated in cervical cancer tissues compared with normal tissues. Downregulation of GTSE1 induced significant repressions in cell colony formation, viability and migration, and Warburg effect, as well as reduced expression of lactate dehydrogenase isoform A (LDHA) at mRNA and protein levels. Additionally, downregulation of GTSE1 weakened the tumorigenesis of HeLa and SiHa cells in vivo. This study demonstrated that downregulation of GTSE1 led to significant inhibitions in cell proliferation, migration, tumorigenesis, and Warburg effect in cervical cancer by blocking the expression of LHDA.

Repeatability of MR fingerprinting in normal cervix and utility in cervical carcinoma.

Magnetic resonance fingerprinting (MRF) is a fast-imaging acquisition technique that generates quantitative and co-registered parametric maps. The aim of this feasibility study was to evaluate the agreement between MRF and phantom reference values, scan-rescan repeatability of MRF in normal cervix, and its ability to distinguish cervical carcinoma (CC) from normal cervical tissues. An International Society of Magnetic Resonance in Medicine/National Institute of Standards and Technology (ISMRM/NIST) phantom was scanned using MRF 15 times over 65 days. Agreement between MRF and phantom reference T1 and T2 values was assessed by linear regression. Healthy volunteers and patients with suspected CC were prospectively recruited. MRF was repeated twice for healthy volunteers (MRF1 and MRF2). Volumes of interest of normal cervical tissues and CC were delineated on T1 and T2 maps. MRF scan-rescan repeatability was evaluated by Bland-Altman plots, within-subject coefficients of variation (wCV), and intraclass correlation coefficients (ICC). T1 and T2 values were compared between CC and normal cervical tissues using Mann-Whitney U test. Receiver operating characteristic (ROC) analysis was performed to evaluate diagnostic efficiency. Strong correlations were observed between MRF and phantom (R2=0.999 for T1, 0.981 for T2). Twelve healthy volunteers (28.7±5.1 years) and 28 patients with CC (54.6±15.2 years) were recruited for the in-vivo experiments. Repeatability of MRF parameters were wCV <3% for T1, <5% for T2 and ICC ≥0.92 for T1, ≥0.94 for T2. T1 value of CC (1,529±112 ms) was higher than normal mucosa [MRF1: 1,430±129 ms, MRF2: 1,440±130 ms; P=0.031, area under the curve (AUC) ≥0.717] and normal stroma (MRF1: 1,258±101 ms, MRF2: 1,276±105 ms; P<0.001, AUC ≥0.946). T2 value of CC (69±9 ms) was lower than normal mucosa (MRF1: 88±16 ms, MRF2: 87±13 ms; P<0.001, AUC ≥0.854), but was not different from normal stroma (P=0.919). Excellent agreement was observed between MRF and phantom reference values. MRF exhibited excellent scan-rescan repeatability in normal cervix with potential value in differentiating CC from normal cervical tissues.

Designing DNA cage-based immuno-fluorescence strategy for rapid diagnosis of clinical cervical cancer tissues

Exploiting a tissue diagnosis method to abstain the involuted operating and consume valuable reagents while realizing high-speed and inexpensive pathological grading technology to supply a better scheme for cancer therapy is a significant method of cancers detection. A promising immuno-fluorescence strategy was rationally designed and synthesized by loading ruthenium complex into cervical cancer-targeted DNA-cage, which was well used to realize high-speed and inexpensive diagnosis of clinical cervical cancer tumor tissues avoiding the traditional multi-stage process, thus demonstrating high application potential in clinical pathological grading and surgical judgment. Moreover, it has been finding that Apts-DNA@Ru can enrichment in the tumor region, interestingly, no enrichment in normal cervical cancer tissue. It has the potential to realize the integration of in vivo diagnose and further synchronous treatment in the near future. Thence, this study demonstrates a strategy for integration of cancer-targeted DNA-cage and fluorescent RuPOP as alternative IHC reagents for next-generation more rapid convenient cancer detection.

CircSPIDR acts as a tumour suppressor in cervical adenocarcinoma by sponging miR-431-5p and regulating SORCS1 and CUBN expression.

To identify circular RNAs (circRNAs) with tumor suppressor activity against cervical adenocarcinoma, we compared the circRNA levels of cervical adenocarcinoma and normal cervical tissues. We found that circSPIDR was dramatically downregulated in cervical adenocarcinoma tissues. In cervical adenocarcinoma cells, overexpression of circSPIDR reduced cell viability, inhibited colony formation and promoted apoptosis, whereas knockdown of circSPIDR exerted the opposite effects. CircSPIDR overexpression also suppressed the tumorigenicity of cervical adenocarcinoma cells in a xenograft mouse model. CircSPIDR was found to sponge miR-431-5p, thereby de-repressing sortin-related VPS10 domain-containing receptor 1 (SORCS1) and cubilin (CUBN) and inhibiting the development of cervical adenocarcinoma. In clinical cervical samples, circSPIDR expression correlated negatively with miR-431-5p expression and positively with SORCS1 and CUBN expression. These results demonstrated that circSPIDR suppresses cervical adenocarcinoma by competitively binding to miR-431-5p, thus upregulating SORCS1 and CUBN. These findings suggest circSPIDR could serve as a novel therapeutic target for treatment of cervical adenocarcinoma patients.

Effects of miR-202-5p silencing PIK3CA gene expression on proliferation, invasion, and epithelial-mesenchymal transition of cervical cancer SiHa cells through inhibiting PI3K/Akt/mTOR signaling pathway activation.

To explore the mechanism of miR-202-5p targeting the expression of PIK3CA and mediating the activation of PI3K/Akt/mTOR signaling pathway on the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of cervical cancer. The objects of study were 105 cases of cervical cancer and their corresponding normal tissues. qRT-PCR was used to detect the expression of miR-202-5p and PIK3CA in adjacent normal tissue and cervical cancer tissue. Dual luciferase reporter assay was used to verify the targeting relationship between miR-202-5p and PIK3CA gene. Human cervical cancer cell lines HPV-16E6, SiHa, HeLa, and CaSki were purchased for our cell experiments. The expression levels of PIK3CA in the cells were detected by qRT-PCR. The cell line with higher expression levels was selected to complete the follow-up experiment. The cultured cells were transfected and divided into the miR-202-5p mimic NC group, miR-202-5p mimic group, miR-202-5p inhibitor NC group, miR-202-5p inhibitor group, siRNA-PIK3CA NC group, siRNA-PIK3CA group, miR-202-5p inhibitor NC + siRNA-PIK3CA NC group, miR-202-5p inhibitor + siRNA-PIK3CA NC group, and miR-202-5p inhibitor + siRNA-PIK3CA group. QRT-PCR was used to detect the expression of miR-202-5p. Western blot and qRT-PCR were applied to detect the mRNA and protein expression levels of related pathway proteins (PIK3CA, PI3K, PTEN, p-Akt1, and p-mTOR) and epithelial-mesenchymal transition-related factors (N-cadherin, E-cadherin, and vimentin). Cell proliferation was detected by plate colony formation assay. Transwell assay was used to detect the invasion ability of each group. When compared with the adjacent tissues, PIK3CA mRNA expression level was significantly increased and miR-202-5p expression level was significantly decreased in cervical cancer tissues (all P < 0.05). PIK3CA was a target gene of miR-202-5p. The mRNA expression level of PIK3CA in SiHa cervical cancer cells was significantly higher than that in CaSki, HeLa, and HPV-16E6 cells (all P < 0.05), and SiHa cervical cancer cells were selected to complete the follow-up experiments. When compared with the corresponding NC group, the expression of miR-202-5p in miR-202-5p mimic group was increased. In addition, the mRNA and protein expression levels of E-cadherin and PTEN in miR-202-5p mimic and siRNA-PIK3CA groups were increased, and the protein expression of p-Akt1 and p-mTOR was decreased, and also, the mRNA and protein expression levels of PIK3CA, PI3K, N-cadherin, and vimentin were decreased (all P < 0.05); in miR-202-5p inhibitor group, the expression levels of miR-202-5p, E-cadherin, and PTEN decreased, the protein expression of p-Akt1 and p-mTOR increased, and the mRNA and protein expression of PIK3CA, PI3K, N-cadherin, and vimentin increased in miR-202-5p inhibitor group (all P < 0.05); in miR-202-5p inhibitor + siRNA-PIK3CA group, the expression of miR-202-5p decreased (P < 0.05), but the mRNA and protein expression of PIK3CA, PI3K, p-Akt1, p-mTOR, N-cadherin, E-cadherin, and vimentin had no significant changes (all P > 0.05). When compared with the corresponding NC group, the number of cell clones in miR-202-5p mimic group and siRNA-PIK3CA group was decreased, and the invasion ability of miR-202-5p inhibitor group was increased, and the invasion ability was enhanced (all P < 0.05); miR-202-5p inhibitor + siRNA-PIK3CA group showed no significant change in the number of cell clones and the rate of invasion (P > 0.05). In conclusion, the overexpression of miR-202-5p can suppress PIK3CA gene expression and the activation of PI3K/Akt/mTOR signaling pathway to suppress the proliferation, invasion, and EMT of cervical cancer.

Abstract 2538: Retinol binding protein-1 and E6-E7 HR-HPV mRNA expression in normal cervical tissue and high grade squamous intraepithelial lesions

Abstract Background: Gene expression differences between cervical healthy tissue and premalignant lesions could contribute to identify potential biomarkers for each step of this tumor progression. In a previous cDNA microarray analysis, we identified the gene Retinol Binding Protein 1 (RBP1), differentially expressed between normal cervix and a high grade squamous intraepithelial lesion (HSIL) with HPV-18 transforming infection. RBP1 codifies a retinol-binding protein implied in differentiation of epithelia and may play a role on inhibition of E2F-dependent gene expression and suppress cell growth. Design: The expression of RBP1 mRNA was evaluated using RNAscope chromogenic in situ hybridization (CISH) in a tissue microarray constructed from specimens (conization and hysterotomy) obtained from 169 patients, with mean age of 35.37 years (SD ±10.4). Stained sections from donor blocks were reviewed by two pathologist, who chose representative areas of normal cervix, low grade squamous intraepithelial lesion (LSIL), and HSIL. RNAscope 2.5HD Red RBP1 assay, and RNAscope 2.5HD Red HPV-HR18 , that detect E6/E7 mRNA for eighteen different high-risk (HR) HPV genotypes, were performed, the stainings were visually scored by two pathologist from 0 to 4 in a semi-quantitative analysis, based on the average number of red dots per cell. The mean score values for each tissue group were compared using GraphPad Prism® 7.0. A p&amp;lt; 0.05 was considered statistically significant. Results: The in situ expression of RBP1 mRNA was determined in a cervical TMA including, normal cervix: 66, LSIL: 74, and HSIL: 130, tissue cores. There was not a statistically significant difference (p=0.965) between the RBP1 mRNA expression score in the squamous epithelium from normal cervix (0.42, 95% CI:0.23-0.61), and LSIL (0.51, 95% CI:0.28-0.74). But, its expression was higher in HSIL (1.30, 95% CI:1.09-1.50), with a very significant difference compared to squamous epithelium from normal cervix (p&amp;lt;0.0001). Mean HPV E6/E7 mRNA expression in HSIL was significantly higher than mean HPV E6/E7 mRNA expression in LSIL (p&amp;lt;0.0001). There was positive-moderate correlation between RBP1 mRNA expression and E6/E7 HR-HPV mRNA expression (Spearman r =0.55). Conclusions: The in situ mRNA expression of RBP1 is similar in LSIL and squamous epithelium lining normal cervix, but the significantly different expression between healthy cervical tissue and HSIL, considered as the real premalignant cervical lesion, confirms our previous findings. There is a moderate correlation with the expression of E6/E7 mRNA for eighteen different HR-HPV. We could hypothesized that this overexpression represent a failed mechanism of regulation induced by the HR-HPV infection in cervical carcinogenesis. Citation Format: Ines Benedetti, Reinhard Rodríguez, Lia Barrios. Retinol binding protein-1 and E6-E7 HR-HPV mRNA expression in normal cervical tissue and high grade squamous intraepithelial lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2538.

Abstract 2153: KRT16 and E6-E7 HR-HPV mRNA in situ expression in normal cervical tissue, and low-grade and high-grade squamous intraepithelial lesions

Abstract Background: KRT16 codifies for acidic-intermedium cytokeratin constitutively expressed in squamous epithelia. Its role in carcinogenesis may be related to its expression in the hyperproliferative phase on epidermal cultures, but yet need to be clarified. In a previous cDNA microarray analysis, the KRT16 gene was differentially expressed between healthy cervical tissue and a high grade squamous intraepithelial lesion (HSIL) with confirmed HPV-18 transforming infection. Gene expression profile differences between uterine cervix healthy tissue, low grade, and high grade squamous intraepithelial lesion (LSIL, HSIL) could contribute to identify potential biomarkers for cervical premalignant lesions progression. Design: A chromogenic in situ hybridization (CISH) assay, (RNAscope), was performed to evaluate the KRT16 mRNA in situ expression in a tissue microarray (TMA) constructed from excision specimens (conization and hysterotomy) from 169 patients, with a mean age of 35.37 years (SD ±10.4), with representative areas from normal cervix tissue, LSIL and HSIL. The RNAscope 2.5HD Red KRT16 assay, and the RNAscope 2.5HD Red E6/E7 HPV-HR18 assay, designed to detect E6/E7 mRNA for eighteen different high-risk (HR) HPV genotypes, were performed. Stainings were visually scored from 0 to 4 by two pathologist based on the average number of dots per cell according to a semi-quantitative analysis; score values were compared using GraphPad Prism 7.0 software. A p&amp;lt; 0.05 was considered statistically significant. Results: The KRT16 mRNA in situ expression in healthy cervical tissue, LSIL and HSIL was determined by CISH in a cervical TMA including, normal cervix: 84, LSIL: 77, and HSIL: 157, tissue cores. The mean KRT16 mRNA expression scores in squamous epithelium lining normal cervix (0.95, 95% CI: 0.70-1.20), LSIL (0.88, 95% CI:0.60-1.15), and HSIL (1.11, 95% CI:0.89-1.33), were not significantly different (p=0.573). Mean HPV E6/E7 mRNA expression score in HSIL was significantly higher than mean HPV E6/E7 mRNA expression in LSIL (p&amp;lt;0.0001). There was a positive-low correlation between KRT16 mRNA expression and E6/E7 HR-HPV mRNA expression (Spearman r =0.27). Conclusions: Although previous cDNA microarray analysis identified differential expression of KRT16 between healthy cervical tissue and a HSIL with confirmed HPV-18 transforming infection, this was not confirmed when comparing its in situ mRNA expression between squamous epithelium lining normal cervix, LSIL and HSIL. Also, there is not a strong correlation with the expression of E6/E7 mRNA for eighteen different HR-HPV. The fact that KRT16 is constitutively expressed in basal cells may help to elucidate this phenomenon. The role of KRT16 in proliferative phase of basal cells and cervical carcinogenesis remains unclear, further studies are required to define it. Citation Format: Ines Benedetti, Reinhard Rodríguez, Lia Barrios. KRT16 and E6-E7 HR-HPV mRNA in situ expression in normal cervical tissue, and low-grade and high-grade squamous intraepithelial lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2153.

Abstract 479: mRNA in situ expression of candidate genes for progression risk tissue biomarkers, in premalignant cervical lesions

Abstract Background Comparing the in situ differences in mRNA expression of candidate genes between normal cervical tissue and premalignant lesions, could contribute to detect progression risk biomarkers. Use of RNAscope chromogenic in situ hybridization (CISH) that amplifies specific mRNA signals and detects molecules as points that can be quantified, could help the detection of these biomarkers. Methods A tissue microarray was constructed from cervical samples (conization and histerectomy). Representative areas of normal cervix, low-grade (LSIL), and high-grade squamous intraepithelial lesions (HSIL) were chosen. RNAscope 2.5HD Red HPV-HR18 assay that detect E6/E7 mRNA for eighteen high-risk (HR) HPV genotypes, and RNAscope 2.5HD Red assays to detect mRNA from: KRT16, TMEM45A and RBP1, were performed. The stainings were scored by two pathologist from 0 to 4 in a semi-quantitative analysis based on the average number of dots per cell; mean score values were compared using GraphPad Prism 7.0. A p&amp;lt;0.05 was considered statistically significant. Results A total of 390 cervical tissues cores from 169 patients with mean age 36.08 years (SD ±10.48) were included. Mean mRNA expression of HR-HPV E6/E7, TMEM45A and RBP1 were significantly higher in HSIL compared to LSIL and normal cervix squamous epithelium. KRT16 mRNA expression scores in normal cervix squamous epithelium, LSIL, and HSIL, were not significantly different (Table 1). Conclusions CISH allowed to detect E6/E7 mRNA molecules for eighteen HR-HPV genotypes in routinely processed tissues, and to evaluate the expression of the described candidate genes in their context without destroying the tissue morphology. Identification of differential expression between normal cervical tissue and HSIL supports the possible role of TMEM45A and RBP1 in cervical cancer progression and its potential use as progression risk tissue biomarkers. Table 1.Biomarkers candidate genes expression profiles in normal cervix, LSIL and HSILCISH scoresMean (±SD)95% CINormal cervixLSILHSILp valueHR-HPV E6/E7 mRNAn=640.4098(±0.7611)0.2149-0.6048n=640.4098 (±0.7611)0.2149-0.6048n=1381.294 (±1.189)1.078-1.51&amp;lt;0.0001*TMEM45A mRNAn=761.105 (± 1.322)0.8031-1.407n=691.232 (±1.477)0.8771-1.587n=2381.514(± 1.491)1.264-1.7650.046*RBP1 mRNAn=660.4242(±0.7658)0.236-0.6125n=740.5135 (±0.9826)0.2859-1.159n=1301.3 (±1.186)1.094-1.506&amp;lt;0.0001*KRT16 mRNAn=840.9524(±1.15)0.7028-1.202n=770.8831 (±1.214)0.6076-1.159n=1571.115 (±1.41)0.8924-1.3370.573* Citation Format: Ines Benedetti, Felipe Ruiz, Reinhard Rodríguez. mRNA in situ expression of candidate genes for progression risk tissue biomarkers, in premalignant cervical lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 479.

Abstract 2530: Differences in mRNA expression of a candidate gene, between normal cervical tissue and cervical premalignant lesions

Abstract Background: Cervical cancer is the fourth most common cancer in women worldwide, with around 85% of cases occurring in underdeveloped countries. The diagnosis of premalignant lesions is ​​the objective of cervical cancer prevention programs, aimed at increasing the chances of cure and survival time. In a previous study of our group cDNA of TMEM45A gene was detected as differentially expressed between healthy cervical tissue and a high grade squamous intraepithelial lesion (HSIL) with confirmed HPV-18 transforming infection. TMEM45A gene family is highly expressed in squamous epithelium, playing roles in differentiation, senescence and chemoresistance in some sort of epithelial carcinomas. The overexpression of this product may confer antiapoptotic pathways, contributing to the development of cervical squamous cell carcinoma. We compared the TMEM45A in situ mRNA expression in healthy cervical tissue, low grade squamous intraepithelial lesion (LSIL) and HSIL, using RNAscope chromogenic in situ hybridization (CISH). Design: TMEM45A expression was evaluated by CISH using a tissue microarray (TMA) constructed from cervical excision samples (conization and histerectomy) from 169 patients, with a mean age of 35.37 years (SD ±10.4), from a reference center for cervical pathology in the Colombian Caribbean. Stained sections from the donor blocks were reviewed by two pathologist, verifying the original sign-out diagnosis, and representative areas from normal cervix, LSIL and HSIL were selected. RNAscope 2.5HD Red TMEM45A assay was performed as a technical service in Advanced Cell Diagnostics´s laboratories (Newark, CA). The staining was visually scored by two pathologist from 0 to 4 based on the average number of dots per cell, according to a semi-quantitative analysis. The mean score values for each lesion group were compared using GraphPad Prism 7.0 software. A p&amp;lt; 0.05 was considered statistically significant. Results: The TMEM45A mRNA in situ expression was determined in a cervical TMA including, normal cervix: 76, LSIL: 69, and HSIL: 138, tissue cores. There was a higher mean staining score for TMEM45A mRNA in HSIL (1.51, 95% CI:1.26-1.76), compared to normal cervix (1.10, 95% CI:0.80-1.40). Mean score of TMEM45A in situ mRNA expression in HSIL was significantly higher than mean TMEM45A in situ mRNA expression in the normal cervix lining squamous epithelium (p=0.046). Conclusions: TMEM45A expression analysis identified differential expression between normal cervix lining squamous epithelium and HSIL. This finding supports the possible role of TMEM45A in carcinogenesis of well differentiated epithelial lesions. Additional studies to detect the TMEM45A expression at protein with samples of women from the Colombian Caribbean are in progress to validate these results. Citation Format: Ines Benedetti, Reinhard Rodríguez, Lia Barrios. Differences in mRNA expression of a candidate gene, between normal cervical tissue and cervical premalignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2530.

Identification and Validation of Autophagy-Related Gene Nomograms to Predict the Prognostic Value of Patients with Cervical Cancer.

Autophagy is a process of engulfing one's own cytoplasmic proteins or organelles and coating them into vesicles, fusing with lysosomes to form autophagic lysosomes, and degrading the contents it encapsulates. Increasing studies have shown that autophagy disorders are closely related to the occurrence of tumors. However, the prognostic role of autophagy genes in cervical cancer is still unclear. In this study, we constructed risk signatures of autophagy-related genes (ARGs) to predict the prognosis of cervical cancer. The expression profiles and clinical information of autophagy gene sets were downloaded from TCGA and GSE52903 queues as training and validation sets. The normal cervical tissue expression profile data from the UCSC XENA website (obtained from GTEx) were used as a supplement to the TCGA normal cervical tissue. Univariate COX regression analysis of 17 different autophagy genes was performed with the consensus approach. Tumor samples from TCGA were divided into six subtypes, and the clinical traits of the six subtypes had different distributions. Further absolute shrinkage and selection operator (LASSO) and multivariable COX regression yielded an autophagy genetic risk model consisting of eight genes. In the training set, the survival rate of the high-risk group was lower than that of the low-risk group (p < 0.0001). In the validation set, the AUC area of the receiver operating characteristic (ROC) curve was 0.772 for the training set and 0.889 for the verification set. We found that high and low risk scores were closely related to TNM stage (p < 0.05). The nomogram shows that the risk score combined with other indicators, such as G, T, M, and N, better predicts 1-, 3-, and 5-year survival rates. Decline curve analysis (DCA) shows that the risk model combined with other indicators produces better clinical efficacy. Immune cells with an enrichment score of 28 showed statistically significant differences related to high and low risk. GSEA enrichment analysis showed the main enrichment being in KRAS activation, genes defining epithelial and mesenchymal transition (EMT), raised in response to the low oxygen level (hypoxia) gene and NF-kB in response to TNF. These pathways are closely related to the occurrence of tumors. Our constructed autophagy risk signature may be a prognostic tool for cervical cancer.

MicroRNA‑148a‑3p inhibits the proliferation of cervical cancer cells by regulating the expression levels of DNMT1 and UTF1

MicroRNAs (miRs) serve a key role in carcinogenesis. miR-148a-3p has been demonstrated to act as a tumor suppressor in several tumors, such as epithelial ovarian cancer and esophageal cancer. However, to the best of our knowledge, the role of miR-148a-3p in cervical cancer remains unclear. In the present study, the expression levels of miR-148a-3p measured by reverse transcription-quantitative PCR were significantly decreased in cervical cancer tissues compared with that in normal cervical tissues. Furthermore, overexpression of miR-148a-3p markedly suppressed the proliferation of cervical cancer cells. The luciferase reporter assay demonstrated that DNA methyltransferase 1 (DNMT1) was the target gene of miR-148a-3p and that its expression measured by western blotting was inhibited by miR-148a-3p in cervical cancer cells. Correlation analysis highlighted that the expression levels of the undifferentiated embryonic cell transcription factor-1 (UTF1) were negatively associated with the expression levels of DNMT1 in cervical cancer tissues. Furthermore, DNMT1 knockdown increased the expression of UTF1 and decreased the methylation level of UTF1 promoter. These data demonstrated the expression levels of UTF1 were regulated by DNMT1 methylation in cervical cancer cells. Collectively, the results of the present study suggested that miR-148a-3p may inhibit the proliferation of cervical cancer cells by regulating the expression levels of DNMT1/UTF1, which provides potential therapeutic targets for cervical cancer.

N6-Methyladenosine Associated Silencing of miR-193b Promotes Cervical Cancer Aggressiveness by Targeting CCND1.

ObjectiveCervical cancer is a frequently encountered gynecological malignancy as a major contributor to cancer-related deaths in women. This study focuses on how miR-193b promotes cervical cancer aggressiveness as well as the role of m6A in miR-193b silencing.MethodsCervical cancer samples and the matching adjacent normal cervical tissues were used to determine the significance of miR-193b in cervical cancer. The CCK-8 assay, cell cycle analysis, qRT-PCR, Western blot assay, IHC, RIP, and xenograft models were utilized to explore the impact of miR-193b in cervical cancer and how m6A regulates miR-193b expression. Luciferase reporter assays, qRT-PCR, and Western blotting were enlisted to study the interaction between miR-193b and CCND1.ResultsOur study suggested that lower miR-193b expressions were strongly linked to more advanced cervical cancer stages and the presence of deeper stromal invasion. miR-193b functions as a tumor suppressor that is regulated by m6A methylation in cervical tumors. METTL3 modulates miR-193b mature process in an m6A-dependent manner. Reintroduction of miR-193b profoundly inhibits tumorigenesis of cervical cancer cells both in vivo and in vitro through CCND1 targeting.Conclusionsm6A associated downregulation of miR-193b promotes cervical cancer aggressiveness by targeting CCND1.

Dynamin 3 overexpression suppresses the proliferation, migration and invasion of cervical cancer cells.

Dynamin 3 (DNM3) functions as a tumor suppressor in various malignancies. However, the underlying mechanism of DNM3 in cervical cancer remains to be elucidated. The present study aimed to indicate the function of DNM3 in cervical cancer. The expression of DNM3 in cervical tissues and cells was measured using bioinformatics analysis, immunohistochemistry and reverse transcription-quantitative PCR. The pcDNA3.1 plasmid was used to overexpress DNM3 in SiHa and C33A cells. The effects of DNM3 overexpression on cell proliferation, migration, invasion and apoptosis was detected by the CCK-8, clone formation, Transwell, flow cytometry and western blotting assays. In the present study, it was revealed that DNM3 was expressed at significantly low levels in cervical cancer tissues and cell lines compared with normal cervical tissues and cell lines. In addition, the low expression of DNM3 was significantly associated with high pathological grading of cervical cancer. The overall survival rate of patients with low DNM3 expression was significantly improved compared with patients with high DNM3 expression. In addition, the overexpression of DNM3 significantly inhibited the proliferation, migration and invasion of cervical carcinoma cells and induced cell apoptosis. The findings of the present study further revealed that the overexpression of DNM3 may inhibit cell migration and invasion by inactivating the epithelial mesenchymal transition process. In summary, the present study demonstrated that DNM3 was a tumor suppressor in cervical cancer progression and that it may serve as a potential prognostic biomarker for patients with cervical carcinoma.

FOXP3‑induced LINC00885 promotes the proliferation and invasion of cervical cancer cells.

Cervical cancer (CC) is the fourth most common type of cancer in women worldwide. LINC00885, a novel oncogenic type of long non-coding RNA, is upregulated in various types of cancer, but its expression in CC remains unknown. The aim of the present study was to investigate the expression of LINC00885 in CC, and its effect on the proliferation and invasion of CC cells in vitro. The expression levels of LINC00885 and forkhead box protein 3 (FOXP3) from The Cancer Genome Atlas (TCGA) were selected to analyze the differences between CC tissues and normal cervical tissues using bioinformatics analyses. Reverse transcription-quantitative (RT-q)PCR was used to detect the relative expression of LINC00885 in CC cell lines, and its expression was knocked down. Cell Counting Kit-8 assays and EdU staining were used to detect the changes in cell proliferative capacity of cells. Transwell experiments were used to examine cell invasion. Dual-luciferase reporter and chromatin immunoprecipitation assays were performed to examine the interactions between LINC00885 and FOXP3. FOXP3 and epithelial-mesenchymal transition (EMT)-related proteins were detected using western blotting. The expression of LINC00885 and FOXP3 in CC tissues and CC cells was significantly higher compared with the normal cervical tissues and normal cervical epithelial cells. FOXP3 could specifically interact with the promoter of LINC00885 and activate LINC00885 transcription. Knockdown of LINC00885 and silencing of FOXP3 significantly inhibited proliferation, invasion and EMT of CC cells. Overexpression of LINC00885 reversed the inhibitory effects of FOXP3 knockdown on the proliferation and invasion of CC cells. Collectively, LINC00885 and FOXP3 may serve as biomarkers for the early diagnosis of CC and as a potential therapeutic target for reversing the malignant phenotype of CC.

Family with sequence similarity 83 member A promotes tumor cell proliferation and metastasis and predicts poor prognosis in cervical cancer

Family with sequence similarity 83 member A (FAM83A) is a member of the FAM83 family and is proven to have oncogenic properties in several cancers. However, the mechanisms of FAM83A in human cervical cancer (CC) progression are unknown. Here, we found that FAM83A is highly expressed in CC tissues and cell lines through western blot and qRT-PCR. We utilized GEO datasets to assess FAM83A expression in CC in comparison to the normal cervical tissue (NCT) (GSE6791), and similarly, in lymph node positive CC compared to the lymph node negative CC (GSE26511). Immunohistochemistry (IHC) was used to quantify FAM83A expression in 20 NCT and 105 CC patient samples. FAM83A expression is upregulated in early-stage CC and correlates with aggressive clinicopathologic features. Moreover, both our hospital’s and TCGA datasets revealed that patients of early-stage CC with higher FAM83A expression had a poorer prognosis. Subsequently, CCK-8 and transwell assays verified that FAM83A promotes proliferation, migration, and invasion of CC cells. Additionally, Gene Set Enrichment Analysis (GSEA) revealed that FAM83A is not only involved in cell development, differentiation, and proliferation but is also correlated with cell junction assembly and cell matrix adhesion. It might also be affiliated with the regulation of tumor necrosis factor-mediated signaling pathway and the regulation of the ErbB signaling pathway in CC. These results indicate that FAM83A promotes tumor cell proliferation, migration, and metastasis. Our study provides novel evidence FAM83A may act as a promising therapeutic target for CC.

Discovery of key genes as novel biomarkers specifically associated with HPV-negative cervical cancer

Cervical cancer is a common female malignancy that is mainly caused by human papillomavirus (HPV) infection. However, the incidence of HPV-negative cervical cancer has shown an increasing trend in recent years. Because the mechanism of HPV-negative cervical cancer development is unclear, this study aims to find the pattern of differential gene expression in HPV-negative cervical cancer and verify the underlying potential mechanism. Differentially expressed genes were compared among HPV-positive cervical cancer, HPV-negative cervical cancer, and normal cervical tissues retrieved from TCGA. Subsequently, dysregulated differentially expressed genes specifically existed in HPV-negative cervical cancer tissues and HPV-negative cell lines were validated by qRT-PCR, western blotting, and immunohistochemical staining. We found seventeen highly expressed genes that were particularly associated with HPV-negative cervical cancer from analysis of TCGA database. Among the 17 novel genes, 7 genes (preferentially expressed antigen in melanoma [PRAME], HMGA2, ETS variant 4 [ETV4], MEX3A, TM7SF2, SLC19A1, and tweety-homologs 3 [TTYH3]) displayed significantly elevated expression in HPV-negative cervical cancer cells and HPV-negative cervical cancer tissues. Additionally, higher expression of MEX3A and TTYH3 was associated with a shorter overall survival of patients with HPV-negative cervical cancer. Our study implies that these seven genes are more likely to provide novel insights into the occurrence and progression of HPV-negative cervical cancer.

Integrated Analysis of ceRNA Regulatory Network Associated With Tumor Stage in Cervical Cancer.

ObjectiveTo analyze the abnormally expressed genes involved in cervical cancer occurrence and development.Materials and MethodsIntegrated bioinformatics methods were used to analyze differentially expressed (DE) RNAs, including mRNAs, microRNAs (miRNAs), and long non-coding RNAs (lncRNAs), in stage I, II, III, and IV cervical cancer patients from the TCGA database to fully reveal the dynamic changes caused by cervical cancer.ResultsFirst, DE RNAs in cervical cancer tissues from stage I, II, III, and IV patients and normal cervical tissues were identified and divided into different profiles. Several DE RNA profiles were down-regulated or up-regulated in stage I, III, and IV patients. GO and KEGG analysis of DE mRNA profile 1, 2, 4, 5, 6 and 22 which were significantly down-regulated or up-regulated showed that DE mRNAs are involved in cell division, DNA replication, cell adhesion, the positive and negative regulation of RNA polymerase ll promoter transcription. Besides, DE RNA profiles with significant differences in patient stages were analyzed to perform a competing endogenous RNA (ceRNA) regulatory network of lncRNA, miRNA, and mRNA. The protein-protein interaction (PPI) network of DE mRNAs in the ceRNA regulatory network was also constructed. The network had nine central genes (up-regulated genes: CDKN2A, GSK3B, BIRC5, CYCS, MAD2L1; down-regulated genes: PTEN, FOXO3, CCND2, TGFBR2). Survival analysis found that 5 lncRNAs, 9 mRNAs, and 4 miRNAs can be used as prognostic indicators of cervical cancer. Finally, combined with cluster analysis results, we further screened 2 DE RNAs (AMZ2P1 and HDAC5) using clinical samples, suggesting that AMZ2P1, and HDAC5 may act as diagnostic biomarkers for the development of cervical cancer.ConclusionThis research provides new effective targets and reliable biological markers for the diagnosis and prognosis of cervical cancer.

Discovery and validation of novel biomarkers for detection of cervical cancer.

AimsTo investigate novel biomarker for diagnosis of cervical cancer, we analyzed the datasets in Gene Expression Omnibus (GEO) and confirmed the candidate biomarker in patient sample.Materials and methodsWe collected major datasets of cervical cancer in GEO, and analyzed the differential expression of normal and cancer samples online with GEO2R and tested the differences, then focus on the GSE63514 to screen the target genes in different histological grades by using the R‐Bioconductor package and R‐heatmap. Then human specimens from the cervix in different histological grades were used to confirm the top 8 genes expression by immunohistochemical staining using Ki67 as a standard control.ResultsWe identified genes differentially expressed in normal and cervical cancer, 274 upregulated genes and 206 downregulated genes. After intersection with GSE63514, we found the obvious tendency in different histological grades. Then we screened the top 24 genes, and confirmed the top 8 genes in human cervix tissues. Immunohistochemical (IHC) results confirmed that keratin 17 (KRT17) was not expressed in normal cervical tissues and was over‐expressed in cervical cancer. Cysteine‐rich secretory protein‐2 (CRISP2) was less expressed in high‐grade squamous intraepithelial lesions (HSILs) than in other histological grades.ConclusionFor the good repeatability and consistency of KRT17 and CRISP2, they may be good candidate biomarkers. Combined analysis of KRT17, CRISP2 expression at both genetic and protein levels can determine different histological grades of cervical squamous cell carcinoma. Such combined analysis is capable of improving diagnostic accuracy of cervical cancer.

Guanosine monophosphate synthase upregulation mediates cervical cancer progression by inhibiting the apoptosis of cervical cancer cells via the Stat3/P53 pathway.

Guanosine monophosphate synthase (GMPS) participates in chromatin and gene regulation in multiple types of organisms, and is highly expressed in a variety of human malignancies. The purpose of the present study was to explore the expression of GMPS and its role in cervical cancer (CC), and to provide ideas for improving the clinical efficacy of CC treatment. In the present study, immunohistochemistry, reverse transcription-quantitative PCR analysis, Cell Counting Kit-8 assay, 5-ethynyl-2′-deoxyuridine assay, flow cytometry, western blotting and immunofluorescence assays were conducted to detect the expression of GMPS in normal cervical tissues, CC tissues, para-cancerous tissues and CC cell lines. Moreover, the present study detected the effect of GMPS knockdown on CC cell proliferation, clonal formation ability, aging and apoptosis, as well as on the expression levels of apoptosis-related proteins in tumor cells. The present results demonstrated that the expression level of GMPS in CC was significantly higher compared with that of adjacent tissues; the expression rate of GMPS in CC was 57.36%. GMPS expression was found to successively and gradually increase from that in normal cervical tissues, to that in cervical intraepithelial neoplasia and CC tissues. The abnormal expression of GMPS was positively associated with the degree of CC differentiation and the depth of early invasion. Small interfering (si) RNA knockdown of GMPS inhibited proliferation and colony formation, and promoted aging and apoptosis of CC cells. Furthermore, subcutaneous injection of GMPS-knockdown tumor cells in nude mice resulted in a decrease in the proliferative ability of the tumor. The animal experimental results showed that the tumor growth rate of the short hairpin (sh) RNA-GMPS group was significantly slower than that of the HeLa sh-negative control group. It was identified that GMPS may inhibit CC cell senescence and apoptosis via the Stat3/P53 molecular pathway. Collectively, the present results suggested that GMPS may be a marker of unfavorable prognosis of CC, and it may also be a potential therapeutic target for CC.