Small interfering RNAs for treatment of neurosurgical diseases, specifically involving neuropathic pain and neural tumors
Date Issued
2016
Date
2016
Author(s)
Tsai, Ming-Dar
Abstract
RNA interference (RNAi) describes a conserved biological response to double stranded RNA (dsRNA), which results in the degradation of homologous messenger RNA. This process of sequence-specific, post-transcriptional gene silencing has become a key technique for rapidly assessing gene function in both plants and mammals. For target RNA recognition to occur, the small interfering RNA (siRNA) duplex unwinds, allowing binding of one siRNA strand to the target mRNA. The advantage of RNAi to an organism is that siRNA, which specifically binds to target mRNA, prevents damage to other tissues. Adopting this approach may increase a treatment’s therapeutic effect and reduce side effects in patients receiving treatment. The first part of this dissertation is RNA interference of bradykinin B2 receptor reducing the neuropathic pain caused by sciatic nerve injury. In the condition of cell injury, several inflammatory mediators release from damaged cells. Some of these mediators cause local effect results in increased sensitivity to pain. The hypersensitivity of this sensation is partly due to inflammatory mediators such as prostaglandins, histamine, bradykinin, substance P, and serotonin, which cause a local effect of nociception, and partly some neurotrophic factors. Some mediators transmit the sensation of pain, induced by damage of surrounding cells or even nociceptive neurons. The sensitization of nociception caused by neural damage is normally known as neuropathic pain. Other mediators, such as nerve growth factor and other neurotrophic factors, regulate the progress of neuron regeneration. However, changes in expression of receptors for allogeneic substances such as bradykinin may also be involved, causing a long-term effect. The result shows the nociception caused by neuropathy was reduced by bradykinin B2 receptor siRNA. We therefore supposed that inhibit bradykinin B2 expression may reduce the nociceptive sensation caused by neuropathy. In our preliminary studies, we screened several inflammatory mediators and found that the nociception caused by neuropathy can be decreased by blocking the transmission of bradykinin. We therefore constructed the RNA interference (RNAi) of bradykinin B2 receptor and applied on the neuropathic animal models. The spared nerve injury models will be used to demonstrate the neuropathic nociception and the mechanical sensitivity behavior test were used to evaluate the degree of neuropathic nociception. Bradykinin B2 receptor expression was upregulated after sciatic nerve crush, while this upregulation was reversed by application of siRNA of bradykinin B2 receptor. This result confirmed that inhibit bradykinin B2 gene expression reduce the nociception caused by neuropathy. The second part of this dissertation is nuclear condensation and cell cycle arrest induced by telomerase siRNA in neuroblastoma cells. Neuroblastoma is a type of malignant extracranial tumor that occurs in children. Advanced neuroblastoma, and tumors with MYCN amplification in particular, has poor prognoses. Therefore, it is important to find an effective cure for this disease. Small interfering RNA (siRNA) disrupts gene function by specifically binding to target mRNA. In this study, we used siRNA against telomerase to treat neuroblastoma, to evaluate any anti-proliferative effect on these cells. We evaluated cell viability by WST-1 assay on neuroblastoma cells treated with or without telomerase siRNA. Nuclear condensation, an indicator for apoptotic cells, was determined by DAPI labeling following siRNA treatment. The effectiveness of telomerase siRNA on altering the neuroblastoma cell cycle was detected by flow cytometry. Our results indicated that telomerase siRNA reduces the viability of neuroblastoma cells and increases the percentage of cells in the cell cycle’s sub-G1 phase. We found that telomerase siRNA increases the percentage of condensed DNA in neuroblastoma cells. In conclusion, using siRNA against telomerase could be further developed as a therapy for the treatment of neuroblastoma. The third part of this dissertation is involvement of Smac, p53, and caspase pathways in induction of apoptosis by gossypol in human retinoblastoma cells. Retinoblastoma is a malignant tumor of the retina usually occurring in young children. To date, the conventional treatments for retinoblastoma have been enucleation, cryotherapy, external beam radiotherapy or chemotherapy. Most of these treatments, however, have possible side effects, including blindness, infections, fever, gastrointestinal toxicity and neurotoxicity. More effective treatments are therefore imperative. Gossypol has been reported as a potential inhibitor of cell proliferation in various types of cancers, such as prostate cancer, breast cancer, leukemia, and lung cancer. This study investigates the possible anti-proliferative effect of gossypol on retinoblastoma. The human retinoblastoma cells were cultured with various concentrations of gossypol and checked for cell viability with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nuclear condensation caused by cell apoptosis was detected by staining retinoblastoma cells with 4'', 6-diamidino-2-phenylindole (DAPI), counting those with condensed nuclei, and determining the percentage of apoptotic cells. In addition, the stages of apoptosis and phases in cell cycles were examined with flow cytometry. The possible signal transduction pathways involved were examined with a protein array assay and western blot analysis. Our results indicated that after incubation, the cell survival rate was significantly lower after treatment with 5, 10, and 20 μM of gossypol. The maximum antisurvival effect of gossypol was observed at 20 μM, and the number of apoptotic cells was higher in the preparations cultured with 10 and 20 μM of gossypol. The results in flow cytometry indicated that at concentrations of 10 and 20 μM, gossypol increased the proportion of early- and late-apoptotic retinoblastoma cells and induced cell arrest of retinoblastoma cells at the same concentrations. This anti-proliferative effect was later confirmed by upregulating the expression of death receptor 5 (DR5), caspase 8, caspase 9, caspase 3, cytochrome C, tumor protein 53 (p53), and second mitochondria-derived activator of caspases (Smac) in the signal transduction pathways. We concluded that gossypol has an anti-proliferative effect on retinoblastoma cells.
Subjects
Neuropathic pain
Bradykinin B2 receptor
siRNA
Telomerase
Neuroblastoma
Nuclear condensation
Cell cycle arrest
Gossypol
Retinoblastoma Apoptosis
SDGs
Type
thesis
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