Small Interfering RNA Targeting Survivin Sensitizes Lung Adenocarcinoma Cell To Soluble FasL

RNA interference (RNAi) is the sequence-specific gene silencing induced by double-strand RNA (dsRNA). This phenomenon is conserved in a variety of organisms: caenorhabditis elegans, drosophlia, plants, and animal. RNAi is mediated by short interfering RNA that is produced from long dsRNA of exogenous or endogenous. The dsRNA is cleaved by an RNase III like enzyme (Dicer) into siRNA.. The siRNA are incorporated into a multisubunit protein complex, the RNA-induced silencing complex (RISC), which direct the siRNA to the appropriate mRNA containing a sequence identical to that of siRNA.. This complex, when activated, can specifically silence or down-regulate gene expression. However, in most mammalian cell, dsRNA longer than 30 nucleotides activate an interferon (IFN) response leading to nonspecific degradation of RNA transcripts and a shutdown of host cell protein translation. This nonspecific effect can be circumvented by the use of synthetic siRNA that are 21 nucleotides long with short 3'over-hangs. So far, RNAi has been used to inhibite virus-induced disease, oncogene-induced tumorigenesis and cancer caused by viral infection. Chemically synthesized siRNA have some defects. The expression of siRNA from DNA template offers several advantages over chemically synthesized siRNA delivery. Hairpin RNA transcribes from vector (for example retroviral-based approach) have been proved to suppress the expression of target gene more efficiently, less expensively and more easily than chemically synthesized siRNA.. PsilencerTM4.1- CMV neo is a plasmid employ a powerful modified Cytomegalomavirus (CMV) promoter to drive high level expression of cloned hairpin siRNA template in a variety of cell type. PsilencerTM4.1- CMV neo vector can stably express in cell for about six months, it has the advantage that the expression of target gene can be reduce for a longer time than that of transient transfection. This vector contain a geneticin(G418) resistance gene to enable antibiotic selection. Antibiotic selection can be used to enrich for cell that were successfully transfected with PsilencerTM4.1- CMV neo by killing off cells that lack the plasmid. For long-term gene knockdown studies the Geneticin resistance gene make it possible to select cell population, or clonal cell, that stably express the hairpin siRNA.Lung cancer is the leading cause of cancer-related deaths in the developed country and many big cities in our country. Nonsmall cell lung cancer (NSCLC) histology accounts for approximately 75~80% of these malignancies. Lung cancer patients have poor prognosis because majority of patients presents with locally advanced or metastatic disease at diagnosis. The median survival time of patients with untreated metastatic NSCLC is only 4~5 month, with a 1-year survival rate of approximately 11~18%. Chemotherapy is the important treatment to lung cancer, but NSCLC cell can develop drug resistance quickly resulting in the failure of therapy. So treatment of these patients is a great challenge and new therapeutic modalities are needed.Like many other malignant tumor, there are a series of imbalance between oncogenes and tumor suppressor genes in the course of lung cancer initiation, development and metastasis. The overexpression of apoptosis inhabited gene is a major reason in lung cancer formation. Apoptosis, also named programmed cell death, is a physiological suicide playing a crucial role in the regulation of tissue homeostasis in multicellular. Cell with genetic abnormality can be eliminated by apoptosis. Therefore, diminished or disordered apoptosis result in accumulation of genetic mutation which may lead to tumor initiation, progression and therapeutic resistance.Apoptosis is influenced by a wide variety of regulatory stimuli. Among the death receptor (TNF-R1 DR-3/TRAMP TRAIL-R1 and TRAIL-R2), Fas is one of the most potent inducers of apoptosis. Fas is a type I member protein belonging to tumor necrosis factor (TNF) receptor family that induces a death signal when bound to its ligand. FasL is the ligand of Fas. Interaction of with its cognate receptor Fas induces receptor trimerization, which in turn result in the recruitment of the adaptor protein FADD and Caspase8.Subsequent activation of downstream leads to irreversible cell damages. Several intracellular protein including IAP (inhibitor of apoptosis protein) and BCL-2 family members are known to interfere with the Fas death signaling cascade thus render cell less sensitive to apoptosis. Survivin is a member of IAP family. Survivin gene exists on chromosome 17q, the molecular weight of Survivin is 16.5 KD with a single baculovirus inhibitor of apoptosis (IAP) repeat (Bir) domain and a coiled-coil region at its C terminus. Because of the presence of the Bir domain, Survivin has been placed in the IAP family. Survivin is present during fetal development but undetectable in terminally differential normal tissue. Importantly, Survivin is abundantly expressed in transformed cell line and in most of common cancer. Survivin is essential for the viability of proliferating cells because knocking out or interfering with its activity results in abnormal cytokinesis,polypliodization and eventual cell death. Murine gene-target studies have confirmed that Survivin is an essential protein because homozygous knockout embryos display gross cellular degeneration, lacked an inner cell mass, and failed to progress beyond embryonic days. Although the exact mechanism of anti-apoptotic function is unclear, some studies had confirmed that Survivin has a capacity to inhibit caspase 3, 7 and 9 in cell receiving an apoptotic stimulus. Because Survivin expression in many malignant tumor, not usually presently in normal tissue and is rarely found in mature tissue, thus Survivin is a good gene therapeutic target in the treatment of cancer. In this study our aim have four: One is to construct Survivin specific small interfering RNA (siRNA) expression vector. Two: To transfect it into A549 cell and select clonal cell that stably express the hairpin siRNA. Three: To test its suppressing effect on Survivin expression in A549 cell and test its antiproliferative and proapoptositic effect to A549 cell. Four: Because FasL is toxic to cell, it may be dangerous to use it to treat cancer patients. We want to find a way to enhance the sensitivity of lung cancer cell to FasL in order to reduce the concentration of FasL which can efficiently induce apoptosis of cancer cell.Chapter I Construction of Survivin specific small interfering RNA expression vectorObjective To construct the Survivin small interfering RNA (siRNA) expression vector. Methods Survivin specific oligonucleotides were designed and synthesized. Two oligonucleotides were annealed to form the double strand DNA fragment. This short double strand DNA was cloned into PsilencerTM4.1-CMV neo vector. The recombinant Survivin-siRNA expression vector was conformed by sequencing. Results Survivin-siRNA expression vector was constructed successfully, DNA sequencing proved that the construction of recombinant expression vector was right. Conclusions: The recombinant Survivin-siRNA expression vector can be constructed successfully. It can express stably. Chapter II The suppressing effect of Survivin specific small interfering RNA expression vector on the expression of Survivin gene in lung adenocarcinoma A549Objective To transfect Survivin small interfering RNA (siRNA) expression vector into lung adenocarcinoma A549 cell and test its suppressing effect on Survivin expression. Methods The Survivin siRNA expression vector was transfected into A549 cell and G418(Geneticin) was used for selecting clonal stable cell line. The inhibitory effect of Survivin-siRNA was tested by semi-quantitative and real-time fluorescent quantitative reverse transcription polymerase chain reaction PCR( FQ RT-PCR) , western blot and immunohistochemistry. Results Survivin-siRNA expression vector was constructed and transfected into A549 cell successfully, it can effectively reduce the mRNA and protein level of Survivin (76.4% and 84.5% respectively). Immunohistochemistry also indicate the lower expression of Survivin. Conclusions: The Survivin-siRNA expression vector can inhibit the expression of Survivin gene of A549 cell.Chapter III Antiproliferative and proapoptositic effects of Survivin specific small interfering RNA expression vector on lung adenocarcinoma A549Objective To test the antiproliferative and proapoptositic effect of Survivin specific small interfering RNA(siRNA)expression vector on lung adenocarcinoma cell A549. Methods Apoptotic cell was observed by acridine orange fluorescen staining. The cell proliferation and apoptosis rate was assayed by tetrazolium bromide (MTT) colorimetry and flow cyclometry. Results Acridine orange fluorescen staining can find apoptotic A549 cell. The A549 cell transfected with Survivin-siRNA have a lower proliferation rate at every time point than that of positive control,negative control and control group A549 cell, the difference is significant(P<0.01).The apoptosis rate of A549 cell transfected with Survivin-siRNA[(10.09±1.31)%]is higher than that of positive control [(1.03±0.22)%],negative control[(0.98±0.26)]%and control group A549 cell[(1.14±0.21)%] , the difference is significant(P<0.01).Conclusions The Survivin-siRNA expression vector can inhibit the proliferation and induce the apoptosis effectively in lung adenocarcinoma cell A549.Chapter IV Small interfering RNA targeting Survivin sensitizes lung adenocarcinoma cell to soluble FasLObjective To test the sensitivity of lung adenocarcinoma cell A549 with Survivin specific RNA interference to soluble FasL. Methods After treated with soluble FasL, proliferation and apoptosis of the lung adenocarcinoma cell A549 with Survivin specific RNA interference was assayed by tetrazolium bromide (MTT) colorimetry and flow cytometry. Results: Survivin-siRNA A549 cell treated with sFasL had lower proliferation rate at every time point than that of single sFasL group,siRNA group and control group cell, the difference is significant(P<0.05), the apoptotic rate (18.6±2.93)% is also higher than that of sFasl group(10.12±2.31)%,siRNA group(10.09±1.31)%and control group(1.14±0.21)% , the difference is significant(P<0.01). Conclusions: The Survivin siRNA can enhance the sensitivity to soluble FasL in lung adenocarcinoma. In this study, we use PsilencerTM4.1- CMV neo plasmid to construct Survivin specific siRNA expression vector. After transfected it into lung adenocarcinoma cell A549 and selected for stably express clonal cell line by G418(geneticin), it successfully reduce the expression of Survivin gene and induce apoptosis in some A549 cell. The Survivin specific RNA interfering A549 cell treated with SFasL can more efficiently induce apoptosis than that of single Survivin specific RNA interfering A549,sFasL treating A549 cell and nomal A549 cell. This also demonstrated that knockdown of gene Survivin can enhance the sensitivity of lung adenocarcinoma cell A549 to apoptosis induction factors. Our works imply that Survivin RNA interference associated with sFasL may be a hopeful method to the clinical use of sFasL.