Suppression of miR-22, a tumor suppressor in cervical cancer, by human papillomavirus 16 E6 via a p53/miR-22/HDAC6 pathway

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that function to down-regulate gene expression involving in various cellular processes related to carcinogenesis. Recently, miR-22 was identified as a tumor-suppressing miRNA in many human cancers. However, the regulatory mechanism and the specific function of this miRNA in cervical cancer remain unclear. In the present study, we carried out gene transfection, western blot and quantitative RT-PCR to explore the regulatory mechanism and the functional role of miR-22 in cervical cancer. We verified that miR-22 was down-regulated in cervical cancer tissues and cervical cancer cell lines relative to matched non-tumor tissues and normal human cervical keratinocyte line (HCK1T). By contrast, histone deacetylase 6 (HDAC6) was inversely correlated with miR-22 in both cervical tissues and cancer cell lines. Mechanically, HDAC6 was down-regulated by miR-22 at the post-transcriptional level, via a specific target site within the 3'UTR, identified by a luciferase reporter assay. Moreover, we also showed that the correlation between miR-22 and HDAC6 expression was regulated by an E6/p53 pathway in HCK1Ts expressing HPV16 E6. For functional study, an ectopic expression of miR-22 could inhibit cell proliferation and migration, and could induce apoptosis of cervical cancer cell lines. These findings demonstrated that miR-22 was down-regulated in cervical cancer and inversely collated with its downstream target HDAC6. MiR-22 acts as tumor suppressor by inhibiting proliferation and migration, and by inducing apoptosis of cervical cancer cell lines by targeting the 3'UTR of HDAC6. This newly identified E6/p53/miR-22/HDAC6 regulatory network might be a candidate therapeutic target for cervical cancer.

Fig 1. miR-22 was down-regulated in cervical cancer tissues and cell lines.

A. The relative levels of miR-22 in the different grades of fresh-frozen cervical tissues. B. Comparison of miR-22 expression levels between lesion tissues and adjacent normal tissues in HSIL and SCC FFPE samples. C. The relative levels of miR-22 in four cervical cancer cell lines and one immortalized human cervical keratinocyte line (HCK1T). The levels of miR-22 were measured by qRT-PCR and normalized with U44 small nuclear RNA. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 2. HDAC6 was up-regulated in cervical cancer tissues and cell lines.

A. The relative levels of HDAC6 in the different grades of fresh-frozen cervical tissues. B. The relative levels of HDAC6 in four cervical cancer cell lines and one immortalized human cervical keratinocyte line (HCK1T). The levels of HDAC6 were measured by qRT-PCR and normalized with GAPDH. C. The expression levels of HDAC6 protein in four cervical cancer cell lines and one immortalized human cervical keratinocyte line (HCK1T) were measured by western blotting. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3. HDAC6 is a target of miR-22.

A. miR-22 inhibited the expression of HDAC6 in cervical cancer cells. B. Predicted duplex formation between human HDAC6 and miR-22. C. The relative luciferase activities in 293FT and C33A cells were determined after the HDAC6 3’UTR plasmid was co-transfected with miR-22. Error bars indicate the standard error of the mean (SEM) of triplicated independent experiments. *P < 0.05, ***P < 0.001.

Fig 4. E6, p53, miR-22 and HDAC6 expression levels in HCK1T cells expressing HPV16 E6.

1x10⁶ cells were seeded in 60-mm tissue culture dishes and maintained in growth medium with/without 1 μg/ml doxycycline. Expression levels of E6 mRNA (A), HDAC6 mRNA (B) and miR-22 (C) at different time points were examined by qRT-PCR. D. p53 and HDAC6 protein levels were analyzed by western blot. Error bars indicate the standard error of the mean (SEM) of triplicated independent experiments. *P < 0.05, ***P < 0.001.

Fig 5. Overexpression of miR-22 inhibited proliferation and migration of cervical cancer cell lines.

A. Transfection of miR-22 expression plasmid to C33A and HeLa cells increased the expression of miR-22 detected by real-time quantitative RT-PCR. B. Cell proliferation of these cells transfected as in (A) was measured at the indicated time periods using the MTT assay. C. Overexpression of miR-22 led to a slower closing of scratch wounds, compared with negative control transfected cells. Error bars indicate the standard error of the mean (SEM) of triplicated independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 6. miR-22 induced cervical cancer cells apoptosis.

Flow cytometric assay showed the increased fractions of apoptotic cells in C33A and HeLa cell lines transfected with miR-22 (A). Mean fraction of apoptosis in C33A (B) and HeLa (C) cells. The values represent apoptotic cells. Error bars indicate the standard error of the mean (SEM) of triplicated independent experiments. *P < 0.05, **P < 0.01. D. Hypothetical mechanism by which HPV E6 induces cervical cancer development/progression through the p53/miR-22/HDAC6 pathway. Modulation of tumor suppressor miR-22 and its down-stream target HDAC6 by the HPV E6/p53 pathway is involved in the development process of cervical cancer.