HPV-positive and HPV-negative SCCHN have distinctly different clinical outcomes, and the expression and mutation status of important cell cycle control proteins are very different.1, 3
Our data show that several cellular miRNAs are also differentially expressed in HPV-positive SCCHN cell lines as compared to HPV-negative SCCHN cell lines. Many of these miRNAs were also found to be differentially expressed between the HPV-positive SCCHN cell lines and transformed oral keratinocytes lacking HPV DNA. Similarly, expression of the high-risk HPV-16 E6 oncogene in HFKs was strongly associated with changes in miRNA expression similar to that seen in the HPV-positive SCCHN cell lines, while siRNA knockdown of HPV-16 E6 reversed this effect for miR-363. These results suggest that HPV-16, and in particular the E6 oncogene, may be involved in altering the levels of several cellular miRNAs.
Altered regulation of cellular miRNAs has been observed in several types of human cancers6, 7, 25
as well as upon oncogenic viral infections, including hepatitis B and C,26
human T-cell lymphotrophic virus 1,28
and HPV-16.25, 29, 30
Recent studies have analyzed miRNA expression in SCCHN,8, 9, 12, 31-33
and found that miRNA profiles in SCCHN are different compared to normal oral tissue. However, currently there is no information available on differential miRNA expression between HPV-positive and HPV-negative SCCHN. Since HPV infection has been shown to play a significant role in the etiology and prognosis of SCCHN,1, 3
we wished to study the effect of HPV-16 infection in cellular miRNA dysregulation in SCCHN.
Very few HPV-positive SCCHN cell lines have been described in the literature. Of four such cell lines that are available, we utilized two (UD-SCC-2 and UPCI:SCC90) to compare their miRNA expression profiles to that of two HPV-negative SCCHN cell lines (PCI13 and PCI30) by microarray analysis. We further utilized the two additional HPV-16 positive SCCHN cell lines (UM-SCC47 and 93-VU-147T) to validate the miRNA data obtained in the above comparison. MiRNA microarray analysis showed that three miRNAs (human miR-363 and miR-33, and rat miR-497) were upregulated and eight known and one predicted miRNAs were downregulated in the HPV-positive SCCHN cell lines compared to the HPV-negative SCCHN cell lines ( and ). The miRNA microarray analysis was used as a screening tool, and the data was subsequently validated via qRT-PCR. Similar to our current results, we have previously found that qRT-PCR analysis is much more sensitive than the miRNA microarrays and the fold-difference in qRT-PCR assays is usually much greater.34
MiR-363 was specifically upregulated in HPV-16 positive SCCHN cell lines compared to the HPV-negative SCCHN cell lines as well as NOK cells (, ), suggesting a possible role of HPV-16 in altering the levels of this miRNA. Furthermore, experiments with HFKs showed that expression of the HPV-16 E6 oncogene increased the levels of miR-363 () and siRNA knockdown of E6 reversed this effect (). Interestingly, miR-363 is part of the oncogenic miR-17~92 family of clusters, which is composed of three clusters of miRNAs, miR-17~92, miR-106a~363, and miR-106b~25 and thought to have evolved through a series of deletions and duplications.35
Other members of this family of miRNA clusters have been implicated in cancers, including small cell lung cancer,36
B cell lymphoma,37
and T cell leukemia.38
MiRNAs in this family have similar or identical seed sequences,35
and since the seed sequence of a mature miRNA contributes significantly to its specificity for its target mRNA5
, it has been hypothesized that miRNAs in the miR-17~92 family may have similar functions.35
MiR-363 has identical seed sequences to miR-92-1, miR-92-2, and miR-25.24
MiR-92-2 and miR-25 are also overexpressed in pancreatic, prostate, and stomach cancers.39
Recently, miR-25 has been shown to be upregulated in gastric cancers where it targets p57, an essential tumor suppressor.40
Since miR-363 and miR-25 have the same seed sequence, and miR-25 is involved in cell cycle disruption,40
it is possible that miR-363 may also be involved in the dysregulation of cell cycle in HPV-associated SCCHN. The qRT-PCR analysis for miR-106a and miR-92a did not show any differences in expression between the HPV-positive and HPV-negative SCCHN cell lines (data not shown). This is not surprising since many miRNAs in a cluster have independent promoters. Eric Rassart's group has shown that the miR-106-363 cluster of miRNAs in mice is located downstream of the Kis2
This gene has three different transcription start sites and it appears to encode the primary miRNAs of the 106-363 cluster. Also, the radiation leukemia virus (RadLV) is commonly integrated close to the Kis2
locus. In mice, RadLV-induced tumors had varied expression of miRNAs in the miR-106-363 cluster, indicating that they may not be transcribed from the same promoter.38
Also, in gastric cancer, miR-363 was shown to be downregulated compared to the normal tissue, whereas all of the other miRNAs in the miR-106a~363 cluster were upregulated.40
Thus, while miR-363 is overexpressed in HPV-positive SCCHN cells compared to HPV-negative SCCHN cells, it is not surprising that we did not see a difference in expression of miR-106a and miR-92a between these cell lines.
Our results also show downregulation of several miRNAs in HPV-associated SCCHN cell lines as compared to both HPV-negative SCCHN and NOK cell lines, including miR-155, miR-181a, miR-218, and miR-29a (, and and ). We have recently demonstrated that the HPV-16 E6 oncogene downregulates miR-218 expression in HPV-16 positive cervical carcinomas.34
Furthermore, we showed that miR-218 targets LAMB3, and downregulation of miR-218 by the E6 oncogene results in overexpression of LAMB3
in cervical carcinoma cells.34
We found that expression of HPV-16 E6 in HFK cells also reduced the levels of miR-218 (). The downregulation of miR-218 in both HPV-positive cervical and oropharyngeal cancer cell lines suggests that HPV-16 may target cellular pathways common to these two types of cancers. Although it is documented that p53 expression activates miR-34a41
and miR-34a levels are reduced in HPV-16 positive cervical cancer,30
we did not find a statistically significant difference between miR-34a levels between HPV-positive and HPV-negative SCCHN cell lines. While all the HPV-positive cell lines used in our study are p53 wt, the HPV-negative cell line PCI-13 has a p53 mutation while PCI-30 has wt p53 (). There are several possible reasons for our observations on miR-34a. For example, since the p53 pathway is complex, it is possible that a single miRNA may be subject to multiple regulatory mechanisms.
Viral infections have been implicated in altered cellular miRNA expression. In human B lymphocytes infected with the Epstein Barr Virus (EBV), elevated levels of miR-155 help in viral persistence by reducing NF-κB signaling.42
It is intriguing that miR-155 was found to be downregulated in the presence of HPV-16 in our studies (, and and ). There have been other studies on miRNA expression in head and neck cancers that have found miR-155 and miR-181a to be upregulated in oral cancer compared to normal oral tissue.9, 31, 32
However, when we compared HPV-positive and HPV-negative SCCHN cell lines, these miRNAs were downregulated in the presence of HPV-16 DNA. Future studies should define the relationship between reduced levels of these miRNAs in HPV-positive SCCHN.
Our studies showed that miR-181a and miR-29a were downregulated in HPV-positive SCCHN cells compared to HPV-negative SCCHN and NOK cells (, and ). The levels of these two miRNAs also decrease upon expression of the HPV-16 E6 oncogene in HFKs (), suggesting a role for E6 in downregulation of these miRNAs. The miR-181 family is known to be highly expressed in the brain43
and is involved in thymocyte development,44
but its role in other tissues is less well-understood. Our data is the first to show a downregulation of miR-181a and miR-181b in HPV-positive SCCHN cell lines compared to HPV-negative SCCHN cell lines (, and ). Overexpression of miR-181a and miR-181b has been shown to induce apoptosis and inhibit growth and invasion in glioma cells.45
Further studies on the roles of the miR-181 family may elucidate roles of these miRNAs in the different characteristics seen in HPV-positive and HPV-negative SCCHN. MiR-29a has been shown to interact with viral proteins. For example, miR-29a targets the HIV-1 Nef protein and interferes with viral replication.46
MiR-29a also targets p85 and CDC42, which are negative regulators of p53.47
Since the HPV-16 E6 protein promotes the degradation of the p53 protein,48
it is possible that downregulation of miR-29a by E6 may further reduce p53 levels upon persistent HPV infection. The precise role of HPV infection in cellular miRNA dysregulation, and the role of HPVs in SCCHN development which also contributes to a better prognosis for these cancers as compared to their HPV-negative counterpart will be the subject of future studies.