Low expression levels of putative HPV encoded microRNAs in cervical samples

Abstract

Using small RNA sequencing of libraries established from cervical samples and cervical cancer cell lines, we have previously reported identification of nine and validation of five putative microRNA species encoded by human papillomaviruses (HPV) including five microRNAs encoded by HPV 16. Here we have studied the expression of HPV 16 encoded microRNAs in cervical samples and in HPV 16 containing cell lines. Different sample matrices were collected for the study: 20 paraffin embedded cervical tissue samples, 16 liquid cytology samples, and 16 cervical cell samples from women attending colposcopy due to cervical abnormalities, as well as four HPV 16 containing cell lines. Total RNA was extracted, the samples were spiked with small synthetic control RNAs, and the expression of five HPV 16 encoded microRNAs was assessed by real-time PCR amplification. HPV encoded microRNAs could be frequently detected, albeit at high cycle counts. HPV16-miR-H1 was detected in 3.6 %, HPV16-miR-H3 in 23.6 %, HPV16-miR-H5 in 7.3 %, and HPV16-miR-H6 in 18.2 % of all valid samples. True positive signals for HPV16-miR-H2 could not be detected in any of the samples. Viral microRNAs were detected most frequently in paraffin-embedded samples: in one sample representing normal squamous epithelium, in one cervical intraepithelial neoplasia (CIN) grade 1, one CIN2, three CIN3, two squamous cell carcinoma, three adenocarcinoma in situ, and two adenocarcinoma samples. One liquid cytology sample from a patient with CIN3 as well as all four cell lines were positive for HPV16-miR-H3. In all cases HPV encoded microRNAs were expressed at low levels.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3524-3) contains supplementary material, which is available to authorized users.

Identification of virus-encoded microRNAs in divergent Papillomaviruses

“…Our bioinformatic analysis did not predict any candidates for the negative strand of papillomaviruses. Likewise, although their genomes were fully covered in our library, no predictions were made for any of the plus- or minus-strand miRNAs previously suggested for HPV types 6, 16, 18, 38 or 45 [ 38 – 40 ]. While highly unlikely, we nevertheless considered it formally possible that our bioinformatic prediction pipeline had missed all 9 purported candidates in these genomes.…”

“…Further, at least two studies examining transformed cell lines report that HPV16 does not encode miRNAs [ 36 , 37 ]. There have been reports in transformed cells of small RNAs from high risk PVs such as HPVs 16 & 18, however these studies did not demonstrate a connection of PV-derived small RNAs to the miRNA biogenesis (Dicer/Drosha) or effector (RISC) machinery, nor did they confirm a biologically meaningful abundance [ 38 – 40 ]. Consequently, these RNAs are not widely accepted as miRNAs and the signal detected likely represents degradation fragments derived from the turnover of longer transcripts.…”

Diagnostic and prognostic value of microRNA-628 for cancers

“…Recently, due to their roles in tumorigenesis and their correlation with clinical characteristics, miRNAs have attracted wide attention. Further, accumulating evidence supports that their abnormal expression levels are associated with various diseases, such as lung cancer 47 , 48 , prostate cancer 22 , 49 , 50 , and cervical cancer 51 , 52 . More importantly, miRNAs present in various biological materials, like serum, plasma, and tissue, are extremely stable, even in extreme thermal environment 53 , 54 .…”

Human Papillomavirus and carcinogenesis: Novel mechanisms of cell communication involving extracellular vesicles

“…Four out of five HPV (i.e. two from HPV16, one from HPV38 and one from HPV68) miRNAs were validated and their expression was verified in established cell lines as well as in clinical specimens even if at low expression level [64]. Interestingly enough, the analysis of putative targets of two HPV16-codified miRNAs revealed that many target genes involved in cell cycle, immune responses and cell migration are commonly regulated by both miRNAs that have also two target sites in the viral genome (i.e., LCR region and L1 gene respectively) [63].…”