Reference MicroRNAs for RT-qPCR Assays in Cervical Cancer Patients and Their Application to Studies of HPV16 and Hypoxia Biomarkers.

MicroRNA (miRNA) expressions in tumor biopsies have shown potential as biomarkers in cervical cancer, but suitable reference RNAs for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays in patient cohorts with different clinicopathological characteristics are not available. We aimed to identify the optimal reference miRNAs and apply these to investigate the potential of miR-9-5p as human papilloma virus (HPV) 16 biomarker and miR-210-3p as hypoxia biomarker in cervical cancer. Candidate reference miRNAs were preselected in sequencing data of 90 patients and ranked in a stability analysis by RefFinder. A selection of the most stable miRNAs was evaluated by geNorm and NormFinder analyses of RT-qPCR data of 29 patients. U6 small nuclear RNA (RNU6) was also included in the evaluation. MiR-9-5p and miR-210-3p expression was assessed by RT-qPCR in 45 and 65 patients, respectively. Nine candidates were preselected in the sequencing data after excluding those associated with clinical markers, HPV type, hypoxia status, suboptimal expression levels, and low stability. In RT-qPCR assays, the combination of miR-151-5p, miR-152-3p, and miR-423-3p was identified as the most stable normalization factor across clinical markers, HPV type, and hypoxia status. RNU6 showed poor stability. By applying the optimal reference miRNAs, higher miR-9-5p expression in HPV16- than HPV18-positive tumors and higher miR-210-3p expression in more hypoxic than less hypoxic tumors were found in accordance with the sequencing data. MiR-210-3p was associated with poor outcome by both sequencing and RT-qPCR assays. In conclusion, miR-151-5p, miR-152-3p, and miR-423-3p are suitable reference miRNAs in cervical cancer. MiR-9-5p and miR-210-3p are promising HPV16 and hypoxia biomarkers, respectively.