www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 7), pp: 12158-12173 Research Paper Human papillomavirus type 16 E6 suppresses microRNA- 23b expression in human cervical cancer cells through DNA methylation of the host gene C9orf3 Chi Lam Au Yeung1,2, Tsun Yee Tsang1, Pak Lun Yau1, Tim Tak Kwok1 1School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China 2Department of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Chi Lam Au Yeung, email: [email protected] Keywords: HPV-16 E6, miRNA-23b, DNA methylation, C9orf3, cervical cancer Received: July 09, 2016 Accepted: November 21, 2016 Published: January 06, 2017 ABSTRACT Oncogenic protein E6 of human papillomavirus type 16 (HPV-16) is believed to involve in the aberrant methylation in cervical cancer as it upregulates DNA methyltransferase 1 (DNMT1) through tumor suppressor p53. In addition, DNA demethylating agent induces the expression of one of the HPV-16 E6 regulated microRNAs (miRs), miR-23b, in human cervical carcinoma SiHa cells. Thus, the importance of DNA methylation and miR-23b in HPV-16 E6 associated cervical cancer development is investigated. In the present study, however, it is found that miR-23b is not embedded in any typical CpG island. Nevertheless, a functional CpG island is predicted in the promoter region of C9orf3, the host gene of miR-23b, and is validated by methylation-specific PCR and bisulfite genomic sequencing analyses. Besides, c-MET is confirmed to be a target gene of miR-23b. Silencing of HPV-16 E6 is found to increase the expression of miR-23b, decrease the expression of c-MET and thus induce the apoptosis of SiHa cells through the c-MET downstream signaling pathway. Taken together, the tumor suppressive miR-23b is epigenetically inactivated through its host gene C9orf3 and this is probably a critical pathway during HPV-16 E6 associated cervical cancer development. INTRODUCTION transactivation or transrepression in ways similar as that for the protein-coding genes [5]. The transcription factors, MicroRNA (miR), a class of small non-coding such as p53, c-myc, E2F and NFκB, were reported to be single-stranded RNA of 19 to 24 nucleotides in length, involved in the regulation of miRNA expressions [5–8]. is recently believed to participate in the development of On the other hand, the regulation of miRNAs may also be cancer, including cervical cancer. The main function of at the post-transcriptional level, such as miRNA maturation miR is to repress the expression of target mRNA by either process. Discrepancies among the levels of primary cleavage or translational silencing. The silencing action transcript, precursor and mature miRNA species have depends on the degree of complementation of miRNA been reported. The biogenesis of mature miRNAs mainly sequence with the 3’-UTR of target mRNAs [1–3]. involves two RNase III enzymes, Drosha and Dicer. “Knowing” the mechanisms that regulate the Previous studies showed that a large fraction of miRNA expression of miRNAs is critical in understanding the might be regulated at the Drosha processing step [4, 9]. role of miRs in cervical cancer development. So far, the Epigenetic modification such as DNA molecular mechanisms however remain largely unknown. hypermethylation often results in the silencing of genes Several microarray profiling studies showed that the while aberrant DNA methylation is detected in many expression pattern of a large number of miRNAs can be cancers. Thus, epigenetic silencing may also be one attributed to the regulatory sequences present in their of the possible mechanisms that contribute to miRNA promoters [4]. Therefore, miRNAs may probably be regulation, especially for miRNAs that with tumor controlled by nuclear transcription factors through either suppressive function, such as miR-124a and 127 [10, www.impactjournals.com/oncotarget 12158 Oncotarget 11]. Oncogenic viruses, such as human papillomavirus in miR-23b expression, ectopic expression of HPV-16 (HPV), are often thought to be involved in the alteration E6 in HCT116 parental and DK cells was carried out of DNA methylation in the host cells, a process appears by transfecting the cells with HPV-16 E6 cDNA [22]. to be critical in cancer progression [12–14]. In fact, DNA HPV-16 E6 suppressed miR-23b expression in HCT116 methyltransferase 1 (DNMT1), a major enzyme for DNA parental cells but failed in the DK cells, indicating that methylation, was found to be overexpressed in human the suppression effect of HPV-16 E6 on miR-23b was cervical cancer [15]. mediated through DNMTs (Figure 1B). On the other Nearly half of the miRNAs are located within hand, in HPV-16 E6 positive human cervical cancer SiHa the introns of protein-coding genes or non-coding cells, knockdown of HPV-16 E6 by E6 siRNA increased transcriptional units, which are known as the host genes. the miR-23b level in cells. Such increase, however, was miR-23b is located in the intron 14 of the host gene lessened by 84% upon the co-transfection with DNMT1 C9orf3 on chromosome 9. Recent studies showed that the cDNA (Figure 1C). This further indicated that DNMT1 expression of miR-23b could be regulated via the upstream might mediate the suppressive effect of HPV-16 E6 on promoter region of the host gene [5, 16] and may also be miR-23b. epigenetically regulated. One of the target genes identified for miR-23b is Lack of CG rich region in the miR-23b upstream c-MET, which is believed to act as a protooncogene and sequence participate in cell proliferation [17, 18]. c-MET was also overexpressed in many human solid tumors including DNMT1 is known to regulate the expression of uterine cervix carcinomas and its overexpression served genes through DNA methylation. Whether DNMT1 as an important prognostic indicator [19, 20]. may mediate the HPV-16 E6 effect on miR-23b through In the present study, HPV-16 E6 was confirmed to DNA methylation is to be examined. By quantitative regulate miR-23b indirectly through the DNA methylation RT-PCR analysis, a 2-fold increase in the expression of of host gene C9orf3 and thus induce c-MET and inhibit miR-23b was observed in SiHa cells treated with DNA apoptosis in cervical cancer cells. The uniqueness and the demethylating agent, 5-aza-2’deoxycytidine (ADC), significance for indirect epigenetic regulation pathway of indicating the likely involvement of DNA methylation in miRs, from the host gene C9orf3 to miR-23b, is worth the regulation of miR-23b (Figure 1D). further investigation. By then, the upstream sequence of miR-23b spanning from 1kb upstream to the mature miR-23b RESULTS was analyzed for the presence of any CG rich region, where presumably DNA methylation may occur. Using Reduced miR-23b expression in DNMT the conventional online algorithms for CpG island knockout cells prediction, such as Methprimer, no typical CpG island (200 nucleotides or more with CG content greater than miR-23b is suggested to be epigenetically regulated 50%) was detected. Despite the failure in the prediction of in the previous study. miR-23b together with a number of putative CpG island in the miR-23b upstream sequence, miRs were found to be overexpressed in human colorectal the possibility of the sequence being methylated was carcinoma HCT116 double DNMT1 and DNMT3b still examined by the methylation-specific PCR (MSP) knockout (DK) cells as compared to the HCT116 parental analysis. No significant difference was however detected cells [21]. It is therefore believed that these miRs may between the ADC-treated cells and the untreated cells be regulated epigenetically. miR-23b was confirmed to or the HPV-16 E6 knockdown cells and the pSilencer be overexpressed in DK cells by quantitative RT-PCR transfected control cells (Supplementary Figure 1). analysis in the present study (Figure 1A). In addition, Also, when SiHa cells were transfected with the luciferase similar to DK cells, the miR was also overexpressed in reporter containing the miR-23b upstream sequence, DNMT1 knockout (D1) cells (Figure 1A), suggesting the reporter activities in cells with or without the ADC that DNMT1 may suppress the expression of miR-23b, treatment were found to be similar. All the results presumably by methylation, in cells. suggested the lack of CG rich region in the upstream sequence of miR-23b and the unlikely involvement of this DNMT1 mediated HPV-16 E6 suppression of sequence in the DNA methylation regulation of miR-23b. miR-23b HPV-16 E6 and DNA methylation decreased HPV-16 E6 was previously found to upregulate C9orf3 mRNA expression DNMT1 in human cervical cancer cells [22]. Therefore, it is hypothesized that HPV-16 E6 may act through miR-23b is an intronic miRNA and the host gene methylation to regulate the expression of miR-23b. To is C9orf3. Although intronic miRNAs can be regulated investigate the regulatory role of HPV-16 E6/DNMT1 by the miRNAs’ own promoters, they may also be co- www.impactjournals.com/oncotarget 12159 Oncotarget Figure 1: DNMT1 mediated the HPV-16 E6 suppression of miR-23. miR-23b expression level was determined by quantitative RT-PCR analysis. A. the miR-23b expression level in HCT116 parental, D1 and DK cells. B. HCT116 parental or DK cells were transiently transfected with pcDNA3/HPV16E6cDNA (HPV16E6FL) or pcDNA3 empty vector for 24 h. C. SiHa cells were transiently transfected with pSilencer/HPVE6siRNA (HPV16E6i) alone or co-transfected with pGFP-C3/DNMT1cDNA (HPV16E6i+DNMT1) for 24 h. The cells were transfected with pSilencer empty vector in parallel in which miR-23b expression level was designated as 1.0. D. SiHa cells were treated with 5 μM 5-aza-2’-deoxycytidine (ADC) for 24 h.