Small Interfering RNA Expression Vector Research Articles

Construction of RNA interference (RNAi) lentiviral expression vector of DEK gene and its effect on the biological behavior of liver cancer cells

Objective: To construct RNA interference (RNAi) lentiviral expression vector of DEK gene, and to explore its effect on the biological behavior of liver cancer cells. Methods: Double-stranded oligo DNAs were annealed and synthesized according to the interference sequence of DEK gene by RNAi technology. Small interfering RNA expression vector pLKO.1 was cloned after enzymatic digestion. The recombinant lentiviral pLKO.1-sh hDEK was constructed, and then the virus supernatant was collected, packed and infected by 293T cells. Real-time reverse transcription PCR (RT-PCR) and Western blot were used to detect DEK expression in human liver cancer cells Bel-7402, Hu-7, SmMC-7721 and HepG2, and DEK knockdown efficiency in each group of lentivirus-infected cells. Cell proliferation ability, cloning ability, apoptosis and migration ability were detected by cell counting kit-8 (CCK8), flow cytometry and scratch test, respectively. The t-test was used to compare the mean between the two groups, and one-way ANOVA was used to compare the multiple groups. Results: Enzymatic digestion and DNA sequencing results confirmed that the recombinant lentiviral vectors pLKO.1-sh hDEK1 and pLKO.1-sh hDEK3 were successfully constructed. RT-PCR and Western blot results showed that the expression of DEK in human liver cancer cells BEL-7402 and Huh7 cells was higher, and pLKO.1-sh hDEK3 was more effective in inhibiting the DEK gene expression (P < 0.05). Therefore, pLKO.1-sh hDEK3 was selected to infect BEL-7402 and Huh7 cells for subsequent functional experiments. CCK8 cell proliferation test result showed that the cell proliferation ability of BEL-7402 and Huh7 cells infected with recombinant lentivirus was weakened when compared with blank control and negative control group (P < 0.05). Apoptosis results showed that the apoptosis rate of knockdown group was higher than that of blank and the negative control group (P < 0.05). Cell scratch test result showed that the wound healing rate of knockdown group was lower than that of blank control and negative control group (P < 0.05), and the difference was statistically significant; however, there was no statistically significant difference between blank control and negative control group. Conclusion: Targeting DEK expression in silent liver cancer cells can inhibit the cell proliferation, migration ability, and induce apoptosis, which lays the foundation for further study of the role of DEK gene in liver cancer.

Expression of histone deacetylase 1 gene in human lung cancer cell line A549 and the effect on the radiation sensitivity of nude mouse transplantation tumor

Objective To investigate the expression of histone deacetylase 1 (HDAC1) gene in human lung cancer cell line A549 and the effect on the radiation sensitivity of nude mouse transplantation tumor. Methods HDAC1 small interfering RNA (siRNA) and recombinant expression vector pcDNA3.1-HDAC1 were transfected with A549 lung cancer cells, respectively. Stable transfected cell lines were screened by G418 selection. reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression levels of HDAC1 mRNA and HDAC1 protein in the cell; cell proliferation was measured by cell counting kit-8 (CCK-8). Clone formation experiment was used to observe the effect of up-regulation and down-regulation of HDAC1 expression on the radiation sensitivity of A549 cells, cell apoptosis was detected by transferase-mediated dUTP nick-end labelling (TUNEL) method. Nude mice experiment of transplantable tumor was applied for in vivo detection of the influence of up-regulated and down-regulation of HDAC1 expression combined with X-ray irradiation in the inhibition of lung cancer growth. Results The expression of HDAC1 gene and protein were significantly lower to 1.86±0.09 and 2.41±0.10 (P=0.015, 0.006) in HDAC1 cell line A549, and the HDAC1 up-regulation group were significantly higher to 3.65±0.14 and 5.41±0.12 (P=0.012, 0.008); The radiation sensitivity of lung cancer A549 cells was increased in the down-regulation group of HDAC1 (P=0.025), and the radiation sensitivity of lung cancer A549 cells was decreased (P=0.009)in the HDAC1 up-regulation group. Down-regulation of HDAC1 inhibited the proliferation of A549 lung cancer, increased the rate of apoptosis to (42.17±3.19)% (P=0.008), proliferation of A549 cells was significantly enhanced in HDAC1 up-regulated group, associated with reduced percentage of apoptotic cells (9.11±1.46)% (P=0.006); Average volume in the nude mice of HDAC1 down-regulated group (547.37±89.68) mm3 were significantly smaller than the control group (1 238.44±212.36) mm3 (P=0.008); furthermore, mean volume of tumor was greater in the HDAC1 up-regulated group than in the control group (1 681.27±195.37) mm3 (P=0.006). After 20 Gy γ irradiation, the average volume of HDAC1 down-regulated group were significantly decreased (214.15±54.26) mm3 (P=0.011). Compared with that before irradiation, the average volume of the tumor in the nude mice was not significantly changed after irradiation (P=0.264). Conclusion Down-regulated expression of HDAC1 can inhibit the growth of human lung cancer A549 cells in nude mice, enhance the radiation sensitivity of tumor cells, whereas up-regulated expression of HDAC1 can produce tumor radiation resistance. Key words: Lung cancer; A549 cell; Histone deacetylase 1; Transplantable tumor; Nude mice

Lengthening of high-yield production levels of monoclonal antibody-producing Chinese hamster ovary cells by downregulation of breast cancer 1

The establishment process of high-producing Chinese hamster ovary (CHO) cells for therapeutic protein production is usually laborious and time consuming because of the low probability of obtaining stable, high-producing clones over a long term. Thus, development of an efficient approach is required to establish stable, high-producing cells. This study presents a novel method that can efficiently establish sustainably high-producing cell lines by acceleration of transgene amplification and suppression of transgene silencing. The effects of breast cancer 1 (BRCA1) downregulation on gene amplification efficiency and long-term productivity were investigated in CHO cells. Small interfering RNA expression vectors against BRCA1 were transfected into the CHO DG44-derived antibody-producing cell clone. Individual cell clones were obtained after induction of gene amplification in the presence of 400nM methotrexate, which were cultured until passage 20. BRCA1-downregulated cell clones CHO B1Sa and B1Sb displayed 2.2- and 1.6-fold higher specific production rates than the S-Mock clone. Fluorescence in situ hybridization showed that transgene amplification occurred at a high frequency in B1Sa and B1Sb clones. Moreover, B1Sa and B1Sb clones at 20 passages had approximately 3.5- and 5.3-fold higher productivity than the S-Mock clone. Histone modification analysis revealed a decrease in an active mark for transcription, trimethylation of histone H3 at lysine 4 (H3K4), in the transgene locus of the S-Mock clone. However, H3K4 trimethylation levels were not decreased in B1Sa and B1Sb clones during long term culture. Our results suggest that high-producing cells, which maintain their productivity long-term, were efficiently established by BRCA1 downregulation.

GW26-e2452 A Study in Construction of Short Hairpin Small Interfering RNA Expression Vector Target Lectin Like Oxidized Low Density Lipoprotein Receptor-1 Gene and Its Effect on foam cells

To construct the short hairpin small interfering RNA(shRNA) eukaryotic expression vector specific to mouse lectin like oxidized low density lipoprotein receptor 1(LOX-1) Gene and to observe its silencing effect on LOX-1 in RAW264.7 cells. (1) The pLOX-1-shRNA expression vector was constructed by

Expression of vascular endothelial growth factor C in esophageal carcinoma and its clinical significance

Objective To investigate the expression of vascular endothelial growth factor C (VEGF-C) in human esophageal squamous cell carcinoma (ESCC) and its clinical significance.Methods The expression of VEGF-C in ESCC and its correlation with lymph node metastasis were detected by using immunohistochemistry (IHC) and in situ hybridization (ISH).Three small interfering RNA (siRNA) expression vectors targeting VEGF-C were constructed and transformed into EC9706 cells.The inhibitory effect of siRNA on the expression of VEGF-C protein and mRNA in tranfected cells was analyzed by using Western blotting and reverse transcription-polymerase chain reaction (RT-PCR).Stably transfected cells were transplanted in nude mice later.The expression of VEGF-C in transplant tumor was detected by using IHC and ISH.Results VEGF-C was up-expressed in ESCC.Its expression was correlated significantly with lymph node metastasis.The positive expression rate of VEGF-C in lymph node metastasis group and non-lymph node metastasis group was 90.0％ and 62.1％ respectively (P ＜0.01 ).VEGF-C-targeted siRNA could decrease the expression of VEGF-C mRNA and protein in EC9706 cells effectively.There was significant difference between transfected group (0.32 ± 0.02) and un-transfected group (0.91 ± 0.09,P ＜0.01 ).Correspondingly,the expression of VEGF-C was 9.26 ±0.95,8.94 ±0.81,9.19 ±0.92 and 32.47 ±3.25 respectively.There was significant difference among them (P ＜0.01 ).Conclusion Targeting VEGF-C siRNA can inhibit VEGF-C expression of EC9706 in vivo and in vitro effectively. Key words: Esophageal neoplasm; Vascular endothelial growth factor C

Transfection of Hairpin Small Interfering RNA Expression Vector Targeting Rat Nuclear Factor (NF) (κB) Inhibits Rat Cell Proliferation Induced by NF-κB Signal Pathway Activation

Objective The aim of this work was to construct one small interfering RNA (siRNA) eukaryotic expression vector targeting rat nuclear factor (NF)κB p65 and identify its inhibition effect on cell proliferation according to its down-regulation of NF-κB pathway. Methods The p65siRNA expression vector “pGenesil-1.2-p65siRNA” and negative control plasmid “HK” were transfected into the cultured rat cells. After transfection, cells were divided into 4 treatment groups: 1) control cells cultured in complete. Dulbecco modified Eagle medium; 2) lipopolysaccharide (LPS) (1 μg/mL); (3) LPS (1 μg/mL) + HK-transfected; 4) LPS (1 μg/mL) + p65siRNA (pGenesil-1.2-p65siRNA). Thereafter, the protein levels of NF-κB p65 in the cells were detected by Western blotting at 72 hours after LPS stimulation. Furthermore, to observe cell proliferation, the proliferative rate of the cell growth was evaluated by the methylthiazolyl tetrazolium assay (at 24, 48, and 72 hours). The cell cycle distribution at 72 hours was detected by flow cytometry. Results p65siRNA effectively down-regulated the protein level of p65 ( P < .05). Meanwhile, the proliferation of cells transfected with p65siRNA expression vector was significantly inhibited ( P < .05), the ratio of cells at G 0/G 1 stage markedly increased, and the proportion of cells at S stage was significantly decreased among transfected compared with control cells ( P < .05). Conclusions p65siRNA effectively suppressed NF-κB, expression, inhibiting rat cell proliferation induced by NF-κB signal pathway activation.

Small noncoding RNAs encoded within the bovine herpesvirus 1 latency-related gene can reduce steady-state levels of infected cell protein 0 (bICP0).

Following acute infection in mucosal epithelium, bovine herpes virus 1 (BHV-1) establishes lifelong latency in sensory neurons within trigeminal ganglia. The latency-related RNA (LR-RNA) is abundantly expressed in sensory neurons of latently infected calves. Expression of LR proteins is necessary for the latency reactivation cycle because a mutant virus that does not express LR proteins is unable to reactivate from latency after dexamethasone treatment. LR-RNA sequences also inhibit bICP0 expression, productive infection, and cell growth. However, it is unclear how LR-RNA mediates these functions. In this study, we identified a 463-bp region within the LR gene (the XbaI-PstI [XP] fragment) that inhibited bICP0 protein and RNA expression in transiently transfected mouse neuroblastoma cells. Small noncoding RNAs (sncRNAs) encoded within the XP fragment (20 to 90 nucleotides in length) were detected in transiently transfected mouse neuroblastoma cells. Two families of sncRNAs were cloned from this region, and each family was predicted to contain a mature microRNA (miRNA). Both miRNAs were predicted to base pair with bICP0 mRNA sequences, suggesting that they reduce bICP0 levels. To test this prediction, sequences encompassing the respective sncRNAs and mature miRNAs were synthesized and cloned into a small interfering RNA expression vector. Both sncRNA families and their respective miRNAs inhibited bICP0 protein expression in mouse neuroblastoma cells and productive infection in bovine cells. In trigeminal ganglia of latently infected calves, an sncRNA that migrated between nucleotides 20 and 25 hybridized to the XP fragment. During dexamethasone-induced reactivation from latency, XP-specific sncRNA levels were reduced, suggesting that these sncRNAs support the establishment and maintenance of lifelong latency in cattle.

The role of Crk/Dock180/Rac1 pathway in the malignant behavior of human ovarian cancer cell SKOV3

Small GTPases, particularly the Rho family, are key regulators of cell motility and migration. Dock180 was well known for the main target of signal adaptor protein Crk and acted as a guanine-nucleotide exchange factor for small GTPase Rac1. In the present study, Dock180 was found to combine primarily with CrkI other than CrkII, and its association with Elmo1 was also demonstrated in ovarian cancer cell SKOV3. To evaluate the role of Dock180 in human ovarian cancer cell, we performed RNAi-mediated knockdown of Dock180 in SKOV3 cells using small interfering RNA expression vector. In Dock180 knockdown cells, we found that Elmo1 expression and Rac1 activity were decreased simultaneously. By contrast, the expressions of both another Crk-combining molecule C3G and Rap1 activity were observed to increase obviously. Accordingly, all Dock180 knockdown cells present with evident change in cell morphology, reduced cell proliferation, and attenuated cell migration. Taken together, these results suggest that signal transfer of Crk/Dock180/Rac1 is implicated in actin cytoskeleton reorganization and thus in the cell proliferation, motility, invasion, and of human ovarian cancer cell line SKOV3.

Vector‐based Ape1 small interfering RNA enhances the sensitivity of human osteosarcoma cells to endostatin in vivo

Osteosarcoma is a highly vascular and extremely destructive malignancy, and the survival of patients with osteosarcoma has not improved significantly in recent years. Antiangiogenic therapy currently holds great potential in conjunction with conventional treatment modalities for osteosarcoma. However, there are examples of gradual loss of response, and perhaps acquired resistance to antiangiogenic drugs. The acquired resistance of antiangiogenesis may be associated with a lot of hypoxia-response genes. The human apurinic/apyrimidinic endonuclease (Ape1) protein, a bifunctional redox factor and apurinic/apyrimidinic (AP) endonuclease, plays a crucial role in protecting against cell death due to hypoxia. We therefore hypothesized that Ape1 may contribute to the resistance of antiangiogenic therapy. To investigate the effect of Ape1 on the sensitivity of human osteosarcoma cells to endostatin, we constructed an Ape1 small interfering RNA expression vector, pSilenceApe1. Transfection of human osteosarcoma 9901 and HOS cells with pSilenceApe1 resulted in a dose-dependent loss of Ape1 protein. pSilenceApe1 also significantly suppressed the expression of vascular endothelial growth factor (VEGF) protein in the 9901 cells. Combined treatment with pSilenceApe1 and recombinant human endostatin (rhES) showed potent antiangiogenic effects in the transwell chamber invasion assay. Then, 20 nude mice bearing 9901 xenografts were divided into four groups: the phosphate-buffered saline treatment control group; the rhES treatment group (1.5 mg/kg, daily); the pSilenceApe1 treatment group (20 microg, once every 3 days); and the combination of rhES and pSilenceApe1 treatment group. pSilenceApe1 significantly suppressed the expression of Ape1 and VEGF protein in the 9901 xenografts. The tumor-inhibition rate of the pSilenceApe1, rhES, and combination of rhES and pSilenceApe1 treatment groups was 38.23, 35.29, and 62.18%, respectively. Furthermore, a significant decrease in microvessel density with an increase in apoptosis was observed following combined treatment with pSilenceApe1 and rhES, compared with control and either agent alone in 9901 xenografts. These results indicate that Ape1 small interfering RNA could enhance the sensitivity of osteosarcoma cells to endostatin.

Adrenomedullin Inhibits Adipogenesis Under Transcriptional Control of Insulin

We generated preadipocyte cell lines impaired in adrenomedullin production through integration of an adrenomedullin small interfering RNA expression vector. The reduction of adrenomedullin synthesis strongly accelerated adipose differentiation. These results were bolstered when overexpression of active adrenomedullin peptide led to delayed differentiation. Therefore, we propose that adrenomedullin is an antiadipogenic factor. Moreover, we checked whether insulin, a proadipogenic factor, regulates expression of adrenomedullin. We observed that insulin had an inhibitory effect on adrenomedullin expression in isolated human adipocyte cells. This response was dose dependent and was reversed by resistin, a new anti-insulin agent. We quantified circulating adrenomedullin in healthy obese patients and observed a threefold increase of adrenomedullin compared with lean patients. Furthermore, adrenomedullin plasma levels are negatively correlated to plasma insulin levels in these obese patients. The insulin inhibitory response was also observed in vivo in Sprague-Dawley rats but not in the insulin-resistant Zucker rat, suggesting that adrenomedullin expression is upregulated in insulin-resistant adipose cells. Using adrenomedullin promoter-luciferase reporter gene constructs, we have shown that the adrenomedullin response to insulin is mediated by insulin-responsive elements. These findings provide new insight into fat mass development and the relationship between obesity and elevated circulating adrenomedullin levels in diabetic patients.

The Role of Isomerized Protein Repair Enzyme, PIMT, in Cellular Functions

Proteins are subject to various types of spontaneous modifications that can disrupt their structures with sometimes adverse affects on biological activity. The formation of L-isoaspartyl (or D-aspartyl) residues, through either the deamidation of asparagine or dehydration of aspartate, is one of the most frequent types of deterioration occurring under physiological conditions. Protein L-isoaspartate/D-aspartate o-methyltransferase (PIMT) is a conserved and ubiquitous enzyme that participates in the repair of various isomerized proteins. PIMT catalyzes the transfer of the methyl group of S-adenosyl-L-methionine onto the alpha-carboxyl group of an L-isoaspartyl (or the beta-carboxyl group of an D-aspartyl) residue, which initiates the conversion of this residue to an L-aspartyl residue. PIMT-deficient mice have been shown to die at a mean age of 42 days from progressive epileptic seizures with grand mal and myoclonus. Although PIMT-deficiency clearly leads to the accumulation of isomerized proteins, it is currently unclear how this causes progressive epilepsy in PIMT-deficient mice. As a first step towards understanding this, we developed a new assay to measure PIMT activity in cell lysates. Additionally, we isolated PIMT knockdown cells from HEK293 cells that were stably transfected with a PIMT small interfering RNA expression vector. PIMT activities were significantly decreased in the PIMT knockdown cells, and analysis of the transfectants revealed that MEK and ERK were hyperactivated after cell stimulation with epidermal growth factor (EGF). These results indicate that the ability to repair L-isoaspartyl-(or D-aspartyl-) containing proteins is important for the maintenance of normal MEK-ERK signaling.

Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector

CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs.

Small interfering RNA expression vector targeting hypoxia-inducible factor 1 alpha inhibits tumor growth in hepatobiliary and pancreatic cancers

Hepatobiliary and pancreatic carcinomas are hypovascular tumors that can proliferate under hypoxic conditions. Recent reports have demonstrated that hypoxia-inducible factor 1 alpha (HIF1alpha) plays an important role in the survival of these cancers. Given these findings, the inhibition of the HIF1alpha pathway might prove to be a powerful tool in the treatment of these cancers. To inhibit HIF1alpha expression, we used small interference RNA (siRNA) expression vectors in this study. The transient transfection of siRNA expression vectors significantly reduced both HIF1alpha mRNA levels (13% of control) and protein levels (41% of control) and significantly inhibited the growth of cancer cell lines (P<0.05). VEGF, Glut1, and aldorase A expressions were also significantly reduced by transfection with these vectors (P<0.05), and we found that these vectors induced apoptosis but not cell cycle arrest. In a subcutaneous tumor model using nude mice, transfected MIA PaCa-2 cells, stably expressing siRNAs, barely formed tumors compared to control (P<0.05). This study thus demonstrates the usefulness of siRNA expression vector in targeting HIF1alpha and points to a potential clinical role in the treatment of pancreatic and hepatobiliary carcinomas.

Analysis of Double-stranded RNA-induced Apoptosis Pathways UsingInterferon-response Noninducible Small Interfering RNA Expression VectorLibrary

We have developed an original vector library that allowed us to exploit the phenomenon of RNA interference but also allowed us to avoid the confounding effects of the interferon response. In the present work, we used our library of small interfering RNA expression vectors to examine the genes involved in apoptosis that was induced by double-stranded RNA. To our surprise, screening of our library revealed two novel double-stranded RNA-induced apoptotic pathways, a JNK/SAPK-mediated mitochondrial pathway and an ERK2-related pathway, both of which appeared to be independent of the serine-threonine protein kinase-dependent caspase pathway. We also found that MST2 and protein kinase Calpha both activated the pro-apoptotic signal mediated by ERK2. The results of our screening analysis suggested the utility of large scale screenings with libraries of small interfering RNA expression vectors.

Construction of glucosylceramide synthase-specific siRNA expression vector and its efficiency in reversal of drug resistance in breast carcinoma cells

To construct a glucosylceramide synthase (GCS)-specific small interfering RNA (siRNA) expression vector and to investigate the inhibitory effect of this siRNA on GCS expression and drug resistance in breast carcinoma cells. Two GCS gene-specific siRNAs were designed and cloned into the expression vector pSUPER to generate the plasmids pSUPER-GCS1 and pSUPER-GCS2. Human adriamycin (ADM)-resistant breast carcinoma cells of the line MCF-7/ADR and human adriamycin-sensitive breast carcinoma cells of the line MCF-7 were cultured and transfected with pSUPER-GCS1, pSUPER-GCS2, and blank vector pSUPER as controls. The expression of GCS mRNA was assayed by RT-PCR and the expression of GCS protein was observed by flow cytometry. The 50% inhibition concentration of ADM on MCF-7/ADR cells was evaluated by MTT method. Flow cytometry was performed to determine the ratio of apoptosis. Double enzyme digestion analysis and DNA sequencing confirmed that pSUPER-GCS1 and pSUPER-GCS2 were successfully constructed. The GCS protein positive rate of the MCF-7/ADR cells 48 hours after transfection with pSUPER-GCS1 and pSUPER-GCS2 were 8.3% +/- 1.0% and 9.2% +/- 0.8% respectively, significantly lower than that before transfection (68.3% +/- 0.6%), with a inhibition rate of 89.4% and 88.5% respectively (both P < 0.01). Forty-eight hours after transfection with pSUPER-GCS1 and pSUPER-GCS2, the relative reversal rates of sensitivity to ADM of the MCF-7/ADR cells were 93.7% and 91.6%. Flow cytometry showed that the apoptotic rate of the MCF-7/ADR cells was 0.80 +/- 0.06 before transfection, 15.38 +/- 1.16 after transfection with pSUPER-GCS1 and 13.92 +/- 1.73 after transfection with pSUPER-GCS2 (both P < 0.05), and was 0.87 +/- 0.12 in the cells transfected with blank vector (P > 0.05). A GCS-specific small interfering RNA expression vector has been constructed successfully that suppresses the GCS expression and reverses the multidrug resistance in breast carcinoma cells by increasing the ratio of apoptosis in drug-resistant cells.

Identification of a Network Involved in Thapsigargin-induced Apoptosis Using a Library of Small Interfering RNA Expression Vectors*[boxs

We describe here the construction of a library of small interfering RNA expression vectors targeted to a few hundred apoptosis-related genes and the application of this library to an investigation of thapsigargin (TG)-induced apoptosis. Thapsigargin triggers endoplasmic reticulum stress, with subsequent apoptosis, but the molecular mechanisms underlying this process are incompletely understood. Using our library, we identified three anti-apoptotic genes, namely, NOXA, E2F1, and MAPK1, in addition to already characterized genes in the apoptotic pathway. In contrast to proposals by others, our data revealed (i) that TG-induced apoptosis is associated with Apaf1 in a caspase-3- and caspase-9-independent manner; (ii) that the E2F1-PUMA pathway might be involved; and (iii) that the ERK pathway, via MAP3K8 (mitogen-activated protein kinase kinase 8), is required for the induction by TG of apoptosis. Our study demonstrates clearly that unexpected and novel genes can be identified effectively by our method, and it provides evidence for the efficacy and utility of the comprehensive analysis of signaling networks and pathways using a library of small interfering RNA expression vectors.