microRNAs are biomarkers of oncogenic human papillomavirus infections

Significance

Persistent infections with high-risk human papillomaviruses (HPVs) lead to development of cervical, penile, anal, and oropharyngeal cancers. The ability to diagnose HPV infections has been dependent on the detection of viral DNA, on virus-associated cytological and histological abnormalities, and on a few virus-induced host proteins. In this study, we identified a subset of host microRNAs regulated specifically by HPV16 or HPV18 infection in in vitro model systems. The elevated expression of miR-16, miR-25, miR-92a, and miR-378 and the decreased expression of miR-22, miR-27a, miR-29a, and miR-100 were attributed to viral oncoprotein E6 or E7. An expression ratio ≥1.5 of miR-25/92a group over miR-22/29a group was found to be informative in distinguishing normal cervix from cervical intraepithelial neoplasia and cervical cancers.

Abstract

Cellular and viral microRNAs (miRNAs) are the transcriptional products of RNA polymerase II and are regulated by transcriptional factors for their differential expression. The altered expression of miRNAs in many cancer types has been explored as a marker for possible diagnosis and therapy. We report in this study that oncogenic human papillomaviruses (HPVs) induce aberrant expression of many cellular miRNAs and that HPV18 infection produces no detectable viral miRNA. Thirteen abundant host miRNAs were specifically regulated by HPV16 and HPV18 in organotypic raft cultures of foreskin and vaginal keratinocytes as determined by miRNA array in combination with small RNA sequencing. The increase of miR-16, miR-25, miR-92a, and miR-378 and the decrease of miR-22, miR-27a, miR-29a, and miR-100 could be attributed to viral oncoprotein E6, E7, or both, all of which are known to target many cellular transcription factors. The examination of 158 cervical specimens, including 38 normal, 52 cervical intraepithelial neoplasia (CIN), and 68 cervical cancer (CC) tissues, for the expression of these eight miRNAs showed a remarkable increase of miR-25, miR-92a, and miR-378 with lesion progression but no obvious change of miR-22, miR-29a, and miR-100 among the HPV-infected tissues. Further analyses indicate that an expression ratio ≥1.5 of miR-25/92a group over miR-22/29a group could serve as a cutoff value to distinguish normal cervix from CIN and from CIN to CC.

Cervical cancer (CC) is the second most common cancer among women worldwide and is caused by persistent infection with oncogenic human papillomaviruses (HPVs). HPV infection has also been identified as a definitive human carcinogen for the penis, vulva, vagina, anus, and oropharynx (including the base of the tongue and tonsils) (1, 2). To date, 15 mucosal HPV types are deemed as oncogenic or high-risk (HR) HPVs, including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, and HPV82 (3). Among the HR HPVs, HPV16 and HPV18 have a combined worldwide contribution to ∼70% of invasive CC (4).

The productive HPV life cycle is tightly linked to squamous cell differentiation, and organotypic raft cultures have been developed to grow these viruses in vitro (5–7). HPV infection is initiated when viral particles gain entry to undifferentiated basal epithelial cells through an abrasion or a wound, where viral early genes are up-regulated during wound healing. The virus-aided expansion of the infected cell population then establishes the infection. The amplification of the extrachromosomal viral DNA and the expression of viral capsid proteins occur sequentially in the middle and upper spinous and superficial cells (7). Persistent, active infection by HR HPVs can lead to cervical intraepithelial neoplasia (CIN) grade 1, 2, or 3 on the basis of increasing abnormal depths of the proliferative cell compartment in the cervical epithelium (8). Histologically, the abnormal proliferative cells are restricted to the lower one-third of the epithelium in CIN1. CIN2 and CIN3 are distinguished by the expansion of the neoplasia to the lower two-thirds (CIN2) or more (CIN3) of the epithelium. CIN3 may involve the full thickness of the epithelium and is sometimes referred to as cervical carcinoma in situ. CIN2 and CIN3 are developed in 10–20% of women with CIN1, and a fraction of CIN3 may progress to CC when the neoplasia invades into the stroma underneath. Two viral oncoproteins, E6 and E7, of the HR HPVs destabilize, respectively, two major cellular tumor suppressor proteins, p53 and retinoblastoma protein (pRB). Both viral proteins function to support the productive phase of the infection in the postmitotic differentiated spinous cells. However, repeated elevated expression of HR HPV E6 and E7 in the undifferentiated basal epithelial cells or stem cells disrupts cell cycle regulation, inhibits cell differentiation, induces chromosome damage, and prevents apoptosis, resulting in cell immortalization and transformation, the basis of HPV carcinogenesis (9, 10). Indeed, the expression of the HR HPV E6 and E7 is consistently elevated in CC.

MicroRNAs (miRNAs), noncoding regulatory RNAs with a length of ∼21 nt, are derived from RNA polymerase II transcripts of coding or noncoding genes, and their expression is subjected to transcriptional and posttranscriptional regulation. Argonaute proteins, the core components of the RNA-induced silencing complex, bind the mature miRNAs and guide them to imperfect complementary sequences located primarily in the 3′ UTR of target mRNAs, leading to translational repression or target degradation (11, 12). The human genome encodes ∼550 miRNA genes to express about 1,000 miRNAs. miRNAs are differentially expressed in many human cell types (13) and target about 60% of genes (14). Aberrant expression of miRNAs has been implicated in numerous disease states and has been explored as a biomarker for possible diagnosis or prognosis of human diseases (15–17). Previously, our group and others found that HPV infection regulates the expression of keratinocyte miRNAs (18–20) and that aberrant miRNA expression in CC and its derived cell lines is important for cancer cell proliferation (21–24). However, the different approaches used led to varied observations (25, 26). To date, there is no systematic study to identify host miRNAs as specific biomarkers for diagnosis of HPV infection or progression. In this report, by using miRNA array analysis and small RNA sequencing (miRNA-Seq), we conducted a comprehensive examination of miRNA profiles in human foreskin keratinocyte (HFK)- or human vaginal keratinocyte (HVK)-derived raft cultures with or without productive HPV16 or HPV18 infection. We identified a group of host miRNAs that are regulated by HPV18 E6 or E7. Altered expression of a subset of these miRNAs was also observed in HR HPV-infected CIN and CC tissues and may serve as a biomarker for HPV infection or progression.

Results

HPV16 and HPV18 Infection Regulates the Expression of a Subset of Host miRNAs.

To investigate HPV infection-induced genome-wide changes in miRNA expression, we first compared miRNA expression profiles in HFK-derived raft cultures with or without HPV16 infection by miRNA array analysis (Fig. S1). As shown in Fig. 1A (Left, panel 1), HPV16 infection led to decreased expression of 20 host miRNAs (green), including miR-34a (18), and increased expression of 22 host miRNAs (red) (P < 0.05), including miR-16 (25). Northern blot analysis of miR-181a, miR-455-3p, miR-203, miR-375, and miR-16 confirmed the altered expression (Fig. 1B, Upper). To minimize potential experimental variances due to cell types, virus types, or detection methods, we expanded the investigation to HPV18 infection in two types of cells by using two analytical approaches. After initial verification by Northern blotting of altered expression of miR-100 and miR-378 in HFK-derived raft cultures with HPV18 infection (Fig. 2B, Lower), we further compared miRNA expression profiles in HFK- and HVK-derived raft cultures with or without HPV18 infection by using miRNA array analysis and miRNA-Seq (Fig. 1A, Right, panels 1–3). Although each cell type with or without HPV infection displayed a slightly different expression profile of host miRNAs (Fig. 1A and Tables S1–S4), 13 abundant host miRNAs with altered expression were commonly detected by both methods from raft cultures of HVKs or HFKs after HPV16 or HPV18 infection (Fig. 1C). Eight miRNAs had increased expression, and five miRNAs had decreased expression (Table 1). We confirmed by Northern blotting the altered expression of miR-16, miR-25, miR-22, and miR-29 in HVK-derived raft tissues infected with either HPV16 or HPV18 (Fig. S2). It is worth noting that we did not find any HPV18-originated viral miRNAs in HVK- or HFK-derived raft cultures by miRNA-Seq.

Fig. 1.

HPV16 and HPV18 infections regulate the expression of a subset of cellular miRNAs. (A, Left) Panels 1 and 2 illustrate increased (red) or decreased (green) expression of individual miRNAs in HFK or HVK raft cultures without (HFK or HVK) or with HPV16 (HFK16) or HPV18 (HVK18) infection by miRNA array analyses. (A, Right) Panels 3 and 4 illustrate increased (yellow) or decreased (blue) expression of individual miRNAs in HVK or HFK raft cultures without or with HPV18 (HVK18 or HFK18) infection by miRNA-Seq analyses, with the heat map drawn from the top 95% of expressed miRNAs in each sample. (B) Verification of the altered expression of miRNAs in HPV16- and HPV18-infected HFK raft cultures by Northern blot analysis. (C) Venn diagram summarizes a subset of host miRNAs with altered expression in the raft cultures among all four platforms (red arrows in A).

Fig. 2.

HPV18 regulates the expression of host miRNAs over the course of infection. (A) Increased (yellow) or decreased (blue) expression of individual miRNAs in day 8 (D8), D10, D12, and D16 HFK raft cultures without (HFK) or with HPV18 (HFK18) infection and examined by miRNA-Seq. The heat map was drawn from the top 90% of expressed miRNAs in each sample, with red arrows for the miRNAs with altered expression in Table 1 over the course of infection. (B) Expression of selected miRNAs (Table S5) in HFK raft cultures over the course of HPV18 infection. (C) Verification of increased miR-25 and miR-92a expression and decreased miR-27a and miR-29a expression in the 10-d raft tissues derived from HFKs with or without HPV16 or HPV18 infection by Northern blot analysis. Three separate tissue samples labeled as 1, 2, and 3 in each raft tissue group were used for the assay. U6 RNA was used as a loading control. Bar graphs below the blot show the relative level (mean ± SD) of each miRNA, after being normalized to U6 RNA, from three separate raft tissues in each raft group.

Table 1.

Thirteen host miRNAs specifically regulated by HPV infections

Name	Expression	miR-array (HFK16/HFK)	miR-Seq (HFK18/HFK)	miR-array (HVK18/HVK)	miR-Seq (HVK18/HVK)
miR-16	Up	+3.1	+3.2	+1.8	+1.8
miR-25	Up	+4.1	+1.6	+2.7	+2.0
miR-92a	Up	+1.3	+1.9	+2.1	+1.6
miR-93	Up	+3.0	+2.3	+3.6	+2.4
miR-106b	Up	+2.0	+1.9	+1.6	+2.4
miR-210	Up	+2.1	+1.4	+2.3	+2.5
miR-224	Up	+2.7	+4.1	+2.4	+2.4
miR-378	Up	+4.0	+1.9	+4.2	+3.7
miR-22	Down	−10.3	−2.1	−3.8	−2.2
miR-24	Down	−2.0	−1.5	−1.9	−1.4
miR-27a	Down	−3.0	−2.0	−3.7	−2.6
miR-29a	Down	−8.3	−2.5	−3.6	−1.6
miR-100	Down	−1.9	−2.8	−2.7	−1.3

Total RNAs were isolated from day 10 raft cultures of HFKs or HVKs. Thirteen miRNAs were identified by four test platforms (Fig. 1C) that are altered in four HPV-infected raft cultures relative to control raft cultures. The numbers indicate the fold increase (+) or decrease (−) of the signal intensity [miRNA (miR)-array assay] or relative miRNA reads (miR-Seq) of the raft tissue with HPV infection divided by that of the raft tissue without HPV infection. Details are provided in Tables S1 and S2.

HPV18 Regulates Host miRNA Expression over the Course of the Productive Infection.

To validate the observation that the altered expression of host miRNAs is specifically attributable to HPV infection, HFK-derived raft cultures with HPV18 infection on day 8, day 10, day 12, and day 16 were analyzed by miRNA-Seq (Fig. 2A). Viral DNA amplification peaked on days 12–14, whereas viral oncoprotein expression waned (7). Again, we did not detect any HPV18-originated viral miRNAs from the raft tissues at any of the four time points. However, relative to uninfected HFK raft cultures, the altered expression of host miRNAs was obvious and could be classified into four groups (Table S5), with additional miRNAs showing fluctuations in expression over the time course (Dataset S1). The miRNAs in group 1 and group 2 exhibited increased and decreased expression over each of the time points. The miRNAs in group 3 displayed elevated expression in early HPV18 infection but reduced expression at the later time points, and the reverse was true for the miRNAs in group 4, which had decreased expression at the earlier time points but increased expression at the later time of HPV18 infection. Nine of the 13 miRNAs with altered expression in Table 1 were in group 1 and group 2 (Fig. 2A and Table S5) and are selectively graphed in Fig. 2B to show their persistent increase or decrease over the course of HPV18 infection relative to the uninfected raft cultures. We also observed an initial increase, but later decrease, of miR-143, a p53-sensitive target (27, 28) and Ras responder (29). In contrast, miR-203, a skin differentiation marker (30), displayed an initial decrease but a stepwise increase in HFK raft tissues over the time course of HPV18 infection (Fig. 2B), in agreement with the squamous differentiation achieved in these raft cultures (Fig. S3A). Northern blotting confirmed the increased expression of miR-25 and miR-92a and the decreased expression of miR-27a and miR-29a in HFK-derived raft tissues on day 10 after HPV16 or HPV18 infection (Fig. 2C). Together, these data suggest that the miRNAs in the first two groups respond consistently to HPV18 infection; however, the responses of the host miRNAs in group 3 and group 4 to HPV18 infection appear to be regulated by HPV activities (7) and keratinocyte differentiation, as reported (31, 32). Based on these results and on the data shown in Fig. 1C and Table 1, we subsequently chose eight host miRNAs (miR-16, miR-22, miR-25, miR-27a, miR-29a, miR-92a, miR-100, and miR-378) for further investigation. These miRNAs have high signal intensity or reads in all testing platforms (Fig. S4) and are transcribed alone or in a larger cistron together with other miRNAs (cluster).

Host miRNAs and Oncogenic HPV E6 and E7.

To investigate whether the increase or decrease of the eight miRNAs was regulated by viral oncoprotein E6 or E7, raft cultures derived from HFKs transduced with a retrovirus expressing HPV18 E6, HPV18 E7, HPV18 E6E7, or empty control retrovirus were examined by reverse transcription real-time quantitative PCR (RT-qPCR). U6 RNA was used as an internal loading control. miR-34a was used as a positive control for the E6 activity, which reduces miR-34a expression because this miRNA is regulated partially by p53 (18). As expected, E6 decreased the expression of miR-34a, whereas E7 did not (18). In contrast, HPV18 E7 significantly increased the expression of miR-25, whereas HPV18 E6 had no effect (Fig. 3). These observations validated the specificity of E6 and E7 activities. In addition, HPV18 E7 had a stronger positive effect than HPV18 E6 on the expression of miR-16 and miR-378, whereas both viral oncoproteins functioned similarly in inducing a moderate increase of miR-92a expression (Fig. 3). Interestingly, miR-22, miR-27a, miR-29a, and miR-100 were all moderately down-regulated by either E6 or E7, with miR-22 and miR-27a being slightly more susceptible to E7 than to E6 (Fig. 3). These results demonstrate that this set of host miRNAs respond to HPV infection and their altered expression could be attributed to viral oncoprotein E6 or E7.

Fig. 3.

HPV18 E6 and E7 oncoproteins regulate the expression of host miRNAs. Expressions of eight host miRNAs selected from Table 1 were examined by RT-qPCR in 11-d raft cultures of HFKs transduced with HPV18 E6, E7, or E6E7 retrovirus or an empty control retrovirus vector. miR-34a was used as a positive control for HPV18 E6 activity (18), whereas U6 RNA was used as an internal loading control. The bar graph shows the relative fold increase (above 0) or decrease (below 0) (mean ± SD) of each miRNA in the raft tissues expressing E6, E7, or E6E7 over the empty vector after being normalized to U6 RNA from two independent experiments.

Expression of miR-16, miR-22, miR-25, miR-27a, miR-29a, miR-92a, miR-100, and miR-378 in HR HPV-Infected Cervical Lesions.

To evaluate whether the altered expression of miRNAs in oncogenic HPV-infected raft tissues could be observed in CINs and CC infected with HR HPVs, we examined by RT-qPCR the expression of miR-16, miR-25, miR-92a, miR-378, miR-22, miR-27a, miR-29a, and miR-100 in 38 normal cervical tissues without HPV infection and in 13 CIN1+2, 39 CIN3, and 68 CC tissues with HR HPV infection. As shown in Fig. 4A, statistically significant elevation of miR-25, miR-92a, and miR-378 was observed among HPV-infected tissue groups, consistent with the results obtained from HPV-infected raft tissues. Expression of miR-16 also displayed a trend of increase in CIN3 and CC tissues, but this increase might have been distorted by the extremely high levels of miR-16 in one sample in the CIN3 group and three samples in the CC group (Fig. 4A). An increased level of miR-378 was observed in CIN3 (P = 0.021) and CC (P = 0.035) over the normal cervical tissues, but not in CC over CIN3 (P = 0.184). In contrast, we found no obvious change of miR-22, miR-27a, miR-29a, and miR-100 in CIN1+2 and CIN3 or of miR-22, miR-29a, and miR-100 in CC over the normal cervical tissues. However, a higher level of miR-27a was observed in CC compared with CIN3, CIN1+2, or normal cervical tissues. In this and previous studies (22), miR-29a expression was decreased from normal tissue to CIN and CC, and this trend was verified by Northern blotting using RNAs extracted from a small group of randomly selected samples (Fig. S5). However, this decrease was not statistically significant (P = 0.416) (Fig. 4A).

Fig. 4.

Expression of selected miRNAs in normal cervix and cervical lesions. (A) Expression of miR-16, miR-25, miR-92a, miR-378, miR-22, miR-27a, miR-29a, and miR-100 in 38 normal cervical tissues and in 13 CIN1+2, 39 CIN3, and 68 CC tissues were examined by RT-qPCR. U6 RNA was used as an internal control. Each dot in the dot plot represents the detection level of individual miRNA in a sample from each group after being normalized to U6 RNA. Mean indicates an average level of the miRNA in all samples examined in each group. (B) Mean expression levels of two miRNA groups (miR-25/92a and miR-22/29a) in normal cervical, CIN1+2, CIN3, and CC tissues. P < 0.0001 for miR-25/92a progressive expression among the four tissue groups (F test).

Based on the mean values and statistical analysis, miR-25 and miR-92a were subsequently combined as a group; miR-22 and miR-29a were combined as another group; and the individual expression levels were averaged in each group for normal, CIN1+2, CIN3, and CC tissues. Comparison between two groups of miRNA levels showed a stepwise increase of the miR-25/92a from normal (1.6) to CIN1+2 (2.2), CIN3 (3.9), and CC (8.2) (Fig. 4B), whereas the small fluctuation of miR-22/29a did not exhibit a trend. Given that individual miRNA levels varied from one sample to another, we further converted the miRNA expression level from individual tissues to the expression ratio between miR-25/92a and miR-22/29a groups for a better view of the dataset. A threshold ratio was set at 1.5 to evaluate the possibility of using these four miRNAs for diagnosis of CINs and CC. As summarized in Table 2, only 5 of 38 (13.2%) normal cervical tissues had a ratio equal to or higher than 1.5, but 5 of 13 CIN1+2 (38.5%), 19 of 39 CIN3 (48.7%), and 60 of 68 (88.2%) CC tissues had a ratio equal to or above 1.5 (P < 0.0001). This increase was significant even for CIN1+2 tissue samples compared with normal cervical samples (P = 0.047). Collectively, our data strongly suggest that the expression ratio between these two miRNA groups could be useful for the diagnosis of cervical lesions and progression to CC due to oncogenic HPV infections.

Table 2.

Expression ratio of two miRNA groups can be used for diagnosis of CIN and CC

		miR-25/92a vs. miR-22/29a
		<1.5		≥1.5
Pathology	Total samples	Samples	%	Samples	%
Normal	38	33	86.8	5	13.2*
CIN1+2	13	8	61.5	5	38.5
CIN3	39	20	51.3	19	48.7
CC	68	8	11.8	60	88.2

^*P < 0.0001 (F test) among four groups with a ratio equal to or greater than 1.5.

Discussion

In this report, host miRNAs specifically regulated by oncogenic HPV16 and HPV18 infection in HFK- and HVK-derived raft cultures were comprehensively investigated by both miRNA array analysis and miRNA-Seq. We identified 13 host miRNAs responsive to oncogenic HPV regulation, including nine miRNAs from six miRNA clusters (miR-15/16/195/457, miR-106b/93/25, miR-17-5p/18a/19a/20a/19b/92a, miR-224/452, miR-23a/24/27a, and miR-100/let-7a) and four noncluster miRNAs (miR-22, miR-29a, miR-210, and miR-378). The previously reported miRNAs (miR-21, miR-34a, miR-146a, miR-143/145, miR-203, and miR-218) that are altered in some CCs (21, 22, 33) or in HPV infection (18, 20, 34) are not among the 13 miRNAs identified in this study, nor are miRNAs altered in HPV31-infected HFK rafts (35), because none of them were consistently altered in all four of our assay platforms. We note that HPV18 infection did modulate other cellular miRNAs in HFK rafts (Dataset S1), as previously found in HPV31 infection (35). From the panel of 13 miRNAs, eight were selected to represent each cluster and noncluster miRNA for further investigation. The expression of miR-224 displayed fluctuation over the time course of HPV18 infection; thus, miR-224 was excluded from further study. miR-210 was also excluded because its expression is induced by hypoxia (36) and both HR and low-risk HPV E7 proteins enhance HIF-1α stability (37, 38).

We note that miR-25, miR-27a, miR-92a, and miR-378 are oncogenic miRNAs and miR-16, miR-22, miR-29, and miR-100 are tumor-suppressive miRNAs (39–42) and that they are modulated by p53, E2F, and c-Myc (25, 43–45). Although miR-16, miR-25, and miR-92a from three different miRNA clusters and noncluster miR-378 all had increased expression, the expression of the miR-106b/93/25 cluster produced from MCM7 transcript (46) appears to be the most sensitive to oncogenic HPV infection, with increased expression in each of the three miRNAs from this cluster in all assays (Table 1). In contrast, miR-92a and miR-16 were the major miRNAs with significantly increased expression within the respective clusters of miR-17-5p/18a/19a/20a/19b/92a and miR-15/16.

We found that both viral E6 and E7 could enhance miR-92a expression but that only viral E7 was responsible for the up-regulation of miR-25 (Fig. 3). These observations are consistent with our understanding that the expression of both the miR-106b/93/25 and miR-17-5p/18a/19a/20a/19b/92a clusters is suppressed by p53 through an indirect mechanism via inhibition of E2F1 expression (44, 47, 48). Their up-regulation can then be attributed to the abilities of the HR HPV E6 to destabilize p53 and of the viral E7 to target the pRB/p130 protein family, resulting in the release of E2F from suppressive transcriptional complexes. Also, HR HPV E6 and E7 increase MCM7 expression through E2F-dependent and E2F-independent pathways (49), and c-Myc transactivated by E2F1 promotes transcription and expression of the miR-17-92a cluster (47), as well as miR-378 (50). Free E2F1 also transactivates the expression of the miR-15/16 cluster (51).

Both HR HPV E6 and E7 interact with c-Myc and augment c-Myc transactivation activities (52, 53). However, c-Myc suppresses the expression of many miRNAs (44) by binding to miRNA promoter, including those of miR-15/16, miR-23a/24/27a, miR-22, miR-29a, and miR-34a, for example (54–57). Moreover, c-Myc–induced miR-17-5p and miR-20a from the miR-17-92a cluster can inhibit E2F1 translation (43) and affect the expression of E2F1-dependent miRNAs (45, 58). Our data indicate that the expression of oncogenic miRNAs is superimposed over that of the tumor-suppressive miRNAs as a net gain by HR HPV infection. It is possible that the posttranscriptional regulation could also play a role in this net gain, because p53 interacts with Drosha/p68 complex to facilitate Drosha-mediated primary-miRNA processing of certain miRNAs (27) and some miRNAs derived from intron regions are coupled to RNA splicing (59, 60).

We observed increased expression of miR-16, miR-25, miR-92a, and miR-378 in CIN and CC tissues with HR HPV infection, consistent with what we observed in raft tissues with HPV16 or HPV18 infection. In particular, the striking increase of miR-25 and miR-92a correlated with the progression of the cervical lesions, making them credible biomarkers of CINs and CC. Furthermore, we found that the expression ratio of miR-25/92a vs. miR-22/29a increased even more significantly in CIN1+2 tissues over the normal cervical tissues as an early indicator of HPV infection of the cervix.

We note that the expression profiles of miR-22, miR-27a, miR-29a, and miR-100 in clinical samples are slightly different from those in HR HPV-infected and HPV18 oncogene-expressing raft cultures. Several reasons may account for the variation. First, the HPV-infected raft cultures have pathological changes similar to CIN1 only and maintain cell differentiation (Fig. S3A). Cell differentiation could affect miRNA expression and, hence, the regulation of miRNAs in productive HPV infections (20, 25). Indeed, the expression of miR-21, and presumably miR-27a and miR-205, in monolayer cell cultures was altered by cell differentiation in the presence of calcium (Fig. S3 B–D). Second, raft cultures comprise a pure population of keratinocytes, whereas patient tissues contain many additional cell types, including fibroblasts, fat cells, endothelial cells, and infiltrating immune cells. Third, tissue sampling is another contributing factor, particularly for cervical precancer lesions, which are surrounded by normal tissues. The biopsies might be a mixture of normal and diseased tissues, thereby leading to a slightly different miRNA profile from sample to sample and from that of the raft cultures. An analysis of additional patient samples could help to clarify the conclusions. Also, because the sample sizes of CIN1+2 in our study are relatively small, future studies of additional and serial patient specimens over a period of time would be important to determine if there is an miRNA ratio conversion in correlation with CIN progression or regression.

Materials and Methods

Primary keratinocyte cultures and their derived raft tissues with or without HPV16 or HPV18 infection were prepared according to our standard protocols. Total RNA purified from raft tissues was used for miRNA microarray and miRNA-Seq analyses. The resultant miRNA profiles from each tissue were validated by Northern blotting and TaqMan miRNA RT-qPCR assays and were further verified in the institutional review board-approved human cervical tissues with or without HR HPV infections. A detailed discussion of the materials and methods used is provided in SI Materials and Methods.