Small hairpin RNA

About: Small hairpin RNA is a research topic. Over the lifetime, 9279 publications have been published within this topic receiving 285471 citations.

In vivo knockdown of the androgen receptor results in growth inhibition and regression of well-established, castration-resistant prostate tumours

Abstract: Purpose: Progression to the castration-resistant state is the incurable and lethal end stage of prostate cancer, and there is strong evidence that androgen receptor (AR) still plays a central role in this process. We hypothesize that knocking down AR will have a major effect on inhibiting growth of castration-resistant tumors. Experimental Design: Castration-resistant C4-2 human prostate cancer cells stably expressing a tetracycline-inducible AR-targeted short hairpin RNA (shRNA) were generated to directly test the effects of AR knockdown in C4-2 human prostate cancer cells and tumors. Results:In vitro expression of AR shRNA resulted in decreased levels of AR mRNA and protein, decreased expression of prostate-specific antigen (PSA), reduced activation of the PSA-luciferase reporter, and growth inhibition of C4-2 cells. Gene microarray analyses revealed that AR knockdown under hormone-deprived conditions resulted in activation of genes involved in apoptosis, cell cycle regulation, protein synthesis, and tumorigenesis. To ensure that tumors were truly castration-resistant in vivo, inducible AR shRNA expressing C4-2 tumors were grown in castrated mice to an average volume of 450 mm3. In all of the animals, serum PSA decreased, and in 50% of them, there was complete tumor regression and disappearance of serum PSA. Conclusions: Whereas castration is ineffective in castration-resistant prostate tumors, knockdown of AR can decrease serum PSA, inhibit tumor growth, and frequently cause tumor regression. This study is the first direct evidence that knockdown of AR is a viable therapeutic strategy for treatment of prostate tumors that have already progressed to the castration-resistant state.

Reversal of MDR1/P-glycoprotein-mediated multidrug resistance by vector-based RNA interference in vitro and in vivo

Abstract: Overexpression of P-glycoprotein (P-gp) encoded by MDR1 gene in cancer cells results in multidrug resistance (MDR) to structurally and mechanistically different chemotherapeutic drugs, which is a major cause for cancer chemotherapy failures to cancer patients. Recently, there were several reports showing that expression of siRNAs targeting MDR1 gene is able to reverse the P-gp mediated MDR, however, the in vivo reversal effects for MDR have still not been identified. We developed a novel MDR reversal system using RNA interference technique in human epidermoid carcinoma KBv200 cells. The stably expressing MDR1 shRNA cells (KBv200/MDR1sh) were established with transfection of vector pEGFPC2-H1-MDR1shDNA containing MDR1-V siRNA expression cassette, and we found that more than 90% of MDR1 mRNA and P-gp were reduced. KBv200/MDR1sh cells simultaneously showed stably expressing EGFP and kept low MDR1 expression beyond ten passages. Compared KBv200/MDR1sh cells with KBv200 cells, resistance to vincristine and doxorubicin decreased from 62.4-fold to 10.5-fold and from 74.5-fold to 9.5-fold respectively, and intracellular doxorubicin accumulation enhanced from 0.30 +/- 0.08 nmoles/10(6) cells to 0.86 +/- 0.16 nmoles/10(6) cells, and the fluorescence intensity of intracellular Rhodamine 123 accumulation increased from 3.58 +/- 1.63/10(6) cells to 13.96 +/- 3.07/10(6) cells. In the nude mice xenografts, vincristine (0.2 mg/kg of body weight) inhibited the growth of KBv200/MDR1sh solid tumors by 42.0%, but the same dose of vincristine didn't inhibit the growth of KBv200 solid tumors significantly. These results suggest that administration of RNAi targeted MDR1 gene can effectively reverse MDR both in vitro and in vivo models.

A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies

Abstract: Dysregulation of the insulin-like growth factor type I receptor (IGF1R) has been implicated in the progression and therapeutic resistance of malignancies. In acute myeloid leukemia (AML) cells, IGF1R is one of the most abundantly phosphorylated receptor tyrosine kinases, promoting cell growth through the PI3K/Akt signaling pathway. However, little is known regarding the molecular mechanisms underlying IGF1R gene dysregulation in cancer. We discovered a novel intragenic long noncoding RNA (lncRNA) within the IGF1R locus, named IRAIN, which is transcribed in an antisense direction from an intronic promoter. The IRAIN lncRNA was expressed exclusively from the paternal allele, with the maternal counterpart being silenced. Using both reverse transcription-associated trap and chromatin conformation capture assays, we demonstrate that this lncRNA interacts with chromatin DNA and is involved in the formation of an intrachromosomal enhancer/promoter loop. Knockdown of IRAIN lncRNA with shRNA abolishes this intrachromosomal interaction. In addition, IRAIN was downregulated both in leukemia cell lines and in blood obtained from high-risk AML patients. These data identify IRAIN as a new imprinted lncRNA that is involved in long-range DNA interactions.

Prostaglandin E2 Promotes Lung Cancer Cell Migration via EP4-βArrestin1-c-Src Signalsome

Abstract: Many human cancers express elevated levels of cyclooxygenase-2 (COX-2), an enzyme responsible for the biosynthesis of prostaglandins. Available clinical data establish the protective effect of COX-2 inhibition on human cancer progression. However, despite these encouraging outcomes, the appearance of unwanted side effects remains a major hurdle for the general application of COX-2 inhibitors as effective cancer drugs. Hence, a better understanding of the molecular signals downstream of COX-2 is needed for the elucidation of drug targets that may improve cancer therapy. Here, we show that the COX-2 product prostaglandin E(2) (PGE(2)) acts on cognate receptor EP4 to promote the migration of A549 lung cancer cells. Treatment with PGE(2) enhances tyrosine kinase c-Src activation, and blockade of c-Src activity represses the PGE(2)-mediated lung cancer cell migration. PGE(2) affects target cells by activating four receptors named EP1 to EP4. Use of EP subtype-selective ligand agonists suggested that EP4 mediates prostaglandin-induced A549 lung cancer cell migration, and this conclusion was confirmed using a short hairpin RNA approach to specifically knock down EP4 expression. Proximal EP4 effectors include heterotrimeric Gs and betaArrestin proteins. Knockdown of betaArrestin1 expression with shRNA significantly impaired the PGE(2)-induced c-Src activation and cell migration. Together, these results support the idea that increased expression of the COX-2 product PGE(2) in the lung tumor microenvironment may initiate a mitogenic signaling cascade composed of EP4, betaArrestin1, and c-Src which mediates cancer cell migration. Selective targeting of EP4 with a ligand antagonist may provide an efficient approach to better manage patients with advanced lung cancer.

RNA interference and double-stranded-RNA-activated pathways

Abstract: RNAi (RNA interference) has become a powerful tool to determine gene function. Different methods of expressing the short ds (double-stranded) RNA intermediates required for interference in mammalian systems have been developed, including the introduction of si (short interfering) RNAs by direct transfection or driven from transfected plasmids or lentiviral vectors encoding sh (short hairpin) RNAs. Although RNAi relies upon a high degree of specificity, recent findings suggest that off-target non-specific effects can be encountered. We found that transfection of siRNAs can results in an interferon-mediated activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway and global up-regulation of interferon-stimulated genes. This effect is mediated in part by the dsRNA-dependent protein kinase PKR, as this kinase is activated by the 21 bp siRNA, and is required in response to the siRNAs. However, the transcription factor IRF3 (interferon-regulatory factor 3) is also activated by siRNA as a primary response, resulting in the stimulation of genes independent of an interferon response. In cells deficient in IRF3, this response is blunted, but can be restored by re-introduction of IRF3. Thus siRNAs induce complex signalling responses in target cells, leading to effects beyond the selective silencing of specific genes.