Function Of QKI In Chemotherapy Induced Cancer Cell Apoptosis And The Underlying Mechanism

Apoptosis, termed programmed cell death, occurs both in the physiological and pathological situation. As a biological behavior in vivo, apoptosis involves in many physiological and pathological processes, such as development, homeostasis, stress, tumor and etc. Apoptosis signal pathways include extracellular ligands pathway and intracellular stress signals pathway. When extracellular ligands such as Fas, TNFa or TRAIL bind to their receptors, the intracellular death domains of these receptors recruit adaptor proteins (such as FADD and TRADD) and initiator caspase-8 and -10, resulting in the formation of the death-inducing signaling complex (DISC). Caspase-8 and -10 are activated at the DISC, which cleave the executioner caspases, caspase-3, -6 and -7. The latter three caspases are the main proteases that degrade the cell. Intracellular stress signals, such as growth factor withdrawal, DNA damage, oxidative stress or oncogene activation, lead to permeabilization of the mitochondrial outer membrane and release of cytochrome c. The consequent release of cytochrome c leads to the formation of a complex– the apoptosome– which contains cytochrome c, Apaf-1 and initiator caspase-9. Caspase-9 is auto-activated by induced proximity in the apoptosome. Active caspase-9 cleaves and thereby activates the executioner caspases. Complicated and intercrossed signal pathways for apoptosis make the regulation of apoptosis a complex and diverse process.Nowadays, more attention is paid to the post-transcriptional regulation mediated by RNA binding protein and miRNA. Posttranscriptional regulation is believed to play a vital role in the apoptosis. RNA binding protein QKI belongs to the evolutionarily conserved STAR family and has a critical role in the myelination of CNS development. However, the wide expression pattern of QKI in numerous tissues besides CNS implicates a fundamental role of QKI in other biological behavior, such as vascular development, cell apoptosis, cell adhesion, cell growth and organogenesis. The QKI gene produces a diverse set of proteins by alternative splicing. The three well studied isoforms (QKI-5, -6, and -7) appear to have different roles in development. They are constructed with the same 311-amino acid body but have different carboxyl tails consisting of 30, 8, and 14 amino acids, respectively. Because QKI-5 contains a nuclear location signal peptide (NLS), it mainly locates in the cell nuclus. QKI-6 and 7 mainly locate in the cell plasma. But sometimes QKI-5 translocates between cell plasma and cell nuclus. The distinction of location decides that isoforms of QKI have different function. According to the predicted 1400 candidate target mRNAs reported in Nat Stuct Mol Bio, many putative downstream targets are related with apoptosis, such as Bid,FOXO1,Sirt1, leading to the possibility of QKI in apoptosis regulation.To this end, we observed the alteration of QKI expression level during the chemotherapy induced cancer cell apoptosis through Real time PCR, Western Blotting and Dual-luciferase reporter assay system. Then we investigated the influence of the QKI expression changes on apoptosis through PARP detection, Annexin V-FITC/PI flow cytometry and ROS detection. Finally, we elucidate a potential mechanism study on the role of QKI on the chemotherapy induced cancer cell apoptosis. The main findings of our study are as follows:1. We constructed a QKI dominant negative mutation vector.The dominant negative mutation expresses peptide with the dimer sequence and without the RNA bingding ability. In the vector, the C terminal of the DN mutation was fused with the nuclear location signal of QKI-5, which thus has the same localization of QKI-5 isoform and specifically inhibits QKI-5 function.2. The recombinant adenovirus of QKI-6 has been successfully constructed through ViraPower? adenovirus expression system, which has been expressed even at a very low MOI.3. RT-PCR and Western-blot results showed that the mRNA and protein level of QKI decreased under the stimulation of three different chemotherapy drugs. The activity of QKI promoter was downregulated under the stimulation. QKI mRNA stability assay showed that QKI mRNA stability decreased under the stimulation. In addition, proteasome inhibitor could not reverse the downregulation of QKI expression under those stimuli. These results showed that the alteration of QKI expression level occurred on the transcriptional and post-transcriptional level.4. Overexpression of QKI-5 resulted in down-regulation of epricubicin and cisplatin induced apoptosis in Hela cells, while overexpression of QKI-7 resulted in significant more obvious apoptosis. In addition, overexpression of QKI-6 had no obvious apoptosis on epricubicin and cisplatin induced apoptosis. Repression of endogenous of all the QKI isoforms resulted in up-regulation of epricubicin and cisplatin induced apoptosis in Hela cells, which maybe due to the predominant expression of QKI-5 in Hela cells. 5. Overexpression of QKI-7 resulted in 1.4 fold increase of ROS in cells even under the rest stage, and the increase was up to 3 fold during epricubicin and cisplatin induced apoptosis, suggesting QKI-7 promotes the production of ROS especially during the chemotherapy. For the role of QKI-5 in antagonizing apoptosis, we found that FOXO3a 3'UTR contained two QKI response elements by bioinformatics analysis. Western blotting showed that FOXO3a expression decreased when QKI-5 expression increased under the stimulation of ciaplatin in Hela cells. At the same time we observed that overexpression of QKI-5 resulted in the reduction of FOXO3a expression, suggesting a role of FOXO3a in QKI-5 mediated apoptosis regulation. Further study is needed to confirm the speculation.In summary, we first defined an important role of QKI in chemotherapy induced apoptosis. QKI-5 has an anti-apoptosis function possibly through reducing the FOXO3a expression. In contrast, QKI-7 promotes the apoptosis of cells through enhancing the production of ROS during the chemotherapy drugs induced apoptosis. These results indicate that the alteration of QKI isoforms expression may be involved in the apoptosis under the stimulation of chemotherapy drugs. Aberrant QKI alteration under chemotherapy may be responsible for drug resistance, which is of great value for further examination. QKI may become a new target of tumor adjuvant chemotherapy.