Coordinated action of human papillomavirus type 16 E6 and E7 oncoproteins on competitive endogenous RNA (ceRNA) network members in primary human keratinocytes

Human papillomaviruses (HPVs) are small, double-stranded circular DNA viruses with significant oncogenic potential. HPVs infect epithelial cells of skin and mucous membranes mainly in the anogenital and head and neck region. High-risk HPV-types, such as HPV16, 18, 31, 33, 45, 51, 58 etc., are main etiologic factors of numerous different carcinomas including cervical, vulvar, penile, anal and head and neck cancers. Nearly 100% of cervical carcinomas are positive for high-risk HPVs, HPV16 being the most prevalent type detected in these malignancies [1, 2]. E6 and E7 early viral proteins are the major oncoproteins of high-risk HPVs. The transforming activity of E6 and E7 is mainly mediated through binding and inactivation of cellular tumour suppressor proteins p53 and pRb, respectively, although they are able to directly or indiretly interact with numerous cellular factors involved in regulation of cell cycle, cell death or cellular differentiation, thereby promote malignant transformation [3‐5].

High-throughput RNA-seq studies have revealed the great complexity of human transcriptome by demonstrating that only approximately 2% of the human genome encodes protein-coding sequences. A significant portion of the human genes is actively transcribed but not translated into functional proteins suggesting their crucial regulatory role in biological processes under different conditions. Among these transcripts, several classes of non-coding RNA molecules have been identified including microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), PIWI-interacting RNAs (piRNAs) and long non-coding RNAs (lncRNAs) [6, 7]. The approximately 18–25 nt miRNAs are the most intensively studied and best-known subtype of non-coding RNAs. miRNAs downregulate the expression of their targets post-transcriptionally by binding to the 3′-UTR of mRNAs leading to the translational inhibition or degradation of their target. Expression of miRNAs may vary in different tissues, developmental stages or pathological conditions [8‐12]. lncRNAs are a diverse group of non-coding transcripts longer than 200 nt which are thought to have important function in gene expression regulation. Their ability to interact with DNA, RNA or proteins suggests their multiple functions in diverse biological processes. They have been linked to epigenetic mechanisms, splicing, translational regulation and protein stability, although the accurate mechanism of their action remains largely unknown [9, 13‐16].

Emerging evidence shows the aberrant function of non-coding RNAs especially miRNAs and lncRNAs in various diseases and tumours including HPV-induced cervical cancers. The non-coding transcripts involved in cell cycle, differentiation, proliferation, migration and apoptosis tend to have altered expression during the initiation and progression of tumorigenesis [9, 15, 17‐27]. Although several details of the complex pathogenesis of HPV-induced carcinogenesis have been explored primarily by focusing on oncogenic activity of E6 and E7 proteins of high-risk HPV types, the complete mechanism is not fully understood [3, 28]. An increasing number of studies have reported both abnormal upregulation and downregulation of numerous miRNAs in cervical cancer cell lines and tissue samples, strongly suggesting the oncogenic or tumour suppressor function of certain miRNAs in HPV-associated carcinogenesis [29‐38]. As for the lncRNAs, despite the growing body of evidence regarding their importance in oncogenesis, only a limited number of studies have directly linked certain lncRNAs to HPV-associated cervical carcinoma [15, 21, 39‐43]. Thus, the modulation of a wide range of host lncRNAs by E6 and E7 oncoproteins of high-risk HPVs remains to be extensively investigated.

According to the newly established competing endogenous RNA (ceRNA) hypothesis, all types of RNAs, both protein coding and non-coding transcripts are able to “talk to each other” and influence the functions of the others as a part of a complex intracellular regulatory network. This communication is thought to be mediated by miRNAs through binding to microRNA response elements (MREs) on various RNA transcripts [44]. Recent studies have proved that lncRNAs have the ability to regulate miRNA activity by acting as decoys for them, leading to the alteration of protein turnover. In this context, the altered interaction between lncRNAs and miRNAs by perturbation in their levels may contribute to the pathogenesis of cancer [9, 45‐49].

In the current study, we performed high-throughput new generation RNA sequencing to detect cellular lncRNA and miRNA expression changes in primary human keratinocytes expressing HPV16 E6, E7 and E6/E7 oncoproteins. In addition to non-coding RNAs, mRNA expression changes were measured and we constructed a ceRNA network of coding and non-coding genes to suggest a regulatory network associated with HPV16 infection. We also refined the mRNAs of this ceRNA network by key biological pathways involved in cancer development as determined by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.

E6 and E7 oncoproteins of high-risk HPVs cause large-scale gene expression changes in the HPV infected epithelial cells. They interact with numerous signaling molecules, thereby they are able to reprogram multiple cellular regulatory pathways involved in cell cycle, cell death, cell differentiation and migration playing a crucial role in HPV-induced abnormal cell proliferation and malignant transformation [5, 58, 60]. In addition to protein coding genes, E6 and E7 can also alter the expression of non-coding RNAs, including lncRNAs and miRNAs [29, 39].

Host cell’s non-coding RNAs, especially miRNAs and lncRNAs function as key regulators of cellular biological processes both under physiological and pathological conditions, including tumour initiation and progression by multiple types of cancers including HPV-associated malignancies. However, the majority of the studies have focused on high-risk HPV associated expressional changes and/or interactions of either miRNAs or lncRNAs. Several studies indicated that miRNAs and lncRNAs may interact with and regulate cellular processes involved in cancer development through modulation of the expression of their downstream mRNA target [47, 48, 61]. Nevertheless, non-coding RNA molecules, both miRNAs and lncRNAs, have the potential to interact many other RNAs and modulate numerous mRNA targets and vice versa, a single mRNA may be targeted by hundreds of miRNAs. Therefore, the aberrant expression of these RNA molecules will influence multiple signaling pathways in HPV infected cells. Thus, analyzing the global context of coding and non-coding RNAs provides valuable information on the complexity of the structure and function of endogenous RNA network regarding high-risk HPV infection. Therefore our goal was to investigate the global expression changes of miRNAs, lncRNAs and mRNAs driven by HPV16 E6/E7 oncoproteins. We described the network of abnormally expressed non-coding RNAs in stably transduced undifferentiated human primary keratinocytes using small RNA (≤ 200 nt) and large RNA (≥ 200 nt) deep sequencing. To understand the potential impact of expression alterations of miRNAs and lncRNAs modulated by E6/E7 oncoproteins on cellular mRNA abundance and functions, we have paired the expression data of the various RNA populations. Furthermore, additional GO and KEGG analyses were performed to understand the consequences of mRNA expression alterations on biological processes. To our best knowledge, to date this is the first study which describes such an integrated expression data of non-coding RNAs and mRNAs and construct a ceRNA network of miRNAs, lncRNAs and mRNAs in association with high-risk HPV infection. It is important to highlight, that all of our functional analyses were performed on the basis of E6/E7 expressing HFK cells, because this scenario represents best the in vivo HPV16 infection in epithelial cells. Beyond that, we examined the individual effect of E6 and E7 oncogenes on non-coding RNA expression, too. It is well documented, that in high-risk HPV-types, including HPV16, E6 and E7 expression is controlled by alternative splicing resulting in three different E6 isoforms (E6*I, E6*II and E6^E7) besides the unspliced full-length E6. The expression of different HPV16 isoforms may vary among cells and tissues, nevertheless E6*I is reported to be the most abundant isoform in cervical tumors and cells [62]. Two different E6 products were detected in HPV16 E6 specific RT-PCR performed in this study which matched the full-length E6 and an E6 splice isoform. Only the full length E6 transcript is responsible for coding the transforming E6 oncoprotein while different E6 isoforms may have an antagonistic effect in tumorigenesis. However, both the regulation of these isoforms and their effect on non-coding RNA expression is poorly understood, there are limited number of studies reporting relationship between miRNAs and E6 isoforms [63]. Although, we were not able to differentiate the effect of full-length E6 and E6 splice isoform on the expression of different RNA populations in our study, our observations likely show the cumulative effect of different E6 isoforms and E7 oncoprotein on RNA expression in our experimental system which may be significant in tumorigenesis. At the same time, further research needs to be performed to clarify the exact role of E6 isoforms on non-coding RNA expression in HPV16 host cells.

In our study, we revealed that 85 miRNAs and 41 lncRNAs were abnormally expressed in HPV E6/E7 expressing cells compared with controls. When we examined the impact of oncoproteins on RNA alteration profile individually, we found that the vast majority of RNA expression changes was driven primarly by E6 oncoprotein, which was not a surprise as E6 has a strong effect on host cell processes [64]. In total, the expression of 21 miRNA clusters were modulated by HPV16 E6/E7 oncoproteins, affecting 55 differentially expressed miRNAs. According to chromosome location, 38 miRNA members of miRNA clusters on chromosome X and chromosome 14 may have functional importance in HPV16 infected cells, seeing the clustered miRNAs often coordinately regulate certain biological processes. Harden and coworkers also described modulation of multiple miRNA clusters by HPV16 E6/E7 oncoproteins and they found several miRNAs with expression changes on chromosome X and chromosome 14 [29].

This study also revealed that several differentially expressed non-coding RNAs tend to have regulatory interaction with multiple RNA species also altered in the presence of HPV16 oncogenes. We constructed a ceRNA network with members of 15 lncRNAs – 43 miRNAs – 358 mRNAs with significantly altered expressions driven by HPV16 oncoproteins. Among non-coding RNAs, lncRNAs such as MEG3 and H19, and miRNAs such as miR − 20b-5p, miR-143-3p, miR-497-5p, miR-34a-5p and miR-195-5p had the highest number of relationships in the interaction network. In accordance with our results, downregulation of MEG3 and overexpression of H19 lncRNAs were found by several research groups in association with various neoplasms and the link of these lncRNAs with p53 and pRb pathways underlines their significance in E6 and E7 mediated transformation [19, 65, 66]. The relationships between lncRNAs and miRNAs, and miRNAs and mRNAs were predicted by using StarBase v2.0, DianaTools-LncBase v.2 and miRTarBase, which are experiment - supported databases corroborating that the RNA-RNA interactions identified in our experiment would occur not only in silico situation [53‐55]. These supporting data justified the construction of a ceRNA network from the experimental data of this study.

The functional enrichment analyses identified numerous ceRNA member coding genes as cancer related genes and the majority of mRNAs are involved in GO biological processes and KEGG pathways associated with cancer initiation and progression. Furthermore, our comprehensive examination recognized nine miRNAs as key regulatory elements in regulation of cell cycle and cell death, regulation of cell proliferation and regulation of epithelial cell proliferation. Although, all of these miRNAs have been reported in previous studies associated with HPV infection, their functions in such a complex ceRNA network were so far unknown and they are worth to be further investigated as potential biomarkers in disease detection and progression.

In this study, our main goal was to highlight the complexity of regulatory network of cellular non-coding RNAs in HPV16 infected cells. In the last few years, meta-analyses of sequencing data from different databases or analyzing the global context of coding and non-coding RNAs using integrative methods have become a new and widespread direction of current cancer research. Nevertheless, carrying further experiments focusing on real biological impact of certain RNA-RNA interactions is very useful and essential to explore the exact functions and regulations in depth of non-coding RNAs in cancer development.

We hypothesize, that the multiple molecular changes driven by E6 and E7 oncoproteins resulting in the malignant transformation of HPV16 infected host cells may occur, at least in part, due to the abnormal alteration in expression and function of non-coding RNA molecules through their intracellular competing network. Although several lncRNAs and miRNAs in this study have been previously reported regarding both to HPV-associated and non-HPV cancers, to our best knowlege, this is the first study which describes a ceRNA network of lncRNAs-miRNAs-mRNAs in HPV16 infected cells. Functional enrichment analyses carried out in this study assume important regulatory roles of certain miRNAs and lncRNAs in key biological processes involved in tumour development. We believe, that our comprehensive results will provide a basis for further detailed experiments. Based on these expressional data, we plan to perform further experiments in the future to investigate the relationships of certain non-coding RNAs which funtion is so far poorly understood. Furthermore, miRNAs and lncRNAs can serve as promising biomarkers for molecular diagnosis and progression of cervical cancers.