Short Hairpin Ribonucleic Acid Research Articles

HnRNP C1/C2 and Pur-beta proteins mediate induction of senescence by oligonucleotides homologous to the telomere overhang

BackgroundExperimental disruption of the telomere overhang induces a potent DNA damage response and is the target of newly emerging cancer therapeutics. Introduction of T-oligo, an eleven-base oligonucleotide homologous to the 3′-telomeric overhang, mimics telomere disruption and induces DNA damage responses through activation of p53, p73, p95/Nbs1, E2F1, pRb, and other DNA damage response proteins. ATM (ataxia telangiectasia mutated) was once thought to be the primary driver of T-oligo-induced DNA damage responses; however, recent experiments have highlighted other key proteins that may also play a significant role.MethodsTo identify proteins associated with T-oligo, MM-AN cells were treated with biotinylated T-oligo or complementary oligonucleotide, cell lysates were run on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and the protein bands observed after treatment of cells with T-oligo or complementary oligonucleotide were analyzed using mass spectrometry. To study the effect of T-oligo on expression of hnRNP C1/C2 (heterogeneous nuclear ribonucleoprotein C1 and C2) and purine-rich element binding proteins (Pur proteins), cells were treated with T-oligo, and immunoblotting experiments were performed. To determine their role in senescence, cells were treated with shRNA (short hairpin ribonucleic acid) against these proteins, and senescence was studied using the senescence associated beta-galactosidase assay.ResultsUsing mass spectrometry, RNA-binding hnRNP C1/C2 and DNA-binding Pur proteins were found to associate with T-oligo. hnRNP C1/C2 exhibited increased expression (3.6–12.0-fold) in non-small-cell lung cancer (NSCLC) and in melanoma cells (4.5–5.2-fold), and Pur proteins exhibited increased expression of 2.2-fold in NSCLC and 2.0-fold in melanoma cells after T-oligo treatment. Experimental knockdown of hnRNP C1/C2 and Pur-beta completely abrogated T-oligo induced senescence in both MU melanoma and H358 NSCLC cells. Additionally, knockdown of Pur-beta prevented T-oligo-induced phosphorylation of p53, hypophosphorylation of pRb, and upregulation of E2F1, p21, and p53.ConclusionThese novel findings highlight proteins essential to T-oligo’s anticancer effects that may be of interest in telomere biology and cancer therapeutics.

Short Hairpin Ribonucleic Acid Constructs Targeting Insulin-like Growth Factor Binding Protein-3 Ameliorates Diabetes Mellitus-related Erectile Dysfunction in Rats

To investigate the expression of insulin-like growth factor binding protein-3 (IGFBP-3) in penile cavernous of streptozotocin (STZ)-induced DM rats and whether downregulation of IGFBP-3 by intracavernosal injection of short hairpin ribonucleic acid (shRNA) targeting IGFBP-3 could improve the erectile function in DM rats. Diabetes was induced in rats by intraperitoneal injection of STZ, and the expression of IGFBP-3 in the penile tissue of adult normal and DM male rats was assayed using reverse transcriptase-polymerase chain reaction and Western blot. Next, shRNA-targeting IGFBP-3 and a scramble sequence were injected into the penile corpora cavernosa of DM rats. At 12 weeks after shRNA-IGFBP-3 administration, the intracavernous pressure in response to electrical stimulation of the cavernous nerves was evaluated. The expression of IGFBP-3 was assayed by Western blot. The concentration of cyclic guanosine monophosphate in the corpus cavernosum was assayed by enzyme-linked immunosorbent assay. At 12 week after intraperitoneal administration of STZ, IGFBP-3 expression had increased in the penis of the DM rat (P <.05) compared with that of the normal control rats. Among the DM rats, IGFBP-3 expression at the messenger RNA and protein level was significantly inhibited 12 weeks after intracavernous administration of IGFBP-3 shRNA (P <.01). At 12 weeks after shRNA-IGFBP-3 injection, intracavernosal pressure was significantly increased in response to cavernous nerve stimulation (P <.05), and an increase in the concentration of cyclic guanosine monophosphate in the corpus cavernous tissue (P <.01) was detected compared with the "randomer" shRNA treatment group. Gene transfer of shRNA-IGFBP-3 could improve erectile function in STZ-induced DM rats by an increase in the cyclic guanosine monophosphate concentration in cavernous tissue.

Tumor-specific RNA Interference Targeting Pokemon Suppresses Tumor Growth and Induces Apoptosis in Prostate Cancer

To explore the exact mechanism of Pokemon in prostate cancer. Pokemon is a member of the POK family of transcriptional repressors. Its main function is suppression of the p14ARF (alternate reading frame) tumor suppressor gene. Although Pokemon expression has been found to be increased in various types of lymphoma, the exact mechanism of the gene in prostate cancer is not clear. In the present study, prostate cancer cells were transfected with the specific short hairpin ribonucleic acid (RNA) expression vector targeting Pokemon. The expression of Pokemon messenger RNA and its protein was detected by semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The cell growth and cell apoptosis were also examined using the methyl thiazolyl tetrazolium assay and flow cytometry. The results demonstrated that specific RNA interference (RNAi) could decrease the expression levels of Pokemon gene messenger RNA and protein in prostate cancer cells. In addition, that specific RNAi significantly inhibited the cell proliferation and increased the apoptotic rate. In vivo experiments showed that specific RNAi inhibited the tumorigenicity of prostate cancer cells and significantly suppressed tumor growth. Therefore, an RNAi-targeted Pokemon gene strategy could be a potential approach to prostate cancer therapy.

Short Hairpin RNA Suppression of Thymidylate Synthase Produces DNA Mismatches and Results in Excellent Radiosensitization

To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. shRNA suppression of TS was compared with 5-fluoro-2'-deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively. TS shRNA produced profound (≥ 90%) and prolonged (≥ 8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA mismatches underlying radiosensitization. Importantly, shRNA suppression of TS avoids FP-mediated TS elevation and its negative prognostic role. These studies support the further exploration of TS suppression as a novel radiosensitizing strategy.

Nonintegrating Knockdown and Customized Scaffold Design Enhances Human Adipose-Derived Stem Cells in Skeletal Repair

An urgent need exists in clinical medicine for suitable alternatives to available techniques for bone tissue repair. Human adipose-derived stem cells (hASCs) represent a readily available, autogenous cell source with well-documented in vivo osteogenic potential. In this article, we manipulated Noggin expression levels in hASCs using lentiviral and nonintegrating minicircle short hairpin ribonucleic acid (shRNA) methodologies in vitro and in vivo to enhance hASC osteogenesis. Human ASCs with Noggin knockdown showed significantly increased bone morphogenetic protein (BMP) signaling and osteogenic differentiation both in vitro and in vivo, and when placed onto a BMP-releasing scaffold embedded with lentiviral Noggin shRNA particles, hASCs more rapidly healed mouse calvarial defects. This study therefore suggests that genetic targeting of hASCs combined with custom scaffold design can optimize hASCs for skeletal regenerative medicine.

Neuroligins/LRRTMs prevent activity- and Ca2+/calmodulin-dependent synapse elimination in cultured neurons

Neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs) are postsynaptic cell adhesion molecules that bind to presynaptic neurexins. In this paper, we show that short hairpin ribonucleic acid-mediated knockdowns (KDs) of LRRTM1, LRRTM2, and/or NL-3, alone or together as double or triple KDs (TKDs) in cultured hippocampal neurons, did not decrease synapse numbers. In neurons cultured from NL-1 knockout mice, however, TKD of LRRTMs and NL-3 induced an ∼40% loss of excitatory but not inhibitory synapses. Strikingly, synapse loss triggered by the LRRTM/NL deficiency was abrogated by chronic blockade of synaptic activity as well as by chronic inhibition of Ca(2+) influx or Ca(2+)/calmodulin (CaM) kinases. Furthermore, postsynaptic KD of CaM prevented synapse loss in a cell-autonomous manner, an effect that was reversed by CaM rescue. Our results suggest that two neurexin ligands, LRRTMs and NLs, act redundantly to maintain excitatory synapses and that synapse elimination caused by the absence of NLs and LRRTMs is promoted by synaptic activity and mediated by a postsynaptic Ca(2+)/CaM-dependent signaling pathway.

Stable knockdown of S100A4 suppresses cell migration and metastasis of osteosarcoma

S100A4, a 10-12 kDa calcium-binding protein, plays functional roles in tumor progression and metastasis. The present study aimed to investigate the function of S100A4 in osteosarcoma (OS) metastasis, using a loss-of-function approach. Our previous expression profiling analysis revealed that S100a4 was preferentially expressed in the highly metastatic mouse OS cell line, LM8. Introducing a short hairpin ribonucleic acid (shRNA) targeting S100a4 using a newly established vector containing insulators and transposons, we established stable LM8 subclones with almost 100% silencing of endogenous S100a4 protein. These transfectants showed a significant suppression of cell migration in vitro as well as a marked reduction in their ability to colonize the lung and form pulmonary metastases in vivo following intravenous inoculation, whereas there was no significant change in cell proliferation or cell attachment to fibronectin, laminin, and type I collagen. Expression and phosphorylation of ezrin, an emerging OS metastasis-associated factor, and expression of MMPs, remained the same in S100a4-shRNA clones. In 61 human OS, immunohistochemical analysis showed that lesional cells in 85.2% samples expressed S100A4 protein, and the immunoreactivity was primarily cytoplasmic, but it also showed occasional nuclear localization. Chondroblastic and osteoblastic OS subtypes expressed more S100A4 than fibroblastic subtypes. The causative role of S100A4 in OS lung metastasis shown in the murine xenograft model, together with the high proportion of primary human OS expressing S100A4, suggest that S100A4 protein represents an important potential target for future OS therapy.

G protein-coupled receptor kinase 5, overexpressed in the α-synuclein up-regulation model of Parkinson's disease, regulates bcl-2 expression

G protein-coupled receptor kinase 5 (GRK5) has been reported to accumulate in Lewy bodies (LBs), a histological hallmark of Parkinson's disease. Recent findings propose that GRK5 might function in Parkinson's disease via phosphorylation of α-synuclein, a major component of LBs. In this study, the changes of the expression levels of GRK5 and its possible effects in Parkinson's disease were evaluated in cell lines and transgenic mice model of α-synuclein overexpression. Both the expression levels of cytoplasmic and nuclear distributed GRK5 were induced an increase via α-synuclein overexpression in vivo and in vitro. The observations that the levels of α-synuclein phosphorylated at Ser-129 (pS129-α-synuclein) remain unchanged despite the downregulation of GRK5 by short hairpin ribonucleic acid (shRNA) transfection suggest that GRK5 is not the sole kinase involved in phosphorylating α-synuclein in Parkinson's disease. In addition, the findings that nuclear accumulation of GRK5 inhibits bcl-2 transcription and expression, at least in part by enhancing histone deacetylase (HDAC) activity, show an unexpected role for nuclear GRK5 in the regulation of an apoptosis-related gene. The present study suggests that GRK5 may be extensively involved in the mechanism of Parkinson's disease.

Dual silencing of epidermal growth factor and insulin-like growth factor 1 receptors significantly limits growth of nasopharyngeal carcinoma in nude mice

We examined the effects of dual silencing of epidermal growth factor and insulin-like growth factor 1 receptors on the growth of nasopharyngeal carcinoma in nude mice; we also assessed potential side effects in these animals. Short hairpin ribonucleic acid expression vectors targeting epidermal growth factor and insulin-like growth factor 1 receptors were constructed. Short hairpin ribonucleic acid plasmids targeting one or both receptors were transfected into human nasopharyngeal carcinoma cells in nude mice. We then assessed epidermal growth factor receptor and insulin-like growth factor 1 receptor expression and also cellular apoptosis. Peripheral blood was collected and subjected to haematological and biochemical analysis. The findings demonstrated that transfection with dual plasmids (targeting both epidermal growth factor receptor and insulin-like growth factor 1 receptor) resulted in tumour cell growth inhibition of 84.78 per cent, and a significant increase in the number of necrotic and apoptotic cells, compared with single plasmid treatment. The short hairpin ribonucleic acid had no significant side effects on the heart, liver, kidney, spleen or blood system in this experimental model. These results indicate that, in nude mice, dual silencing of both epidermal growth factor and insulin-like growth factor 1 receptors results in more apoptosis and greater nasopharyngeal cancer cell growth inhibition, compared with silencing of either epidermal growth factor receptor alone or insulin-like growth factor 1 receptor alone. This occurred without significant side effects in the experimental animals.

Noxa/Mcl-1 Balance Regulates Susceptibility of Cells to Camptothecin-Induced Apoptosis

Although camptothecin (CPT) has been reported to induce apoptosis in various cancer cells, the molecular details of this regulation remain largely unknown. In this study, we demonstrate that 131-113-only protein Noxa is upregulated during CPT-induced apoptosis, which is independent of p53. In addition, we show that phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is responsible for Noxa's induction. Luciferase assay, cAMP response element binding protein (CREB) knockdown experiments further demonstrate that CREB is involved in the transcriptional upregulation of Noxa. Moreover, blocking Noxa expression using specific small interfering ribonucleic acid (siRNA) significantly reduces the apoptosis in response to CPT, indicating that Noxa is an essential mediator for CPT-induced apoptosis. Interestingly, antiapoptotic Mcl-1 was also upregulated through PI3K/Akt signaling pathway upon CPT treatment. Using immunoprecipitation assay, Noxa was found to interact with Mcl-1 in the presence or absence of CPT. Knockdown of Mcl-1 expression by short hairpin ribonucleic acid (shRNA) was shown to potentiate CPT-induced apoptosis. Consistently, ectopic overexpression of Mcl-1 rescued cells from apoptosis induced by CPT. Cells coexpressing Noxa, Mcl-1 at different ratio correlates well with the extent of apoptosis, suggesting that the balance between Noxa, Mcl-1 may determine the susceptibility of HeLa cells to CPT-induced apoptosis.

Short hairpin ribonucleic acid targeting the telomerase catalytic unit of messenger ribonucleic acid significantly limits the growth of laryngeal squamous cell carcinoma in nude mice

Telomerase is an attractive molecular target because it is active in most malignant cells but undetectable in most normal somatic cells. Small, interfering ribonucleic acid segments have been shown to be effective tools for inhibiting the expression of a given gene within human cells. In the present study, we examined the effects of short hairpin ribonucleic acid expression vectors on the growth of laryngeal squamous cell carcinoma in nude mice, and we assessed potential side effects in these animals. Short hairpin ribonucleic acid expression vectors targeting the messenger ribonucleic acid of the telomerase catalytic unit were constructed and transfected into Hep-2 human laryngeal squamous cells carcinoma in nude mice. Apoptosis and telomerase catalytic unit expression within tumour cells were evaluated after treating with short hairpin ribonucleic acid. Peripheral blood was collected for haematological and biochemical analysis. The findings demonstrated that short hairpin ribonucleic acid plasmids could inhibit tumour cell growth by 76.5 per cent, and that many tumour cells underwent necrotic or apoptotic cell death. There were no significant side effects of short hairpin ribonucleic acid on the heart, liver, kidney, spleen or blood system in this experimental model. These results indicated that the short hairpin ribonucleic acid expression vector targeted at the telomerase catalytic unit of messenger ribonucleic acid significantly inhibited the growth of laryngeal carcinoma in nude mice, with no significant side effects on the experimental animals.

RIBONUCLEIC ACID INTERFERENCE FOR NEUROLOGICAL DISORDERS

Ribonucleic acid (RNA) interference (RNAi) is a conserved evolutionary defense mechanism that is gaining utility for therapeutic application by modulating gene expression or silencing disease-causing genes. This strategy has recently achieved success in mammalian cells via synthetic small interfering RNA or short hairpin RNA expressed in vectors for gene delivery. The vector-based RNAi strategy has particular potential because of the possibility of targeted gene delivery, long-term gene expression, and the potential means of penetrating the blood-brain barrier. RNAi-based approaches have been proposed for a variety of neurological disorders, including dominant genetic diseases, neurodegenerative diseases, malignant brain tumors, pain, and viral-induced encephalopathies. This review summarizes the current approaches of the RNAi strategy for neurological disorders, focusing on potential targets for therapeutic intervention.

Effect of RNA interference targeting human telomerase reverse transcriptase on telomerase and its related protein expression in nasopharyngeal carcinoma cells

Analysis of the correlation between telomerase and the expression of its related proteins may provide insight into the molecular mechanism of nasopharyngeal carcinogenesis. We investigated the effect of short hair pin ribonucleic acid (RNA) specific for human telomerase reverse transcriptase messenger RNA on the expression of the proteins c-myc (the transcription factor c-myc is a shortlived nuclear phospho-protein involved in cell proliferation and differentiation, belongs to the myc family), proliferating cell nuclear antigen and Caspase-3 in nasopharyngeal carcinoma cells. Short hairpin RNA expression vectors targeting the messenger RNA of human telomerase reverse transcriptase were constructed. Cells were treated with the short hairpin RNA expression vectors targeting human telomerase reverse transcriptase or vectors that included mismatched short hairpin RNA, and telomerase activity was measured by telomeric repeat amplification enzyme-linked immunosorbent assay. Cell viability was examined using the 3-(4,5-dimethyl thizol-2-yl) 2,5-diphenyl tetrazolium bromide assay. The expression of the three proteins (c-myc, proliferating cell nuclear antigen and Caspase-3) was determined by Western blotting. Short hairpin RNA specific for human telomerase reverse transcriptase messenger RNA significantly inhibited telomerase activity. In addition, the expression of and proliferating cell nuclear antigen were both inhibited, while the expression of Caspase-3 was up-regulated. Our results suggest that short hairpin RNA directed against human telomerase reverse transcriptase inhibits cell viability by regulating telomerase activity and its related proteins expression in nasopharyngeal carcinoma cells. Therefore, RNA interference technology may be a promising strategy for the treatment of nasopharyngeal cancer.

Lentiviral Manipulation of Gene Expression in Human Adult and Embryonic Stem Cells

Human stem cells could revolutionize the field of medicine by providing a diverse range of cell types for tissue replacement therapies and drug discovery. To achieve this goal, genetic tools need to be optimized and developed for controlling and manipulating stem cells ex vivo. Here we describe a lentiviral delivery system capable of high infection rates in human mesenchymal and embryonic stem cells. The lentiviral backbone was modified to express mono- and bi-cistronic transgenes and was also used to deliver short hairpin ribonucleic acid for specific silencing of gene expression in human stem cells. We show that lentiviral transduction can be used to alter gene expression without altering the genes' ability to differentiate in vitro. These vectors will enable rapid analysis of gene function in stem cells and permit the generation of knock-in / knock-out models of human disease in the rapidly developing field of gene therapy.