Long non-coding RNA FAM83H-AS1 is regulated by human papillomavirus 16 E6 independently of p53 in cervical cancer cells

Jamie A Barr,Rajaganapathi Jagannathan,Ivan Martinez,Abby D Harold,Tayvia Brownmiller,Paul R Lockman,Saleem Khan,Karen E Hayes

doi:10.1038/s41598-019-40094-8

Jamie A Barr, Rajaganapathi Jagannathan + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-40094-8

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Abstract

High-risk human papillomavirus (HPV) infection is one of the first events in the process of carcinogenesis in cervical and head and neck cancers. The expression of the viral oncoproteins E6 and E7 are essential in this process by inactivating the tumor suppressor proteins p53 and Rb, respectively, in addition to their interactions with other host proteins. Non-coding RNAs, such as long non-coding RNAs (lncRNAs) have been found to be dysregulated in several cancers, suggesting an important role in tumorigenesis. In order to identify host lncRNAs affected by HPV infection, we expressed the high-risk HPV-16 E6 oncoprotein in primary human keratinocytes and measured the global lncRNA expression profile by high-throughput sequencing (RNA-seq). We found several host lncRNAs differentially expressed by E6 including GAS5, H19, and FAM83H-AS1. Interestingly, FAM83H-AS1 was found overexpressed in HPV-16 positive cervical cancer cell lines in an HPV-16 E6-dependent manner but independently of p53 regulation. Furthermore, FAM83H-AS1 was found to be regulated through the E6-p300 pathway. Knockdown of FAM83H-AS1 by siRNAs decreased cellular proliferation, migration and increased apoptosis. FAM83H-AS1 was also found to be altered in human cervical cancer tissues and high expression of this lncRNA was associated with worse overall survival, suggesting an important role in cervical carcinogenesis.

Highlights

High-risk human papillomavirus (HPV) infection (e.g. HPV-16) is one of the most common causes of cervical cancer[1,2,3], as well as a subset of head and neck squamous cell carcinoma (HNSCC)[1]
As an initial screen to identify host long non-coding RNAs (lncRNAs) that are regulated by HPV-16 E6, we developed a system to look at the effect of E6 expression alone in primary human foreskin keratinocytes (HEKa)
After puromycin selection and stable expression of HPV-16 E6 was confirmed by RT-PCR (Fig. S1), RNA was extracted, and samples were analyzed by RNA high-throughput sequencing (RNA-seq) to determine gene expression alterations in long non-coding RNAs

Summary

Introduction

High-risk HPV infection (e.g. HPV-16) is one of the most common causes of cervical cancer[1,2,3], as well as a subset of head and neck squamous cell carcinoma (HNSCC)[1]. We demonstrated that the lncRNA FAM83H-AS1 ( known as onco-lncRNA-3) is up-regulated in primary keratinocytes expressing HPV-16 oncogene E6 as well as HPV-16 positive human cervical cancer cell lines and cervical tumor samples. We found 151 up- and 100 down-regulated host lncRNAs altered greater than 1.5-fold change when HPV-16 E6 was expressed in HEKa cells compared to GFP control (Fig. 1A, Table S1).

Results

Conclusion