Abstract
We previously demonstrated that apoptosis induced by tocotrienols (γ and δT3) in HeLa cells is preceded by Ca2+ release from the endoplasmic reticulum. This event is eventually followed by the induction of specific calcium-dependent signals, leading to the expression and activation of the gene encoding for the IRE1α protein and, in turn, to the alternative splicing of the pro-apoptotic protein sXbp1 and other molecules involved in the unfolded protein response, the core pathway coping with EndoR stress. Here, we showed that treatment with T3s induces the expression of a specific set of miRNAs in HeLa cells. Data interrogation based on the intersection of this set of miRNAs with a set of genes previously differentially expressed after γT3 treatment provided a few miRNA candidates to be the effectors of EndoR-stress-induced apoptosis. To identify the best candidate to act as the effector of the Xbp1-mediated apoptotic response to γT3, we performed in silico analysis based on the evaluation of the highest ∆ in Gibbs energy of different mRNA–miRNA–Argonaute (AGO) protein complexes. The involvement of the best candidate identified in silico, miR-190b, in Xbp1 splicing was confirmed in vitro using T3-treated cells pre-incubated with the specific miRNA inhibitor, providing a preliminary indication of its role as an effector of EndoR-stress-induced apoptosis.
Highlights
Endoplasmic reticulum (EndoR) stress occurs when the capacity of the EndoR to fold proteins becomes saturated
This event is eventually followed by the induction of specific calcium-dependent signals, leading to the expression and activation of the gene encoding for the IRE1α protein and, in turn, to the alternative splicing of the pro-apoptotic protein sXbp1 and other molecules involved in the unfolded protein response, the core pathway coping with EndoR stress
We previously demonstrated that induction of apoptosis in HeLa cells by γ- and δ-tocotrienols (T3s) is associated with EndoR stress and the unfolded protein response (UPR) through Ca2+ release and X-box binding protein 1 (Xbp1)–mRNA alternative splicing [8]
Summary
Endoplasmic reticulum (EndoR) stress occurs when the capacity of the EndoR to fold proteins becomes saturated. Regulators of UPR include transmembrane proteins, such as PKR-like endoplasmic reticulum kinase (PERK), activating transcription factor (ATF6), and inositol-requiring enzyme (IRE1α), which triggers a conformational shift that activates its endoribonuclease domain This nuclease catalyzes a unique cytoplasmic mRNA-splicing reaction, cleaving out 26 nucleotides from X-box binding protein 1 (Xbp1) mRNA [2,3]. To validate results obtained through TaqMan Cards, γT3 incubation of HeLa cells was repeated and modulation of a set of miRNAs was remeasured using miRNA-specific TaqMan probes and the human pseudogene RNU6B as a calibrator [20] This strategy confirmed that three miRNAs, namely miR-190b (p-value = 0.05), miRNA215 (p-value = 0.0009), and miRNA-148a (p-value = 0.001), were significantly upregulated by γT3
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