Open Journal Systems

ORIGINAL CONTRIBUTIONS

Received: October 22^nd 2014

Accepted: May 15^th 2015

Detection of microRNAs seed sequences within human papillomavirus genomes

David Pineda-Gómez,^a Efraín Garrido,^b Pedro Chávez,^b Mauricio Salcedo^a

^aUnidad de Investigación Médica en Enfermedades Oncológicas, Hospital de Oncología, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social

^bDepartamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados, Instituto Politécnico Nacional

Distrito Federal, México

Communication with: Mauricio Salcedo

Telephone: (55) 5627 6900, extensión 22706

Email: maosal89@yahoo.com

In this paper we are reporting for the first time the presence of seed sequences of human and viral microRNAs embedded within both high and low risk human papillomavirus (HPV) genomes. These seed sequences have high oncogenic potential. They were found using an in silico analysis based on the microRNA sequences added to Sanger’s database. Among these sequences, it was observed a potential fingerprint harbouring several repeated sequences of microRNA 297 (miR-297) within the LCR region of HPV types 16, 18, 33, 45 and 52. Further analyses were performed for low risk HPV types 6 and 11 and we observed that the probable fingerprint was absent in HPV11, even when we detected other repeated sequences of miR-363. According to these findings, besides the fact that we detected the presence of microRNA sequences within HPV genomes, we suggest a common putative viral mechanism of gene expression regulation shared among human virus.

Keywords: MicroRNAs, HPV.

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MicroRNAs are small RNA molecules of 22 to 24 nucleotides in length that play an important role in the regulation of gene expression at the translational level.¹ They exert this function through their ability to integrate into translational silencing complexes, which are dependent on interference RNA (RISC). These inhibit translation by binding to specific molecules of messenger RNA (mRNA), silencing them or sending them to degradation. Almost 10,000 microRNA sequences have been detected in a wide variety of species, but their physiological targets have only been identified and characterized in a fraction of these; however, it has been shown that some microRNAs detected in humans have targets, either related to cell cycle regulation, growth factor expression, or individual development.²

MicroRNAs have also been linked with the development of different types of cancer in humans.¹ Their expression has been reported in different viruses that infect humans and develop pathologies.³ Although apparently this type of molecule is absent in the genome of the human papillomavirus (HPV), which is the main etiologic agent of cervical cancer, in this paper we report the existence microRNA seed sequences in the genome of different types of HPV, which have been previously reported in the genome of other families of viruses and in humans.

Methods

The database with microRNA sequences corresponding to version 10.0 was downloaded from the Sanger Institute website (http://www.microrna.sanger.ac.uk/).^4-6 MicroRNA sequences were classified into families according to the similarity in their sequences, and the seed sequences of each one were selected (taking nucleotides 2-8 at the 5' end), redundancy among them was eliminated, whereby only 1253 seeds were obtained, each representative of a family (of these seeds, 280 corresponded to H. sapiens). This database was called Fam_miRNAs. Then, researchers downloaded from the website of the National Center of Biotechnology Information (NCBI) consensus sequences of the genomes of HPV types 16 and 18 (NC_001526.1 and X01505, respectively) and of different reported sequences of the entire genomes of HPV 6 (AF092932), -11 (EU918768, LZod45-11 strain), -33 (EU918766, LZcc12-33 strain), -45 (EF202164, Qv25000 strain), and -52 (X74481), all in Fasta format.  

Researchers proceeded to create a spreadsheet in Excel (part of Microsoft Office suite, Version 2003) that would detect different seeds in the genome of HPV based on the Fam_miRNAs database; the Find function was used, which requires the viral genome sequence of interest and the seeds contained in the Fam_miRNAs database to be entered into the same spreadsheet. The function generated a report, which recorded the seeds that were found and the viral region in which they were located. Based on this information, a new database was created, called miRNAs_VPH X, wherein X represented the viral type in which the detection took place. After selecting the different microRNAs seeds of H. sapiens, they were placed within a genetic map of the viral types; for this, researchers used the gene sequence of the virus loaded into Word (Microsoft Office suite, version 2003) and the Search command (located within the Editing option of the toolbar), in which the seed sequence to be located was introduced, and the program performed detection in the different viral regions. The seeds were then marked with different colors, depending on their origin (viral, other than HPV, and H. sapiens) and the copy number of each was noted, as well as noting which seeds overlapped and which were contiguous with one another.   

Results

In order to work with genomic sequences representative of each of the HPV types, researchers performed the search for sequences in the NCBI Entrez database, and this only found seeds for types 16 and 18. Of the other viral genome sequences searched, only reports of different strains were found, of which those containing complete genomic sequences were selected.

Upon applying the Find function of Excel, variable amounts of microRNA seeds were detected, both for H. sapiens and different viruses in the different HPV types analyzed. The list of seed sequences corresponding to the viral microRNAs are found in Annex 1, while Annexes 2 and 3 list microRNA seed sequences belonging to H. sapiens detected in the HPV types. 

Repetitions of some seeds were also found in different regions of the genomes, of which the most notable was that recorded in the LCR of the seed of miR-297 in types 16, 18, 33, 45, and 52. Annex 4 shows the LCR regions of the different HPV types included in this study, indicating the position of said repetitions.

Discussion

Since the beginning of the 20th century when microRNAs were discovered in C. elegans, an increasing number of microRNA sequences has been reported in different organisms, and it has been shown that they are part of a mechanism for regulation of gene expression that has barely begun to be studied.^7,8 Several microRNA sequences have been detected in a variety of pathogenic viruses, as in the case of the Herpesviridiae family and some members of the polyomavirus and adenovirus families.³ More recently, the presence of microRNA has been reported in the genome of human immunodeficiency virus (HIV).⁹ These data would suggest that viruses also possess this type of gene regulatory elements.

Although there have been several attempts to detect compatible sequences of microRNAs in the HPV genome, different strategies have failed and only cellular microRNAs have been reported found, as in the case of HPV 31¹⁰ and detection attempts in HPV + cell lines.¹¹ After these reports, we identified compatible sequences of cellular microRNA within the HPV genomes. Unfortunately, at the time the data were not accepted for publication (V Villegas, M Salcedo, personal communication, 2011). However, recently the presence of microRNAs in HPV types 16 (six candidates, two of them located in the LCR), 38, 45, and 68 (one candidate in each) has been reported.¹² With this report we demonstrate and suggest that HPV contains microRNA seed sequences shared by human microRNA sequences and those of other viruses.

This paper presents an in silico analysis that was implemented as a detection strategy for potential microRNAs conserved among different species recorded in the Sanger Institute database and different HPV genomes; for this, tools contained in two programs of Windows Office suite were used. The methodology shown is proposed as an alternative to using other programs that require a basic knowledge of programming and creating scripts, as in the case of Perl. This strategy has been successfully used to detect seeds of approximately 100 different microRNAs encoded in H. sapiens and other viruses. It was not possible to detect complete sequences of mature microRNAs or their precursors (pre-miRNAs) in any of the genomes analyzed by comparing the sequences reported in the miRBase database. Given the length of HPV genomes, we think there is the possibility that these viruses have developed the strategy of keeping in their genome only microRNA seeds that can grant them some benefit for survival, negatively regulating genes encoding tumor suppressor proteins or immune system recognition, or indirectly allowing oncogenic protein synthesis through mechanisms such as those reported by Esquela-Kerschner et al.¹ Moreover, research has demonstrated the existence of alternative  intron cutting and exon splicing to generate a variety of regulatory RNAs in HPV 16, as demonstrated above,¹³ so we suggest that, as part of this mechanism, certain regions could be transcribed where different microRNA seeds are located, which would generate an non-coding RNA molecule that would thus allow a possible regulatory function.       

If the above is true, it is likely that these sequences were acquired as the result of the joint evolution of HPV and H. sapiens, as proposed by several authors for HPV 16 and 18,^14-17 and that they may have been preserved in the viral genome as a survival strategy that includes avoidance of the immune system and cell cycle regulation. In addition, the presence of seeds derived from microRNAs of other viruses with human hosts, such as Kaposi sarcoma, Epstein-Barr virus, or cytomegalovirus, is intriguing; one could speculate that these viruses share the same pattern of infection and possible regulation of cellular functions. The low or zero oncogenic capacity of HPV 6 or 11 could be explained, at least in part, by this event, as no link could be shown between the evolution of the virus and the appearance of different races of humans.¹⁸ Moreover, science has not yet demonstrated the existence of possible recombination between different types of HPV,¹⁷ so we think the presence of human microRNA seeds in the viral genome is an event caused by their coevolution.

As for the bioinformatic analysis results, there are clear differences in the number of seeds present in the low- and high-risk types of HPV. The former shows few microRNAs seeds from other viruses, although type 11 does show a high copy number of these. The high-risk types show a higher number of seeds of this type and a smaller number of copies, which may mean that the virus requires a greater number of these seeds to induce a lesion in its host and, possibly, the development of some type of cancer in the cervix. The low number of seeds of other viruses in the low-risk types of HPV may therefore explain, at least in part, that these HPV are only capable of inducing low-grade intraepithelial lesions.  

There are seeds that overlap with each other in the different viral types (as in the case of miR-297), which are present in the LCR region of HPV 16, 18, 33, 45, and 52. This event may have the following meanings: 1) that this overlap leads to the inactivation of the seeds, so that, when the genomic region where they are located is transcribed, a messenger RNA is created encoding the viral protein and not encoding small regulatory RNAs; or, 2) that overlapping seeds may generate a microRNA unique to the virus, which can interact with multiple mRNAs that are target at once. This last event has been shown in some cases when a pre-miRNA is polycistronic, that is, it codes for two or more microRNAs in a single transcript,³ or they are grouped in tandem, as in the case of a set of microRNAs detected on chromosome 19 in the human.¹⁹ Even the length of this hypothetical HPV microRNA could possess additional binding sites to the target mRNA, which could help stabilize the microRNA-mRNA interaction and subsequent regulation. The existence of this type of molecules, and if any of these hypotheses may have some meaning for viral activity, remain to be demonstrated, using molecular techniques.   

As regards microRNA seeds present in H. sapiens, differences are also observed between types of high-risk HPV. The biggest similarities between these allow types 18 and 45 to be grouped, while types 16, 33, and 52 form another group, which is in line with the phylogenetic trees developed based on the L1 protein.²⁰ This may be because the shared seeds are on highly conserved domains or sequences between different viral types, and the same potential for lesion induction may be reflected in the HPV belonging to the two groups.

The presence of repetitions of the miR-297 seeds in the LCR of high-risk viral types may indicate that these repeats make up a cluster that is necessary for the regulation of viral or cellular genome expression, given the particular location of these seeds (LCR). Consistent with the above, the presence of several repetitions of the miR-363 seed in the corresponding LCR of HPV 11 could reflect that the presence of repeats in this region is necessary, regardless of the specific microRNA to which they belong, and this event might point towards a possible genetic fingerprint that could identify the presence of HPV and differentiate it from other viruses that may be present in the same tissue sample.

In summary, this paper reports on a simple methodology for an in silico analysis that allowed us to detect a variety of microRNA seeds in the genome of different HPV types. This detection strategy could also be used to detect microRNA sequences conserved in genomes of other species, even those that are in the process of assembly.

In conclusion, the human papillomaviruses with high oncogenic power theoretically share compatible sequences with human and viral microRNAs, suggesting common mechanisms of viral regulation. These sequences may at least be in clusters within the LCR control region.

Acknowledgments

This paper is derived from a project approved by CONACYT sectoral funds and the IMSS project on the detection of microRNAs in the human papillomavirus.

During the creation of this work, David Pineda Gomez was a grantee of CONACyT in the Molecular Biotechnology and Biomedicine PhD program at ENCB-IPN.

Clarifying note

over two years ago we sent for publication a report on the existence of microRNAs in HPV to two different indexed journals, and both times it was rejected. However, in 2013, an article was published in the journal PLoS ONE that reported the presence of microRNAs in HPV.

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