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.
Highlights
Human papillomavirus (HPV) infections are the established cause of cervical cancer, and they are implicated in other cancers in various anatomical sites of women and of men
Among the original RNA sequencing results the HPV16miR-H1 microRNA had only a few read counts in RNA sequencing while HPV16-miR-H2 read counts were rather high, and both of these were validated by loopedprimer reverse transcription (RT)-PCR and in situ hybridization, suggesting that sequencing read counts cannot be taken as a direct measure of microRNA expression (Qian et al 2013)
We studied the expression of HPV encoded microRNA in a set of 52 cervical samples and four HPV containing cell lines
Summary
Human papillomavirus (HPV) infections are the established cause of cervical cancer, and they are implicated in other cancers in various anatomical sites of women and of men. MicroRNAs (miRNAs) are small, non-coding regulatory RNA molecules, which regulate the expression of numerous if not most human genes. Many viruses encode their own miRNAs, and DNA viruses. Attempts were made to validate five of these, and four HPV encoded miRNAs were successfully validated: two encoded by HPV 16, one by HPV 38, and one by HPV 68. Among the original RNA sequencing results the HPV16miR-H1 microRNA had only a few read counts in RNA sequencing while HPV16-miR-H2 read counts were rather high, and both of these were validated by loopedprimer RT-PCR and in situ hybridization, suggesting that sequencing read counts cannot be taken as a direct measure of microRNA expression (Qian et al 2013)
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