MicroRNAs play important roles in cancer biology. Calcitriol, the hormonal form of vitamin D3, regulates microRNAs expression in tumor cells. In the present study we asked if calcitriol would modify some of the components of the microRNA processing machinery, namely, Drosha and Dicer, in calcitriol-responsive cervical cancer cells. We found that calcitriol treatment did not affect Drosha mRNA; however, it significantly increased Dicer mRNA and protein expression in VDR-positive SiHa and HeLa cells. In VDR-negative C33-A cells, calcitriol had no effect on Dicer mRNA. We also found a vitamin D response element in Dicer promoter that interacts in vitro to vitamin D and retinoid X receptors. To explore the biological plausibility of these results, we asked if calcitriol alters the microRNA expression profile in SiHa cells. Our results revealed that calcitriol regulates the expression of a subset of microRNAs with potential regulatory functions in cancer pathways, such as miR-22, miR-296-3p, and miR-498, which exert tumor-suppressive effects. In summary, the data indicate that in SiHa cells, calcitriol stimulates the expression of Dicer possibly through the vitamin D response element located in its promoter. This may explain the calcitriol-dependent modulation of microRNAs whose target mRNAs are related to anticancer pathways, further adding to the various anticancer mechanisms of calcitriol.
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.
The DEAD box RNA helicase DDX5 is a multifunctional protein involved in the regulatory events of gene expression. Herein, we presented evidence indicating that DDX5 is transcriptionally upregulated by calcitriol, the hormonal form of vitamin D3. In silico analysis revealed the presence of two putative vitamin D response elements (VDREs) in the DDX5 promoter region. Using luciferase reporter assays, we demonstrated that the DDX5 promoter containing these putative VDREs significantly increased the luciferase activity in vitamin D receptor (VDR)-positive SiHa cells upon calcitriol treatment. Electrophoretic mobility shift assays showed the ability of VDR and retinoid X receptor to interact only with the most proximal VDRE, while chromatin immunoprecipitation analysis confirmed the occupancy of this VDRE by the VDR. Finally, we demonstrated that calcitriol significantly increased both DDX5 mRNA and protein in SiHa cells. In summary, this study shows that DDX5 gene is transcriptionally upregulated by calcitriol through a VDRE located in its proximal promoter. Given the importance of DDX5 as a master regulator of differentiation programs, our study suggests that the pro-differentiating properties of calcitriol may be related with the induction of DDX5.
The Clustered regularly interspaced short palindromic repeats (CRISPR) and the associated protein (Cas9) are naturally found in nature as an adaptive immune system in some bacteria and Archaea (Terns & Terns, 2011). The mechanism has been biotechnologically adapted as a tool for precise genetic edition and applied to diverse species including viruses, bacteria, plants and animals (Barrangou & Doudna, 2016). CRISPR-Cas9 system is comprised of a ribonucleoprotein complex formed by the Cas9 protein, an endonuclease that cleaves DNA in a specific manner directed by its associated short guide RNA (sgRNA) (Gasiunas et al., 2012), complementary to the target DNA sequence which is followed by the nucleotides NGG, known as the protospacer adjacent motif (PAM) (Mojica et al., 2009). This system produces double-strand breaks (DSB) that induce alterations (insertions or deletions) in the genomic DNA mainly driven by the error-prone cellular repair mechanisms: non-homologous end joining (NHEJ) or homologydirected repair (HDR) (Ceccaldi et al., 2016;Chang et al., 2017).
MicroRNAs are a class of small non‐coding RNAs that regulate gene expression at post‐transcriptional level. Regulatory RNAs were discovered in the nematode Caenorhabditis elegans, since then, their biological functions have been studied on different animals, including marine organisms. Several species of penaeid shrimp are important in ecology and fishing, including aquaculture. Overcrowding in aquaculture calls for infectious diseases, which threaten the development of shrimp aquaculture worldwide. Given that microRNAs play crucial regulatory roles in a wide variety of biological processes, there is a scientific interest to understand their contribution on shrimp physiology and pathology, specifically during immune and stress response. Increasing evidence has shown that the expression of microRNAs is affected during virus or bacterial infection and upon stress in shrimp. This information provides valuable insights for a better understanding of shrimp biology by means of microRNA regulation to bacterial and viral diseases.
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