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. 2009 Apr;15(4):637-47.
doi: 10.1261/rna.1442309. Epub 2009 Mar 3.

Oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a through viral oncoprotein E6

Xiaohong Wang  1 , Hsu-Kun Wang, J Philip McCoy, Nilam S Banerjee, Janet S Rader, Thomas R Broker, Craig Meyers, Louise T Chow, Zhi-Ming Zheng
Affiliations

Oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a through viral oncoprotein E6

Xiaohong Wang et al. RNA. 2009 Apr.

Abstract

MicroRNAs (miRNA) play pivotal roles in controlling cell proliferation and differentiation. Aberrant miRNA expression in human is becoming recognized as a new molecular mechanism of carcinogenesis. However, the causes for alterations in miRNA expression remain largely unknown. Infection with oncogenic human papillomavirus types 16 (HPV16) and 18 (HPV18) can lead to cervical and other ano-genital cancers. Here, we have demonstrated that cervical cancer tissues and cervical cancer-derived cell lines containing oncogenic HPVs display reduced expression of tumor-suppressive miR-34a. The reduction of miR-34a expression in organotypic tissues derived from HPV-containing primary human keratinocytes correlates with the early productive phase and is attributed to the expression of viral E6, which destabilizes the tumor suppressor p53, a known miR-34a transactivator. Knockdown of viral E6 expression in HPV16(+) and HPV18(+) cervical cancer cell lines by siRNAs leads to an increased expression of p53 and miR-34a and accumulation of miR-34a in G(0)/G(1) phase cells. Ectopic expression of miR-34a in HPV18(+) HeLa cells and HPV(-) HCT116 cells results in a substantial induction of cell growth retardation and a moderate cell apoptosis. Together, this is the first time a viral oncoprotein has been shown to regulate cellular miRNA expression. Our data have provided new insights into mechanisms by which high-risk HPVs contribute to the development of cervical cancer.

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Figures

FIGURE 1.
FIGURE 1.
Reduction of miR-34a in cervical cancer tissues and cervical cancer-derived cell lines. (A) Cervical cancer tissues express a reduced level of miR-34a. Three age-matched pairs of normal cervix and cervical cancer tissues, each positive for a single genotype (HPV16, 82, or 33), were compared for miR-34a expression by Northern blotting. (B) Bar graph showing relative levels of miR-34a in A after being normalized to U6 snRNA for sample loading. (C) A substantial reduction of miR-34a occurs in cervical cancer-derived cell lines or in human keratinocyte line HaCaT cells with a mutant p53. (Top panel) Total RNAs from individual cell lines were examined by miRNA ligation analyses. Paired total RNAs from normal cervix (N Cx), cervical cancer (CxCa) obtained from Ambion and a miRNA-positive (Pos.) control from USB were used as controls for this analysis. (D) Bar graph showing relative levels of miR-34a in each sample in C after being normalized to tRNA (bottom in C) for sample loading.
FIGURE 2.
FIGURE 2.
Reduction of miR-34a expression in oncogenic HPV-immortalized keratinocytes. (A) Down-regulation of miR-34a in raft cultures derived from HVK or HFK immortalized in vitro by HPV16 or HPV18. Total RNA from each raft culture harvested on day 10 was examined by Northern blotting. (B) Bar graph showing relative miR-34a levels in A after being normalized to U6 snRNA for sample loading.
FIGURE 3.
FIGURE 3.
HPV early proteins down-regulate the expression of miR-34a in HPV18-infected raft cultures. (A) Kinetics of the production of HPV18 L1 in HFK raft cultures. L1 was detected by immunohistochemistry (reddish brown) in terminally differentiated keratinocytes and cornified envelopes at 12-d-old and 16-d-old HPV18-containing HFK raft cultures. (B) Reduction of miR-34a expression in raft cultures of HPV18 DNA-containing HFKs (HFK18) without immortalization on day 8, but not on days 12 and 16. Total RNAs from raft cultures of different ages were examined by Northern blotting. (C) Bar graph showing relative miR-34a levels in B after being normalized to U6 for sample loading.
FIGURE 4.
FIGURE 4.
Role of wild-type p53 in activation of miR-34a expression from C33A cells and HFK raft cultures with HPV18 infection. (A) Overexpression of wild-type p53 activates miR-34a expression from C33A, an HPV-negative cervical cancer cell line containing a mutant (mt) form of p53. (Left panel) Shows no detectable expression of miR-34a, but miR-16 in C33A cells in comparison with CaSki (HPV16+) cells containing a wild-type (wt) p53 with detectable expression of both miR-34a and miR-16 by Northern blot analyses. (Right panel) Shows miR-34a expression in C33A cells transactivated by ectopic expression of wild-type p53 at 24 h after transfection. (B) Destabilization of p53 down-regulates the expression of miR-34a in the early, but not in the late HFK raft cultures with HPV18 infection. Raft cultures of HFKs containing HPV18 were harvested on day (D) 8, 12, and 16 and were analyzed for miR-34a expression by Northern blotting and for p53 and p21cip1 production by Western blotting. (A,B) U6 in each sample served as RNA sample loading in Northern blots. (B) Tubulin in each sample served as protein sample loading in Western blots.
FIGURE 5.
FIGURE 5.
Down-regulation of miR-34a by HPV oncoprotein E6 in cervical cancer-derived cell lines. (A) Strategy to silence E6 oncogene expression using an E6 intron-specific siRNA based on splicing of the bicistronic HPV18 E6E7 RNAs (Tang et al. 2006b). (B) Stabilization of p53 and activation of p21cip1 in HPV18 E6 siRNA-treated HeLa cells. Cell samples at 48 h after siRNA transfection were examined by Western blotting with tubulin as a loading control. (C) Knocking down E6 expression by an E6-specific siRNA up-regulates the expression of miR-34a in HeLa cells. Total cell RNA 48 h after an E6-specific siRNA (Tang et al. 2006b) transfection was examined by miRNA ligation analyses (Maroney et al. 2007). (D) Bar graph showing relative miR-34a levels detected in C after being normalized to tRNA for sample loading. (E) Diagram of siRNA targeting to HPV16 E6 (Tang et al. 2006a,b). (F) Knocking down the expression of HPV16 E6 by an E6-specific siRNA promotes the expression of miR-34a in CaSki cells by Northern blotting. (G) Bar graph showing relative miR-34a levels after being normalized to U6 snRNA for sample loading.
FIGURE 6.
FIGURE 6.
Down-regulation of miR-34a expression by HPV18 E6 in HFK raft cultures. (A) S-phase re-entry by differentiated keratinocytes in raft cultures of HFKs transduced with HPV18 E6 or E7 retroviruses. BrdU incorporation was used to show E7-mediated S-phase re-entry. (B) Destabilization of p53 and reduction of miR-34a expression in raft cultures of HFKs acutely transduced with E6 or E6E7, but not by E7, retroviruses. Raft cultures with or without the corresponding retrovirus infection were harvested on day 10 for detection of p53 and p21 by (upper panel) Western (W) blotting or for total RNA preparation and (lower panel) Northern blotting. Tubulin served as protein sample loading in Western blotting. (C) Bar graph showing relative miR-34a levels detected by Northern blotting in B after being normalized to U6 for sample loading.
FIGURE 7.
FIGURE 7.
Accumulation of miR-34a in G0/G1-phase HeLa cells after transfection of E6-specific siRNA. (A) HeLa cells 48 h following treatment with or without an HPV18 E6-specific siRNA (Tang et al. 2006b) were sorted into G0/G1 and S/G2/M subpopulations by flow cytometry, and total RNA from each subpopulation was quantified for miR-34a expression by miRNA ligation analyses. (B) A ratio of miR-34a levels in the two subpopulations from each group was calculated after being normalized to tRNA for sample loading. One representative experiment of two is shown.
FIGURE 8.
FIGURE 8.
Expression of miR-34a in HPV18+ HeLa cells leads to growth retardation, cell cycle arrest, and apoptosis. (A,B) ectopic expression of miR-34a in HeLa cells leads to (A) cell cycle arrest at G2 and (B) apoptosis. Flow cytometry was conducted with cells transfected twice with miR-34a at an interval of 48 h. (Arrows) Indicate the cell numbers at G2. (C,D) Ectopic expression of miR-34a in HeLa cells suppresses cell growth. (C) HeLa cells received miR-34a transfections (arrows mark transfection time points), and total viable cells were counted. (D) Total cell RNA after miR-34 transfection was analyzed by miRNA ligation analyses and tRNA served as a loading control.
FIGURE 9.
FIGURE 9.
A model for miR-34a expression and its role in the development of cervical cancer. (Left) Normal cells express wt p53, which activates the expression of tumor-suppressive miR-34a to control cell proliferation and growth. Viral E6 expression from oncogenic HPVs causes the destabilization of cellular p53 and reduction of tumor-suppressive miR-34a, leading to uncontrolled cell proliferation and development of cancer. (Black box) p53 binding site; (shaded box) miR-34a coding region.
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