MicroRNA Regulation Of Human Choline Kinase Alpha Gene Expression
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Date
2016-12
Authors
Mohamad Ayub Khan, Sharzehan
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
MicroRNA (miRNA) is a small RNA molecule of 22 nucleotides long that has emerged to play a fundamental role in the regulation of gene expression, which affects biological processes such as cell proliferation and differentiation. Choline kinase is the first enzyme in the Kennedy pathway that synthesizes phosphatidylcholine, which is the major phospholipid component in the mammalian cell membrane. Choline kinase is encoded by two separate genes, namely ckα and ckβ having tissue specific characteristic and thus could be modulated by miRNAs. However, the potential regulation of ck gene expression by miRNA has never been explored and experimentally tested. In this study, it is hypothesized that miRNA interacts with CK mRNA and regulates its expression in the cell. Prediction for potential miRNAs that bind to ck mRNA was achieved by using four online softwares and based on the favorable characteristics for miRNA-mRNA interaction, hsa-miR-876-5p and hsa-miR-646 were chosen for further experimental validations. The miRNA concentration and transfection duration were first optimized with miRNA targeting GAPDH. The levels of GAPDH mRNA and protein were analyzed by using real-time PCR and Western detection, respectively. Results showed that 25 nM and 48 hours transfection with miRNA targeting GAPDH produced the strongest downregulation of mRNA and protein levels in HepG2 cells without apparent cytotoxic effect. To experimentally validate the two predicted ck-targeting miRNAs, mimics for hsa-miR-876-5p and hsa-miR-646 were transfected into HepG2 cell line and the level of ckα mRNA was determined by real-time PCR. Only hsa-miR-876-5p
was able to induce significant downregulation of ck mRNA. Subsequently, the concentration and transfection duration of hsa-miR-876-5p were optimized to obtain the strongest downregulation of ck gene expression. The results showed that the strongest downregulation was obtained when HepG2 cells were transfected with 25 nM of hsa-miR-876-5p for 48 hours, which resulted in four times lower levels of ckα mRNA compared to negative control. Effects of the miRNA on HepG2 cell viability and cell morphology were also analyzed by using MTT cell viability assay and scanning electron microscopy, respectively. The viability was approximately 25% lower than negative control and electron micrographs showed sign of apoptosis when the HepG2 cells were transfected with optimum concentration and duration of hsa-miR-876-5p. This study has identified hsa-miR-876-5p as a potential miRNA modulator of ck gene expression. Since ck has been implicated in various cancers, this miRNA could play an important role in tumorigenesis. This study has provided an association between miRNA and ckα gene expression and the knowledge can lead to a better anti-cancer strategy that targets choline kinase.
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Keywords
MicroRNA (miRNA) is a small RNA , in the regulation of gene expression.