Identification of Deregulated Pathways, Key Regulators, and Novel miRNA-mRNA Interactions in HPV-Mediated Transformation.

Next to a persistent infection with high-risk human papillomavirus (HPV), molecular changes are required for the development of cervical cancer. To identify which molecular alterations drive carcinogenesis, we performed a comprehensive and longitudinal molecular characterization of HPV-transformed keratinocyte cell lines. Comparative genomic hybridization, mRNA, and miRNA expression analysis of four HPV-containing keratinocyte cell lines at eight different time points was performed. Data was analyzed using unsupervised hierarchical clustering, integrated longitudinal expression analysis, and pathway enrichment analysis. Biological relevance of identified key regulatory genes was evaluated in vitro and dual-luciferase assays were used to confirm predicted miRNA-mRNA interactions. We show that the acquisition of anchorage independence of HPV-containing keratinocyte cell lines is particularly associated with copy number alterations. Approximately one third of differentially expressed mRNAs and miRNAs was directly attributable to copy number alterations. Focal adhesion, TGF-beta signaling, and mTOR signaling pathways were enriched among these genes. PITX2 was identified as key regulator of TGF-beta signaling and inhibited cell growth in vitro, most likely by inducing cell cycle arrest and apoptosis. Predicted miRNA-mRNA interactions miR-221-3p_BRWD3, miR-221-3p_FOS, and miR-138-5p_PLXNB2 were confirmed in vitro. Integrated longitudinal analysis of our HPV-induced carcinogenesis model pinpointed relevant interconnected molecular changes and crucial signaling pathways in HPV-mediated transformation.

Altered microRNA processing proteins in HPV-induced cancers.

High-risk human papilloma virus (hrHPV) infections are associated with the development of anogenital cancers, in particular cervical cancer, and a subset of head and neck cancers. Previous studies have shown that microRNAs (miRNAs) contribute to the development and progression of HPV-induced malignancies. miRNAs are small non-coding RNAs that exist as multiple length and sequence variants, termed isomiRs. Efficient processing of miRNAs and generation of isomiRs is accomplished by several processing proteins. Deregulation of Drosha, AGO2, and TENT2, among others, has been observed in HPV-induced cancers and was even found at the precancerous stage. This suggests that miRNA processing proteins may be involved during early cancer development and that the generated isomiRs could provide promising biomarkers for early cancer diagnosis.

Complementarity between miRNA expression analysis and DNA methylation analysis in hrHPV-positive cervical scrapes for the detection of cervical disease.

Cervical screening by high-risk HPV (hrHPV) testing requires additional risk stratification (triage), as most infections are transient and only a subset of hrHPV-positive women harbours clinically relevant disease. Molecular triage markers such as microRNAs (miRNAs) and DNA methylation markers are particularly promising, as they can be objectively tested directly on hrHPV-positive scrapes and cervicovaginal self-samples. Here, we evaluated the marker potential of 10 candidate miRNAs in 209 hrHPV-positive scrapes of women with underlying precancer (cervical intraepithelial neoplasia, grade 2-3 (CIN2-3)), cancer, or without disease (CIN0/1). A predictive miRNA classifier for CIN3 detection was built using logistic regression, which was compared to and combined with DNA methylation marker FAM19A4. Markers were correlated to histology parameters and hrHPV genotype. A miRNA classifier consisting of miR-149, miR-20a, and miR-93 achieved an area under the curve (AUC) of 0.834 for CIN3 detection, which was not significantly different to that of FAM19A4 methylation (AUC: 0.862, p = 0.591). Combining miRNA and methylation analysis demonstrated complementarity between both marker types (AUC: 0.939). While the miRNA classifier seemed more predictive for CIN2, FAM19A4 methylation was particularly high in HPV16-positive and histologically advanced CIN3, i.e. CIN3 with high lesion volume. The miRNA classifier, FAM19A4 methylation, and the miRNA/methylation combination were highest in cancer-associated scrapes. In conclusion, a panel of three miRNAs is discriminatory for CIN3 in hrHPV-positive scrapes and can complement DNA methylation analysis for the efficient detection of cervical disease. Combined analysis of the two marker types warrants further evaluation as triage strategy in hrHPV-based screening.

miR-9-5p Exerts a Dual Role in Cervical Cancer and Targets Transcription Factor TWIST1.

Squamous cell carcinoma (SCC) and adenocarcinoma (AC) represent the major cervical cancer histotypes. Both histotypes are caused by infection with high-risk HPV (hrHPV) and are associated with deregulated microRNA expression. Histotype-dependent expression has been observed for miR-9-5p, showing increased expression in SCC and low expression in AC. Here, we studied the regulation and functionality of miR-9-5p in cervical SCCs and ACs using cervical tissue samples and hrHPV-containing cell lines. Expression and methylation analysis of cervical tissues revealed that low levels of miR-9-5p in ACs are linked to methylation of its precursor genes, particularly miR-9-1. Stratification of tissue samples and hrHPV-containing cell lines suggested that miR-9-5p depends on both histotype and hrHPV type, with higher expression in SCCs and HPV16-positive cells. MiR-9-5p promoted cell viability and anchorage independence in cervical cancer cell lines SiHa (SCC, HPV16) and CaSki (metastasized SCC, HPV16), while it played a tumor suppressive role in HeLa (AC, HPV18). TWIST1, a transcription factor involved in epithelial-to-mesenchymal transition (EMT), was established as a novel miR-9-5p target. Our results show that miR-9-5p plays a dual role in cervical cancer in a histotype- and hrHPV type-dependent manner. MiR-9-5p mediated silencing of TWIST1 suggests two distinct mechanisms towards EMT in cervical cancer.

Genome-wide microRNA analysis of HPV-positive self-samples yields novel triage markers for early detection of cervical cancer.

Offering self-sampling for HPV testing improves the effectiveness of current cervical screening programs by increasing population coverage. Molecular markers directly applicable on self-samples are needed to stratify HPV-positive women at risk of cervical cancer (so-called triage) and to avoid over-referral and overtreatment. Deregulated microRNAs (miRNAs) have been implicated in the development of cervical cancer, and represent potential triage markers. However, it is unknown whether deregulated miRNA expression is reflected in self-samples. Our study is the first to establish genome-wide miRNA profiles in HPV-positive self-samples to identify miRNAs that can predict the presence of CIN3 and cervical cancer in self-samples. Small RNA sequencing (sRNA-Seq) was conducted to determine genome-wide miRNA expression profiles in 74 HPV-positive self-samples of women with and without cervical precancer (CIN3). The optimal miRNA marker panel for CIN3 detection was determined by GRridge, a penalized method on logistic regression. Six miRNAs were validated by qPCR in 191 independent HPV-positive self-samples. Classification of sRNA-Seq data yielded a 9-miRNA marker panel with a combined area under the curve (AUC) of 0.89 for CIN3 detection. Validation by qPCR resulted in a combined AUC of 0.78 for CIN3+ detection. Our study shows that deregulated miRNA expression associated with CIN3 and cervical cancer development can be detected by sRNA-Seq in HPV-positive self-samples. Validation by qPCR indicates that miRNA expression analysis offers a promising novel molecular triage strategy for CIN3 and cervical cancer detection applicable to self-samples.

Triage of high-risk HPV-positive women in population-based screening by miRNA expression analysis in cervical scrapes; a feasibility study.

Background: Primary testing for high-risk HPV (hrHPV) is increasingly implemented in cervical cancer screening programs. Many hrHPV-positive women, however, harbor clinically irrelevant infections, demanding additional disease markers to prevent over-referral and over-treatment. Most promising biomarkers reflect molecular events relevant to the disease process that can be measured objectively in small amounts of clinical material, such as miRNAs. We previously identified eight miRNAs with altered expression in cervical precancer and cancer due to either methylation-mediated silencing or chromosomal alterations. In this study, we evaluated the clinical value of these eight miRNAs on cervical scrapes to triage hrHPV-positive women in cervical screening. Results: Expression levels of the eight candidate miRNAs in cervical tissue samples (n = 58) and hrHPV-positive cervical scrapes from a screening population (n = 187) and cancer patients (n = 38) were verified by quantitative RT-PCR. In tissue samples, all miRNAs were significantly differentially expressed (p < 0.05) between normal, high-grade precancerous lesions (CIN3), and/or cancer. Expression patterns detected in cervical tissue samples were reflected in cervical scrapes, with five miRNAs showing significantly differential expression between controls and women with CIN3 and cancer. Using logistic regression analysis, a miRNA classifier was built for optimal detection of CIN3 in hrHPV-positive cervical scrapes from the screening population and its performance was evaluated using leave-one-out cross-validation. This miRNA classifier consisted of miR-15b-5p and miR-375 and detected a major subset of CIN3 as well as all carcinomas at a specificity of 70%. The CIN3 detection rate was further improved by combining the two miRNAs with HPV16/18 genotyping. Interestingly, both miRNAs affected the viability of cervical cancer cells in vitro. Conclusions: This study shows that miRNA expression analysis in cervical scrapes is feasible and enables the early detection of cervical cancer, thus underlining the potential of miRNA expression analysis for triage of hrHPV-positive women in cervical cancer screening.

A Strategy to Find Suitable Reference Genes for miRNA Quantitative PCR Analysis and Its Application to Cervical Specimens.

miRNAs represent an emerging class of promising biomarkers for cancer diagnostics. To perform reliable miRNA expression analysis using quantitative PCR, adequate data normalization is essential to remove nonbiological, technical variations. Ideal reference genes should be biologically stable and reduce technical variability of miRNA expression analysis. Herein is a new strategy for the identification and evaluation of reference genes that can be applied for miRNA-based diagnostic tests without entailing excessive additional experiments. We analyzed the expression of 11 carefully selected candidate reference genes in different types of cervical specimens [ie, tissues, scrapes, and self-collected cervicovaginal specimens (self-samples)]. To identify the biologically most stable reference genes, three commonly used algorithms (GeNorm, NormFinder, and BestKeeper) were combined. Signal-to-noise ratios and P values between control and disease groups were calculated to validate the reduction in technical variability on expression analysis of two marker miRNAs. miR-423 was identified as a suitable reference gene for all sample types, to be used in combination with RNU24 in cervical tissues, RNU43 in scrapes, and miR-30b in self-samples. These findings demonstrate that the choice of reference genes may differ between different types of specimens, even when originating from the same anatomical source. More important, it is shown that adequate normalization increases the signal-to-noise ratio, which is not observed when normalizing to commonly used reference genes.

An evidence based strategy for normalization of quantitative PCR data from miRNA expression analysis in forensic organ tissue identification.

Messenger-RNA (mRNA)-based analysis of organ tissues and their differentiation in complex crime stains has recently been introduced as a potential and powerful tool to forensic genetics. Given the notoriously low quality of many forensic samples it seems advisable, though, to substitute mRNA with micro-RNA (miRNA) which is much less susceptible to degradation. However, reliable miRNA detection and quantification using quantitative PCR requires a solid and forensically relevant normalization strategy. In our study we evaluated a panel of 15 carefully selected reference genes for their suitability as endogenous controls in miRNA qPCR normalization in forensically relevant settings. We analyzed assay performances and expression variances in 35 individual samples and mixtures thereof integrating highly standardized protocols with contemporary methodologies and included several well-established computational algorithms. Based on these empirical results, we recommend SNORD48, SNORD24, and RNU6-2 as endogenous references since these exhibit the most stable expression levels and the least expected variation among the evaluated candidate reference genes in the given set of forensically relevant organ tissues including skin. To account for the lack of consensus on how best to perform and interpret quantitative PCR experiments, our study's documentation is according to MIQE guidelines, defining the "minimum information for publication of quantitative real-time PCR experiments".