Abstract
miRNAs have emerged as important players in the regulation of gene expression and their deregulation is a common feature in a variety of diseases, especially cancer. Currently, many efforts are focused on studying miRNA expression patterns, as well as miRNA target validation. Here, we show that the over expression of miR-23a∼27a∼24-2 cluster in HEK293T cells induces apoptosis by caspase-dependent as well as caspase-independent pathway as proved by the annexin assay, caspase activation, release of cytochrome-c and AIF (apoptosis inducing factor) from mitochondria. Furthermore, the over expressed cluster modulates the expression of a number of genes involved in apoptosis including FADD (Fas Associated protein with Death Domain). Bioinformatically, FADD is predicted to be the target of hsa-miR-27a and interestingly, FADD protein was found to be up regulated consistent with very less expression of hsa-miR-27a in HEK293T cells. This effect was direct, as hsa-miR-27a negatively regulated the expression of FADD 3′UTR based reporter construct. Moreover, we also showed that over expression of miR-23a∼27a∼24-2 sensitized HEK293T cells to TNF-α cytotoxicity. Taken together, our study demonstrates that enhanced TNF-α induced apoptosis in HEK293T cells by over expression of miR-23a∼27a∼24-2 cluster provides new insights in the development of novel therapeutics for cancer.
Highlights
MiRNAs are a class of highly conserved, non-coding singlestranded RNAs (18–24 nt) derived from the endogenously produced pre-miRNA having a hairpin structure [1,2,3]. miRNAs refine gene expression post-transcriptionally either via the cleavage of target mRNAs or the inhibition of translation of target mRNAs depending upon complementarity between miRNA and the 39 untranslated regions (UTRs) of targeted mRNA in animals and plants [4,5,6,7,8]
The emerging role of miRNAs in the regulation of fundamental set of cellular mechanisms such as proliferation, apoptosis, development, differentiation and metabolism [9,10,11,12,13,14,15,16] clearly suggests that any aberration in miRNA biogenesis pathway or its activity contributes to the human disease pathogenesis including cancer [17]
Primer Designing and Cloning The sequences of miRNAs were retrieved from miRbase, the 39 UTR sequence was retrieved from NCBI and for primer designing primer 3 software was used
Summary
MiRNAs are a class of highly conserved, non-coding singlestranded RNAs (18–24 nt) derived from the endogenously produced pre-miRNA (precursor) having a hairpin (stem-loop) structure [1,2,3]. miRNAs refine gene expression post-transcriptionally either via the cleavage of target mRNAs or the inhibition of translation of target mRNAs depending upon complementarity between miRNA and the 39 untranslated regions (UTRs) of targeted mRNA in animals and plants [4,5,6,7,8]. Intrinsic pathway (the mitochondrial-mediated pathway) is activated in response to a variety of stress conditions, such as cytotoxic drugs, UV irradiation or growth factor withdrawal, which induce mitochondrial perturbation and the cytoplasmic release of pro-apoptotic mitochondrial proteins such as cytochrome-c leading to the activation of caspase-9. Both pathways lead to activation of caspase-3, the executioner caspase responsible for the final morphological changes observed during apoptosis. Caspase- independent pathway occurs via the translocation of AIF (Apoptosis Inducing Factor) from the mitochondria towards the nucleus; resulting in apoptosis [23]
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