DNA methylation regulated microRNAs in HPV-16-induced head and neck squamous cell carcinoma (HNSCC)
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Epigenetic modifications have been reported to play an important role in regulating gene expression and these modifications become critical when they have a role in controlling another important layer of epigenetic regulation namely microRNAs. In the present study, we have identified the microRNAs that may be regulated by promoter DNA methylation and histone acetylation in Human papilloma virus-positive head and neck squamous cell carcinoma.
HPV-negative cell line (UPCI:SCC-116) and HPV-16 +ve cell line (UPCI:SCC-090) were treated with methylation inhibitor (5-aza-2′-deoxycytidine, AZA) and acetylation inhibitor (Trichostatin-A, TSA), followed by micro-array analysis. The differentially expressed miRNAs were validated in control (n = 10), HPV-16 +ve (n = 30), and HPV −ve (n = 30) HNC, TCGA (n = 529) tissue samples, and two HPV −ve (SCC116 and Hacat) and two HPV +ve (SCC090 and SiHa) cell lines. Methylation-specific PCR (MSP) and chromatin immunoprecipitation assay (CHIP) were performed to validate their regulation. In silico and in vitro analyses of identified miRNAs were done to study putative pathways they target and their possible role in carcinogenesis.
Among 10 miRNAs specifically up-regulated in microarray analysis of AZA-treated SCC090 cells, we observed significantly decreased expression of hsa-miR-181c-5p, hsa-miR-132-5p, hsa-miR-658 in HPV +ve HNC cohort, TCGA tissue samples, and cell lines as compared to their HPV −ve counterpart, and their promoter region also possesses CpG islands. MSP and analysis of TCGA data (MethHC) revealed increased frequency of methylation at the promoter of hsa-miR-132-5p that is negatively correlated with its expression. In TSA-treated SCC090 cells, out of 7 miRNAs, two namely Hsa-miR-129-2-3p and Hsa-miR-449a were found to be up-regulated as compared to HPV −ve cells. However, the levels of enrichment by anti-acetyl-H3 and anti-acetyl-H4 were significantly low in cell lines compared to respective controls and both were up-regulated in HPV +ve compared to HPV −ve TCGA tissue samples. In silico analysis revealed hsa-miR-132-5p targeted canonical β-catenin/wnt pathway and modulation of down-stream genes of the pathway was observed on over-expression/inhibition of hsa-miR-132-5p.
This study suggests the role of epigenetic modifications in regulating expression of miRNAs in HPV +ve HNSCC.
KeywordsHead and neck cancer HPV-16 Epigenetics MicroRNAs DNA methylation
This study was carried out at Genetics of Complex disorder laboratory, PGIMER, Chandigarh, India. UPCI:SCC-116 and UPCI:SCC-090 cell lines were kind gifts from Dr S Gollin (University of Pittsburgh, PA, USA). The authors are grateful to all individuals who participated in this study and the laboratory staff members for their skillful technical help. The work has been presented in various national and international conferences. Malay K Sannigrahi thankfully acknowledges research fellowship from Indian Council of Medical Research, New Delhi, India. Dr. Rajni Sharma thankfully acknowledges fellowship from DBT, India (BT/Bio-CARe/08/837/2010–2011). We sincerely thank and greatly appreciate the two anonymous reviewers for their comments and suggestions for improving the manuscript.
Compliance with ethical standards
We declare no conflict of interest.
Approved by Institutional Ethics Committee (IEC), Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India.
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