Repeated microendoscopic discectomy for recurrent lumbar disk herniation

OBJECTIVES: To explore the microendoscopic discectomy technique and inclusion criteria for the treatment of recurrent lumbar disc herniation and to supply feasible criteria and technical notes to avoid complications and to increase the therapeutic effect. METHODS: A consecutive series of 25 patients who underwent posterior microendoscopic discectomy for recurrent lumbar disc herniation were included. The inclusion criteria were as follows: no severe pain in the lumbar region, no lumbar instability observed by flexion-extension radiography and no intervertebral discitis or endplate damage observed by magnetic resonance imaging. All patients were diagnosed by clinical manifestations and imaging examinations. RESULTS: Follow-up visits were carried out in all cases. Complications, such as nerve injuries, were not observed. The follow-up outcomes were graded using the MacNab criteria. A grade of excellent was given to 12 patients, good to 12 patients and fair to 1 patient. A grade of excellent or good occurred in 96% of cases. One patient relapsed 3 months after surgery and then underwent lumbar interbody fusion and inner fixation. The numerical rating scale of preoperative leg pain was 7.4± 1.5, whereas it decreased to 2.1±0.8 at 7 days after surgery. The preoperative Oswestry disability index of lumbar function was 57.5±10.0, whereas it was 26.0±8.5 at 7 days after surgery. CONCLUSION: In these cases, microendoscopic discectomy was able to achieve satisfactory clinical results. Furthermore, it has advantages over other methods because of its smaller incision, reduced bleeding and more efficient recovery. .

Lentivirus mediated silencing of Ubiquitin Specific Peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro

BACKGROUND: Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells. RESULTS: In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G2/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells. CONCLUSIONS: All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.

Targeting PPM1D by lentivirus-mediated RNA interference inhibits the tumorigenicity of bladder cancer cells

Protein phosphatase magnesium/manganese-dependent 1D (PPM1D) is a p53-induced phosphatase that functions as a negative regulator of stress response pathways and has oncogenic properties. However, the functional role of PPM1D in bladder cancer (BC) remains largely unknown. In the present study, lentivirus vectors carrying small hairpin RNA (shRNA) targeting PPM1D were used to explore the effects of PPM1D knockdown on BC cell proliferation and tumorigenesis. shRNA-mediated knockdown of PPM1D significantly inhibited cell growth and colony forming ability in the BC cell lines 5637 and T24. Flow cytometric analysis showed that PPM1D silencing increased the proportion of cells in the G0/G1 phase. Downregulation of PPM1D also inhibited 5637 cell tumorigenicity in nude mice. The results of the present study suggest that PPM1D plays a potentially important role in BC tumorigenicity, and lentivirus-mediated delivery of shRNA against PPM1D might be a promising therapeutic strategy for the treatment of BC.

Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

Silencing of CXCR4 Inhibits Tumor Cell Proliferation and Neural Invasion in Human Hilar Cholangiocarcinoma

BACKGROUND/AIMS: To evaluate the expression of CXC motif chemokine receptor 4 (CXCR4) in the tissues of patients with hilar cholangiocarcinoma (hilar-CCA) and to investigate the cell proliferation and frequency of neural invasion (NI) influenced by RNAi-mediated CXCR4 silencing. METHODS: An immunohistochemical technique was used to detect the expression of CXCR4 in 41 clinical tissues, including hilar-CCA, cholangitis, and normal bile duct tissues. The effects of small interference RNA (siRNA)-mediated CXCR4 silencing were detected in the hilar-CCA cell line QBC939. Cell proliferation was determined by MTT. Expression of CXCR4 was monitored by quantitative real time polymerase chain reaction and Western blot analysis. The NI ability of hilar-CCA cells was evaluated using a perineural cell and hilar-CCA cell coculture migration assay. RESULTS: The expression of CXCR4 was significantly induced in clinical hilar-CCA tissue. There was a positive correlation between the expression of CXCR4 and lymph node metastasis/NI in hilar-CCA patients (p<0.05). Silencing of CXCR4 in tumor cell lines by siRNA led to significantly decreased NI (p<0.05) and slightly decreased cell proliferation. CONCLUSIONS: CXCR4 is likely correlated with clinical recurrence of hilar-CCA. CXCR4 is involved in the invasion and proliferation of human hilar-CCA cell line QBC939, indicating that CXCR4 could be a promising therapeutic target for hilar-CCA.

Circadian clock of Aedes aegypti: effects of blood-feeding, insemination and RNA interference

Mosquitoes are the culprits of some of the most important vector borne diseases. A species’ potential as a vector is directly dependent on their pattern of behaviour, which is known to change according to the female’s physiological status such as whether the female is virgin/mated and unfed/blood-fed. However, the molecular mechanism triggered by and/or responsible for such modulations in behaviour is poorly understood. Clock genes are known to be responsible for the control of circadian behaviour in several species. Here we investigate the impact mating and blood-feeding have upon the expression of these genes in the mosquito Aedes aegypti . We show that blood intake, but not insemination, is responsible for the down-regulation of clock genes. Using RNA interference, we observe a slight reduction in the evening activity peak in the fourth day after dstim injection. These data suggest that, as in Drosophila , clock gene expression, circadian behaviour and environmental light regimens are interconnected in Ae. aegypti .

Highly expressed genes are associated with inverse antisense transcription in mouse.

There is a growing evidence, that antisense transcription might have a key role in a range of human diseases. Although predefined sense-antisense pairs were extensively studied, the antisense expression of the known sense genes is rarely investigated. We retrieved and correlated the expression of sense and antisense sequences of 1182 mouse transcripts to assess the prevalence and to find the characteristic pattern of antisense transcription. We contrasted three Affymetrix MGU74A version 1 mouse genome chips to six MGU74A version 2 chips. For these 1182 transcripts, the version 1 chips contain the antisense sequences of the transcripts presented on the version 2 chips. The original data was taken from the GEO database (GDS431 and GDS432). As the Affymetrix data are semiquantitative, the relative expression levels of antisense partners were analysed. We detected antisense transcription, although the average antisense expression is shifted towards smaller expression values (MGU74A version 1, 516; version 2, 1688). An inverse direct correlation between sense and antisense expression values could be observed at high expression values. At a very high relative expression--above 40,000--the Pearson correlation coefficient is getting closer to -1. Transcripts with high inverse expression ratio may be correlated to the investigated gene (major histocompatibility complex class II trans activator). The ratio of sense to antisense transcripts varied among different chromosomes; on chromosomes 14 and 1 the level of antisense expression was higher than that of sense. We conclude that antisense transcription is a common phenomenon in the mouse genome. The hypothesis of regulatory role of antisense transcripts is supported by the inverse antisense gene expression of highly expressed genes.

Grandes alcances de los RNAs pequeños RNA de interferencia y microRNA / Great potential of small RNas: RNA interference and microRNA

Double-stranded RNA (dsRNA) induces a sequence-specific silencing in eukaryotic cells. This silencing process beggins when long dsRNA is cleaved to 21 to 26 long small RNA by means of the RNAse III-type enzyme Dicer. These small dsRNA are included into silencing effector complexes, that are targeted to complementary sequences. Small RNA dependent gene silencing can be achieved by distinct mechanisms based depending mainly on the nature of target sequences and on the proteins present in the effector complex. The route of interference RNA (RNAi) begins when Dicer yields small interference RNA (siR-NA) that bind to complementary mRNA for its degradation, forming the RISC complex. siRNA are naturally formed from transposons and dsRNA viruses during its replication, as well as from other bidirectional transcribed repetitive sequences. Some of the enzymes thar are part of the RNAi machinery, including Dicer, are encoded by multigene families in many species, that also play a role in other mechanisms of RND-dependent gene silencing. MicroRNA's (miRNA) are other small RNA's that can induce gene silencing at the mRNA level. These are formed in a general manner when Dicer process hairpin structures resulting from the transcription of non-coding sequences from plant and animal genomes. miRNA's are integrated into a RISC-like complex, after which, depending on their degree of complementarity with target mRNA, can either repress translation or induce mRNA degradation. miRNA-dependent silencing is essential for the development of multicellular organisms. Artificial RNAi induction by means of siRNA or miRNA is being used as a tool to inactivate gene expression in culture cells and in living organisms. This review focuses on the progress in the understanding of the mechanisms involved in gene regulation by RNA in animals and details some current efforts to apply theses phenomena as a tool in research and in the therapeutic of human diseases.

El RNA de doble cadena puede inducir un silenciamiento secuencia-específico en eucarionte. Este proceso de silenciamiento se inicia cuando el RNAdc largo es procesado a RNA pequeño de 21 a 26 nucleótidos mediante la enzima RNAsa III Dicer. Estos RNA pequeños se incorporan a complejos efectores de silenciamiento, que son guiados a secuencias complementarias blanco. Existen diferentes tipos de silenciamiento, cuyas diferencias se basan principalmente en la naturaleza de la secuencia blanco y en la composición proteica de los complejos efectores. La ruta del RNA de interferencia (RNAi) se inicia cuando Dicer genera pequeños RNA de interferencia (siRNA) que se unen por complementariedad al mRNA para su degradación, utilizando el complejo RISC. De manera natural, los siRNA se originan de transposones y virus que producen RNAdc durante su replicación, así como también de otras secuencias repetidas transcritas bidireccionalmente. Algunas de las enzimas que conforman la maquinaria del RNAi como Dicer, entre otras, son codificadas por familias multigénicas en varias especies y también participan en otros mecanismos de silenciamiento mediado por RNA. Los microRNA son otros RNA pequeños que pueden inducir silenciamiento al unirse al mRNA. Éstos se generan de manera general cuando Dicer procesa estructuras de horquilla compuestas de regiones no codificantes, en genomas de plantas y animales. Los miRNA se incorporan a un complejo similar a RISC y, dependiendo de su grado de complementariedad con el mRNA blanco, pueden tener represión traduccional o bien digerir el mRNA. El silenciamiento mediado por miRNA es esencial para el desarrollo de plantas y animales. La inducción artificial del RNAi mediante siRNA o miRNA ha sido adoptada como una herramienta para inactivar la expresión génica, tanto en células en cultivo como en organismos vivos. En esta revisión se muestra el gran progreso en el entendimiento de los mecanismos que participan en la regulación génica mediada por RNA en animales y detalla algunos esfuerzos actuales para encauzar a estos mecanismos como una herramienta en la investigación y como posible terapia en enfermedades.

The RNA interference revolution

The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.

RNA interference: a novel and physiologic mechanism of gene silencing with great therapeutic potential

The post-genomics scientific era has evolved rapidly while achieving advanced understanding of the structure and function of the genes responsible for both the phenotypic characteristics of higher organisms and the pathophysiology of several genetic diseases. Researchers in the fields of oncology and infectious diseases have become more convinced of the great potential of molecular biology approaches to further develop highly specific diagnostic and less toxic therapeutic strategies. During the last two decades, approaches for the specific silencing of essential viral genes and cellular oncogenes were evaluated with optimism for developing directed therapies. However, there were drawbacks in the use of antisense oligonucleotides as a practical mechanism of achieving gene silencing both in vitro and in vivo. Recently, a novel role for post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA) was discovered in the experimental model of C. elegans. This mechanism, termed RNA interference (RNAi) has also been found in other eukaryotes, from plants to mammals, including humans. RNAi is presently being explored both in vitro and in vivo in functional genomics studies and possible therapeutic uses due to its highly specific and physiologic mode of gene silencing. This article focuses on the most current information available regarding the RNAi mechanism and its uses in models of cancer and infectious diseases.

Silenciamento de genes com RNA interferência: um novo instrumento para investigação da fisiologia e fisiopatologia do córtex adrenal / Gene silencing with RNA interference: a novel tool for the study of physiology and pathophysiology of adrenal cortex

A inativação de genes por knock-out ou por bloqueio da tradução de seus transcritos (silenciamento) constitui uma estratégia extremamente poderosa tanto para atribuir função aos genes como para mapear a inter-relação dos diferentes componentes das vias regulatórias intracelulares. Um dos meios para se obter o silenciamento pós-transcricional consiste na ativação de um mecanismo mediado por RNAs fita-dupla (dsRNA) conhecido como RNA interferência (RNAi). O RNAi se mostrou um instrumento extremamente versátil em pesquisa biomédica, podendo ser utilizado em experimentos de silenciamento pontual de genes ou ser adaptado para estudos em larga escala de genômica funcional, podendo, inclusive, ser utilizado como meio de terapia gênica. Neste trabalho, os autores discutem as vias intracelulares envolvidas no RNAi, bem como as principais estratégias e limitações técnicas para se obter o silenciamento em células de mamíferos. Fazem, também, uma revisão das principais aplicações do RNAi na terapêutica de doenças humanas e na investigação de fenômenos fisiológicos e fisiopatológicos do córtex adrenal.