A cis-acting element in the promoter of human ether à go-go 1 potassium channel gene mediates repression by calcitriol in human cervical cancer cells

Abstract

The human ether à go-go 1 potassium channel (hEAG1) is required for cell cycle progression and proliferation of cancer cells. Inhibitors of hEAG1 activity and expression represent potential therapeutic drugs in cancer. Previously, we have shown that hEAG1 expression is downregulated by calcitriol in a variety of cancer cells. Herein, we provided evidence on the regulatory mechanism involved in such repressive effect in cells derived from human cervical cancer. Our results indicate that repression by calcitriol occurs at the transcriptional level and involves a functional negative vitamin D response element (nVDRE) E-box type in the hEAG1 promoter. The described mechanism in this work implies that a protein complex formed by the vitamin D receptor-interacting repressor, the vitamin D receptor, the retinoid X receptor, and the Williams syndrome transcription factor interact with the nVDRE in the hEAG1 promoter in the absence of ligand. Interestingly, all of these transcription factors except the vitamin D receptor-interacting repressor are displaced from hEAG1 promoter in the presence of calcitriol. Our results provide novel mechanistic insights into calcitriol mode of action in repressing hEAG1 gene expression.

Calcitriol increases Dicer expression and modifies the microRNAs signature in SiHa cervical cancer cells

“…The PCR thermocycling parameters consisted of an initial denaturation step at 95°C for 10 min, followed by 45 cycles at 94°C for 10 s, 60°C for 30 s, and 72°C for 1 s, as described in the kit protocol from Roche. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize gene expression, given that it is not affected by calcitriol treatment in SiHa, HeLa, and C33-A cervical cancer cells (Avila et al 2010;Cázares-Ordoñez et al 2015). Data were analyzed using a relative quantification analysis with the LightCycler Software 4.05 (Roche, Germany).…”

“…Western blotting was performed as described previously (Cázares-Ordoñez et al 2015). Proteins were separated onto 10% denaturing polyacrylamide gels and transferred by wet electroblotting overnight at 25 volts to nitrocellulose membranes.…”

“…Protein concentration was determined by the bicinchoninic acid protein assay. EMSAs were performed using the Gel Shift Assay System (Promega, Madison, Wisconsin, USA), as described by Cázares-Ordoñez et al (2015), using double-stranded probes generated by mixing the complementary oligonucleotides (Integrated DNA Technologies, Inc., Coralville, Iowa, USA), shown in Table 2. EMSAs were performed with 32 P end-labeled probes containing a canonical DR3-type VDRE as a positive control (DR3-VDRE) (Qi et al 2002), the putative VDRE in the human Dicer gene promoter (Dicer-VDRE) or a mutant form of this VDRE (Dicer-VDRE-Mut).…”

“…Indeed, ether-à -go-go 1 is an early marker of cervical cancer (Farias et al 2004) and a promising anticancer target (Bai et al 2013). Recent results from our group have demonstrated that calcitriol, through downregulating the ether-à -gogo 1 potassium channel (Cázares-Ordoñez et al 2015), prevented cell proliferation of cervical cancer cells (Avila et al 2010). These observations suggest that part of the calcitriol effects on cell proliferation may be through the upregulation of specific miRNAs, as mentioned above.…”

“…Thus, the aim of the present study was to investigate if calcitriol affects the expression of Drosha and Dicer in calcitriol-responsive cervical cells and to evaluate its effects on miRNA differential expression. SiHa and HeLa cervical cell lines were considered calcitriol-responsive because they express the VDR (Avila et al 2010;Kloss et al 2010), whereas C33-A cervical cell line was considered calcitriol-unresponsive because it lacks the VDR (Cázares-Ordoñez et al 2015).…”