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Abstract Backgrounds Although the role of high‐risk human papillomavirus (HPV) E6 and E7 in cellular malignant transformation has been elucidated， the function of both genes in cellular homeostasis is still unknown. Autophagy functions in maintenance of cellular homeostasis play a key role in the initiation and development of cancer and infectious disease. Methods Cervical cancer cell lines SiHa and CaSki were utilized in this study. Results We found that HPV 16E6/E7 (16E6/E7) downregulation inhibited autophagy, and consequently suppressed cell proliferation and promoted early apoptosis. Transcriptome sequencing demonstrated that Atg9B and LAMP1 were downregulated in 16E6/E7 knockdown cells. Gene function experiments revealed that 16E6/E7 downregulation depressed Atg9B and LAMP1, and Atg9B and LAMP1 overexpression compensated, at least partially, autophagy blockage induced by 16E6/E7 knockdown. Immunoprecipitation assay showed that 16E7 interacted with Atg9B and dual‐luciferase reporter system revealed that 16E6 most likely regulated −1750 to −2000 nt in Atg9B and −1800 to −2000 nt in LAMP1 promoter region. Conclusions Our findings verified that 16E6/E7 activated autophagy via accelerating autophagosome formation and degradation, and Atg9B and LAMP1 were involved in the process of 16E6/E7 modulating autophagy, suggesting that targeting autophagy may be a potential approach in cervical cancer therapeutics.

RNA methylation has recently emerged as an important category of epigenetic modifications, which plays diverse physiopathological roles in various cancers. Recent studies have confirmed the presence of 5-methylcytosine (m5C) modification on mammalian mRNAs, mainly modified by NOP2/Sun RNA methyltransferase family member 2 (NSUN2), but little is known about the underlying functions of m5C. Gynecologic cancers are malignancies starting from women's reproductive organs. The prevalence of gynecologic cancers leads to a massive economic burden and public health concern. In this study, we investigated the potential biological functions of NSUN2 in common gynecologic cancers including cervical cancer, ovarian cancer, and endometrial cancer. Remarkably, distinct scenarios were found. The levels of NSUN2 did not show alteration in endometrial cancer, and in ovarian cancer, depletion of upregulated NSUN2 did not reduce carcinogenesis in cancer cells, suggesting that the upregulated NSUN2 might be an incidental effect. On the contrary, NSUN2 played a role in tumorigenesis of cervical cancer; depletion of upregulated NSUN2 notably inhibited migration and invasion of cancer cells, and only wild-type but not catalytically inactive NSUN2 rescued these malignant phenotypes of cancer cells. Mechanistically, NSUN2 promoted migration and invasion by leading to m5C methylation on keratin 13 (KRT13) transcripts, and methylated KRT13 transcripts would be recognized and stabilized by an m5C reader, Y-box binding protein 1 (YBX1). Collectively, these results not only displayed the nature of diversity among human malignancies, but also demonstrated a novel NSUN2-dependent m5C-YBX1-KRT13 oncogenic regulatory pathway. [ABSTRACT FROM AUTHOR]

Purpose: This study aims to validate the value of microRNA (miRNA) detection for triaging human papillomavirus (HPV)-positive women in the general population. Patients and Methods: miR-375 detection in cervical exfoliated cells has been demonstrated to have the superior value to cytology in triaging primary HPV-positive women in the hospital population. In this study, residual samples of cervical exfoliated cells from 10,951 women in a general population were used to detect miRNA. The performance efficiency of miRNA detection in identifying high-grade cervical intraepithelial neoplasia (CIN) was evaluated. Pearson chi-square test and McNemar pairing test were used to compare miRNA detection and cytology. Results: In valid 9,972 women aged 25–65, miR-375 expression showed a downward trend along with an increase in cervical lesion severity. The expression level of miR-375 ≤1.0 × 10-3 was identified as positive. In the HPV-positive and 12 HPV genotypes other than 16/18 (HR12)-positive women, miR-375 detection showed equivalent sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) to that of cytology (≥ASC-US) and higher or similar sensitivity and NPV but lower specificity and PPV than that of cytology (≥ASC-H) in identifying CIN3+ and CIN2+. In HPV 16-positive women, miR-375 positivity had higher sensitivity and NPV but lower specificity and PPV than that of cytology (≥ASC-H and HSIL) in identifying CIN3+ and CIN2+. The immediate CIN3+ risk of miR-375 positivity was 19.8% (61/308) in HPV-positive, 10.8% (22/204) in HR12-positive, and 43.5% (37/85) in HPV16-positive women, respectively. Conclusion: The detection of miR-375 in cervical exfoliated cells may be an optional method for triaging primary HPV-positive women in population-based cervical cancer screening. [ABSTRACT FROM AUTHOR]

Background: The literature reports conflicting results regarding the effect of human papillomavirus (HPV) genotype 16 (HPV-16)/18 (HPV-18) positivity on cervical cancer (CC) prognosis. Aim: To conduct a meta-analysis to examine the effect of HPV-16/18 positivity on the prognosis of patients with CC. Methods: PubMed, Embase, and the Cochrane Library were searched for available papers published up to March 2020. The main outcome was the hazard ratio (HR) of overall survival (OS) or disease-free survival (DFS) comparing HPV-16 or HPV-18 positivity and negativity. The random-effects model was used for synthesizing survival outcomes. Results: Nine studies and 2,028 patients were included. Four studies reported OS in HPV-16 positivity, and no association was found between HPV-16 positivity and OS to CC (HR = 0.79, 95% CI: 0.26–2.39, P = 0.675). Three studies reported DFS in HPV-16 positivity, and no association was found between HPV-16 positivity and DFS to CC (HR = 0.80, 95% CI: 0.30–2.11, P = 0.654). Two studies reported DFS in HPV-18 positivity, and no association was found between HPV-18 positivity and DFS to CC (HR = 0.99, 95% CI: 0.55–1.78, P = 0.984). One study reported progression-free survival (PFS) in HPV-18 positivity, and an association was observed between HPV-18 positivity and PFS to CC (HR = 2.66, 95% CI: 1.44–4.94, P = 0.002). The sensitivity analyses showed that one study biased the analysis of the association between HPV-16 and OS, and another study biased the association between HPV-16 and DFS. Conclusion: The presence of HPV-16 and HPV-18 positivity appears to have no significant association with prognosis in CC in either OS or PFS. The presence of HPV-16 or HPV-18 positivity has no significant association with prognosis in CC in either OS or PFS. [ABSTRACT FROM AUTHOR]

Background Deep learning has presented considerable potential and is gaining more importance in computer assisted diagnosis. As the gold standard for pathologically diagnosing cervical intraepithelial lesions and invasive cervical cancer, colposcopy-guided biopsy faces challenges in improving accuracy and efficiency worldwide, especially in developing countries. To ease the heavy burden of cervical cancer screening, it is urgent to establish a scientific, accurate and efficient method for assisting diagnosis and biopsy. Methods The data were collected to establish three deep-learning-based models. For every case, one saline image, one acetic image, one iodine image and the corresponding clinical information, including age, the results of human papillomavirus testing and cytology, type of transformation zone, and pathologic diagnosis, were collected. The dataset was proportionally divided into three subsets including the training set, the test set and the validation set, at a ratio of 8:1:1. The validation set was used to evaluate model performance. After model establishment, an independent dataset of high-definition images was collected to further evaluate the model performance. In addition, the comparison of diagnostic accuracy between colposcopists and models weas performed. Results The sensitivity, specificity and accuracy of the classification model to differentiate negative cases from positive cases were 85.38%, 82.62% and 84.10% respectively, with an AUC of 0.93. The recall and DICE of the segmentation model to segment suspicious lesions in acetic images were 84.73% and 61.64%, with an average accuracy of 95.59%. Furthermore, 84.67% of high-grade lesions were detected by the acetic detection model. Compared to colposcopists, the diagnostic system performed better in ordinary colposcopy images but slightly unsatisfactory in high-definition images. Implications The deep learning-based diagnostic system could help assist colposcopy diagnosis and biopsy for HSILs. [ABSTRACT FROM AUTHOR]

Purpose: To determine whether early proteins from high-risk human papillomavirus (HPV) have the capacity to maintain cellular stemness. Patients and methods: First, we isolated cancer stem cell like cells from two cervical cancer cell lines, SiHa and CaSki, using non-adhesive culture with serum-free medium. Second, we knocked down HPV16 E7 in SiHa sphere cells and overexpressed HPV16 E7 in U2OS sphere cells. Third, we used RNA-seq analysis and Western blotting to screen and identify the expression of differentially expressed genes in SiHa cells with HPV16 E7 knockdown. Results: We found that both SiHa and CaSki cells grew as cell spheres (oncospheres) and shared the properties of cancer stem cells, including high expression of stem cell marker OCT4 and SOX2, self-renew, and resistance to chemotherapeutic drugs. The stem-like properties were deprived when HPV16 E7 was knocked down in SiHa sphere cells and maintained when HPV16 E7 was over-expressed in U2OS sphere cells. APH1B was up-regulated, among differential expression genes, in SiHa cells with HPV16 E7 knockdown and modulated cellular stemness and SiHa sphere cells with APH1B knockdown regained the stem-like properties deprived by E7 inhibition. Conclusion: HPV16 E7 possesses the capacity to maintain cellular stemness and APH1B may participate in this process in cervical cancer sphere cells. [ABSTRACT FROM AUTHOR]

Cytology-based cervical cancer screening is restricted because of a lack of cytologists. Thus, HPV-based instead of cytology-based screening may be a more suitable strategy in China. Here, we assessed the effectiveness of HPV testing (Cobas® 4800 Test, Roche) and HPV-based programs to detect high-grade cervical intraepithelial neoplasia ( CIN) or cancer compared with cytology (Thinprep, Hologic) and cytology-based programs through a cross-sectional study in 11,064 Chinese women aged 21-65 years who were enrolled from Longyou County in Zhejiang Province, China. The rates of HPV positivity and cytology abnormality were 9.8% and 6.1%, respectively. The HPV positivity rate had two age peaks, 21-24 (15.4%) and 60-65 (14.4%) years. According to adjusted data, HPV testing demonstrated significantly higher sensitivity and negative predictive value ( NPV) than cytology for detecting CIN2 or worse (90.0% vs. 66.7%, 99.9% vs. 99.5%), and there was an acceptable specificity (91.3%) and positive predictive value ( PPV, 12.5%). Furthermore, primary HPV testing with type 16/18 genotyping showed the highest sensitivity (78.6%) and NPV (99.7%) among four screening strategies, and there was similar specificity (96.8%) and PPV (23.9%) compared with co-testing screening to detect CIN2+, while there were fewer colposcopies (4.2) and tests (106.3) performed than with co-testing and primary cytology screening to detect a case of high-grade CIN. The differences in effectiveness were approximately similar when CIN3+ was the identifying target. Our findings suggest that primary HPV testing with type 16/18 genotyping has a higher sensitivity and NPV, possesses optimal cost/effectiveness in the first round of screening and is a feasible strategy of cervical cancer screening for Chinese women. [ABSTRACT FROM AUTHOR]

Background: The integration of HR-HPV genome into host DNA is regarded as a key step for the development of cervical cancer. However, HR-HPV genome indeed exists as episome except for integrant. It may be alternative mechanisms in episome-associated carcinogenesis, although, by which HPV 16 episome induces cervical carcinogenesis is unclear now.Methods: Ninety-three invasive cervical cancer tissues with HPV16 positive were collected. Viral physical status was calculated from comparing E2 to E6-copies and detection of viral load was made with realtime-PCR using copy numbers of E6. HPV16 E6 mRNA transcript levels were measured by realtime-PCR. The methylation frequency of HPV16 promoter was detected by PCR and pyrosequencing.Results: In 93 samples, 21.5% (20/93) presented purely integrated viral genome, 53.8% (50/93) mixed viral genome, and 24.7% (23/93) purely episomal viral genome. Mean E6 expression in samples with purely episomal viral genomes was 7.13-fold higher than that with purely integrated viral genomes. Meanwhile, viral load in samples with purely episomal viral genomes was 4.53-fold higher than that with purely integrated viral genomes. E6 mRNA expression increased with the viral load in purely episomal cases. There were no differences of mean methylation frequency between purely episomal and integrated virus and among five CpG positions of HPV16 promoter for all samples. And there also was no correlation between E6 mRNA expression and methylation of HPV16 promoter among all samples with purely HPV16 episomal virus.Conclusions: HPV16 with the purely episomal viral genomes exists in a definite proportion of invasive cervical cancer, and episomal HPV16 also overexpresses E6 mRNA, probably through a high level of viral load. [ABSTRACT FROM AUTHOR]

Cervical cancer is the most common cause of cancer-related deaths in women from developing countries. Identification of novel prognostic predictors or therapeutic targets may improve patient prognosis. In the current study, we demonstrated by real-time PCR that miR-224 expression was significantly upregulated (1.82-fold, P = 0.0025) in cervical cancer tissues (n = 126) compared with in normal cervical tissues (n = 64). Higher expression of miR-224 was significantly associated with poorer prognostic factors, including advanced FIGO stage, nodal metastasis, larger tumor size, vascular involvement and deep stromal invasion (all P < 0.05). Enforced expression of miR-224 promoted cell proliferation, migration and invasion in SiHa and CaSki cancer cell lines. Bioinformatic analysis indicated that RASSF8 (RAS-association domain family 8) was a potential target of miR-224. Western blot analysis and luciferase reporter assay showed that overexpressed miR-224 inhibited RASSF8 protein expression and decreased the activity of a luciferase reporter containing the 3′ untranslated region (UTR) of RASSF8, respectively. Further, RASSF8 knockdown by specific RNAi showed similar effects in cervical cancer cells transfected with miR-224 mimic. Our findings suggest that miR-224 directly targets RASSF8 and thereby acts as a tumor promoter in cervical cancer progression. [ABSTRACT FROM AUTHOR]

Aberrant expression of classical cadherins has been observed in tumor invasion and metastasis, but its involvement in cervical carcinogenesis and cancer progression is not clear. We investigated E-, P- and N-cadherin expression and its significance in cervical squamous cell carcinoma (SCC) and cervical intraepithelial neoplasia (CIN). This retrospective study enrolled 508 patients admitted to Women's Hospital, School of Medicine, Zhejiang University with cervical lesions between January 2006 and December 2010. Immunochemical staining was performed in 98 samples of normal cervical epithelium (NC), 283 of CIN, and 127 of early-stage SCC. The association of cadherin staining with clinical characteristics and survival of the patients was evaluated by univariate and multivariate analysis. We found gradients of decreasing E-cadherin expression and increasing P-cadherin expression from NC through CIN to SCC. Aberrant E-cadherin and P-cadherin expression were significantly associated with clinical parameters indicating poor prognosis and shorter patient survival. Interestingly, we found very low levels of positive N-cadherin expression in CIN and SCC tissues that were not related to CIN or cancer. Pearson chi-square tests showed that E-cadherin expression in SCC was inversely correlated with P-cadherin expression (E-P switch), and was not correlated with N-cadherin expression. More important, patients with tissues exhibiting an E-P switch in expression had highly aggressive phenotypes and poorer prognosis than those without E-P switch expression. Our findings suggest that E-cadherin and P-cadherin, but not N-cadherin staining, might be useful in diagnosing CIN and for predicting prognosis in patients with early-stage SCC. [ABSTRACT FROM AUTHOR]

Acquired chemo-resistance is one of the key causal factors in cancer death. Emerging evidences suggest that miRNA and epithelial–mesenchymal transition play critical roles in the chemo-resistance in cancers. Here, we showed the association of paclitaxel-resistance with miR-375 over-expression and epithelial–mesenchymal transition inducement in cervical cancer. Using different cervical cancer cell models, we found that paclitaxel transiently induced up-regulation of miR-375 expression, proliferation inhibition, transition from epithelial to mesenchymal phenotype, and consequently impaired paclitaxel sensitivity. Forced over-expression of miR-375 may suppress Ecadherin expression by a directly targeting pathway, which led to paclitaxel resistance. Contrarily, re-expression of Ecadherin partly reversed epithelial–mesenchymal transition phenotype and miR-375 induced paclitaxel-resistance. Our findings suggest that paclitaxel-induced miR-375 over-expression facilitates epithelial–mesenchymal transition process via directly targeting Ecadherin, proliferation inhibition, and consequently results in chemo-resistance in cervical cancer cells. A reversion of miR-375 or Ecadherin expression may be a novel therapeutic approach for overcoming chemo-resistance in cervical cancer. [ABSTRACT FROM AUTHOR]

To identify new genetic risk factors for cervical cancer, we conducted a genome-wide association study in the Han Chinese population. The initial discovery set included 1,364 individuals with cervical cancer (cases) and 3,028 female controls, and we selected a 'stringently matched samples' subset (829 cases and 990 controls) from the discovery set on the basis of principal component analysis; the follow-up stages included two independent sample sets (1,824 cases and 3,808 controls for follow-up 1 and 2,343 cases and 3,388 controls for follow-up 2). We identified strong evidence of associations between cervical cancer and two new loci: 4q12 (rs13117307, Pcombined, stringently matched = 9.69 × 10−9, per-allele odds ratio (OR)stringently matched = 1.26) and 17q12 (rs8067378, Pcombined, stringently matched = 2.00 × 10−8, per-allele ORstringently matched = 1.18). We additionally replicated an association between HLA-DPB1 and HLA-DPB2 (HLA-DPB1/2) at 6p21.32 and cervical cancer (rs4282438, Pcombined, stringently matched = 4.52 × 10−27, per-allele ORstringently matched = 0.75). Our findings provide new insights into the genetic etiology of cervical cancer. [ABSTRACT FROM AUTHOR]

NEDD9, a focal adhesion scaffolding protein, has been recently proposed to regulate invasion and metastasis in some cancer types, but unknown in cervical cancer. The aim of this study was to determine if NEDD9 was involved in the progression and metastasis of cervical cancer. The experimental results showed NEDD9 protein was overexpressed in cervical cancer compared with normal cervical epithelium tissues. Overexpression of NEDD9 was correlated with histological grading, lymph node metastasis, and FIGO stage of cervical cancer. Silencing NEDD9 resulted in tyrosine dephosphorylation of FAK and SRC oncoproteins, and decreased cell migration and invasion in the cervical carcinoma SiHa and HeLa cells. Overexpression of NEDD9 led to tyrosine phosphorylation of FAK and SRC oncoproteins, and increased cell migration and invasion. Moreover, tyrosine phosphorylation of NEDD9 was significantly decreased via suppressing tyrosine phosphorylation of FAK or SRC, suggesting a positive feedback loop of tyrosine phosphorylation between NEDD9 and FAK or SRC. In addition, our data showed that silencing NEDD9 decreased Vimentin expression and increased E-cadherin expression in cervical cancer cells, and vice versa. E-cadherin was subject to regulation of NEDD9, FAK and SRC, but altered neither tyrosine-phosphorylated nor total NEDD9. Our findings suggest that NEDD9 is overexpressed in cervical cancer tissues and cells, and overexpressed NEDD9 promotes migration and invasion in cervical carcinoma cells, probably via a positive feedback loop of tyrosine phosphorylation between NEDD9 and FAK or SRC. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Neoadjuvant chemotherapy (NACT) for cervical cancer still remains controversial. NACT was evaluated to establish selection criteria. Methods: A matched-case comparison was designed for the NACT group (n =707) and primary surgery treatment (PST; n =707) group to investigate short-term responses and high/intermediate risk factors (HRFs/IRFs). The 5-year disease-free survival (DFS) and overall survival (OS) rates were stratified by NACT response, HRFs/IRFs, International Federation of Gynecology and Obstetrics (FIGO) stage and tumour size, respectively. Results: The clinical and pathological response rates were 79.3% and 14.9% in the NACT group. In comparison to the PST group, IRFs but not HRFs were significantly decreased (P <0.05), and the 5-year DFS rate was significantly improved in the NACT group (88.4% versus 83.1%, P =0.021). Moreover, the 5-year DFS and OS rates were favourably increased in the clinical responders in comparison to the PST group and the clinical non-responders (P< 0.05). Compared to those of clinical non-responders, the 5-year DFS and OS rates of clinical responders, with or without HRFs, were also significantly increased (P< 0.01). In stage IB2, the 5-year DFS and OS rates were significantly increased, whereas operation duration declined in the NACT group (P< 0.05). For patients with stage IB tumours of 2–5cm, the 5-year DFS and OS rates of clinical responders were significantly improved (P <0.05). Conclusions: NACT is a suitable option for patients with cervical cancer, especially for NACT responders and patients with stage IB, which provides a new concept of fertility preservation for young patients. [Copyright &y& Elsevier]

Abstract: Objective: Transcriptional silencing of HPV oncogenes using short interfering RNA (siRNA) blocks E6/E7 expression. Our objective was to estimate the effective value of E6/E7 specific siRNA-induced transcriptional gene silencing as a potential therapeutic strategy for cervical cancer. Methods: In vitro studies were performed by employing two categories of siRNA targeting promoter of E6/E7 gene and E7 transcript, respectively, and inhibitory effect of both siRNAs was further observed in vitro and on xenograft in BALB/c mice that were inoculated with siRNA transfected SiHa cells and parental SiHa cells followed by siRNA intratumoral injection in vivo. Tumor volume and growth curves were assessed. Furthermore, cellular proliferation and apoptosis of inoculated tumors were determined by immunohistochemistry staining and TUNEL assay. Results: The two most active siRNA sequences specifically knockdown E6/E7 expressions at mRNA level in HPV16 positive Siha cells, increased p53 and decreased p16 expressions at protein level, inhibited cell proliferation, and induced cell apoptosis in vitro. Furthermore, both siRNAs effectively inhibited tumor formation and growth no matter in mice with siRNA transfected cells in vitro or with siRNA intratumoral injection in vivo. TUNEL staining and FCM assay consistently showed that tumor retardation was through induction of cellular apoptosis. Conclusion: RNAi targeting the promoter of HPV16 E6/E7 acts effectively in vitro and in vivo, especially through intratumoral delivery, and may be a candidate therapeutic strategy for cervical cancer. [Copyright &y& Elsevier]