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.

miR-644-5p carried by bone mesenchymal stem cell-derived exosomes targets regulation of p53 to inhibit ovarian granulosa cell apoptosis

Abstract: This article aims to reveal the therapeutic effects and potential mechanisms of bone mesenchymal stem cell (BMSC)-derived exosomes on premature ovarian failure (POF). Exosomes were collected from BMSCs and were used to treat cisplatin-induced POF mouse models. Pathological changes of ovarian tissue were detected by using HE staining and by Western blot that detected the expression of apoptosis-related proteins. In cisplatin-induced primary granulosa cell injury, exosomes were co-cultured with the granulosa cells. The apoptosis or viability of granulosa cells was analyzed by flow cytometry or MTT, respectively. In Target scan and microT-CDS databases, an intersection of miRNAs targeting to p53 was found. The expressions of miRNAs in BMSC-derived exosomes were detected by qRT-PCR. Besides, miR-664-5p targeted to p53 of cells was verified by dual-luciferase reporter assay. BMSC-derived exosomes improved the follicular morphology of POF mice and inhibited the expression of apoptosis-related protein. By co-culture of exosomes and primary granulosa cells, BMSC-derived exosomes repressed cisplatin-induced granulosa cells apoptosis and increased cells viability, while these effects were abrogated after the exosome-containing RNA was degraded by RNase. By Target scan, microT-CDS and qRT-PCR, miR-664-5p was regarded as the dominated RNA in BMSC-derived exosomes. By dual-luciferase reporter assay, miR-664-5p negatively regulated p53 luciferase activity. After shRNA interfering miR-664-5p of BMSC, BMSC-derived exosomes exerted no protective effect on cisplatin-induced granulosa cell apoptosis. Our results indicated that miR-644-5p carried by BMSC-derived exosomes inhibited the apoptosis of ovarian granulosa cell by targeting p53 of cells, suggesting that miR-644-5p had the potential to treat POF and restore ovarian function.

The metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), upregulates p21 via p53-independent mechanisms.

Abstract: The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), has been shown to markedly reduce metastasis of numerous tumors. The current study was focused on further elucidating the molecular mechanisms behind the antitumor function of NDRG1. We have identified for the first time that NDRG1 upregulates the potent cyclin-dependent kinase inhibitor, p21. This effect was observed in three different cancer cell types, including PC3MM and DU145 prostate cancer cells and H1299 lung carcinoma cells, and occurred independently of p53. In addition, reducing NDRG1 expression using short hairpin RNA in PC3MM and DU145 cells resulted in significantly reduced p21 protein levels. Hence, p21 is closely correlated with NDRG1 expression in these latter cell types. Examining the mechanisms behind the effect of NDRG1 on p21 expression, we found that NDRG1 upregulated p21 via transcriptional and posttranscriptional mechanisms in prostate cancer cells, although its effect on H1299 cells was posttranscriptional only. Further studies identified two additional NDRG1 protein targets. The dominant-negative p63 isoform, ΔNp63, which has been found to inhibit p21 transcription, was downregulated by NDRG1. On the other hand, a truncated 50 kDa MDM2 isoform (p50 MDM2 ), which may protect p21 from proteasomal degradation, was upregulated by NDRG1. The downregulation of ΔNp63 and upregulation of p50 MDM2 are potential mechanisms by which NDRG1 increases p21 expression in these cells. Additional functional studies identified that NDRG1 inhibits cancer cell migration, suggesting that p21 is a molecular player in its antimetastatic activity.

Interleukin-1β induces ICAM-1 expression enhancing leukocyte adhesion in human rheumatoid arthritis synovial fibroblasts: Involvement of ERK, JNK, AP-1, and NF-κB

Abstract: Interleukin-1beta (IL-1beta) has been shown to induce the expression of adhesion molecules on various cell types and contributes to inflammatory responses. However, the molecular mechanisms by which IL-1beta induced intercellular adhesion molecule (ICAM)-1 expression remain unclear in human rheumatoid arthritis synovial fibroblasts (RASFs). Here, we demonstrated that IL-1beta induces ICAM-1 gene expression via the de novo protein synthesis through transcription and translation, which is attenuated by pretreatment with actinomycin D and cycloheximide, respectively. IL-1beta-induced ICAM-1 expression, extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) phosphorylation, AP-1 activation, and nuclear factor-kappaB (NF-kappaB) p65 translocation were attenuated by the inhibitors of MEK1/2 (U0126), JNK (SP600125), AP-1 (tanshinone IIA), and NF-kappaB (helenalin) or transfection with respective short hairpin RNA plasmids. Moreover, IL-1beta-stimulated NF-kappaB p65 translocation was blocked by helenalin, but not by U0126 or SP600125, revealing that MAPKs and NF-kappaB pathways were independent on these responses. IL-1beta-stimulated AP-1 activation was blocked by U0126 or SP600125, revealing that ERK and JNK linked to AP-1 on these responses. IL-1beta-stimulated ICAM-1 gene expression was attenuated by pretreatment with U0126, SP600125, tanshinone IIA, or helenalin, revealed by ICAM-1 promoter assay and real-time RT-PCR analysis. Finally, up-regulation of ICAM-1 enhanced the adhesion of leukocytes to RASFs exposed to IL-1beta. These results suggest that in human RASFs, activation of ERK, JNK, AP-1, and NF-kappaB are essential for IL-1beta-induced ICAM-1 expression and leukocyte adhesion.

Notch1 regulates the expression of the multidrug resistance gene ABCC1/MRP1 in cultured cancer cells

Abstract: Multidrug resistance (MDR) is a barrier to successful cancer chemotherapy. Although MDR is associated with overexpression of ATP-binding cassette (ABC) membrane transporters, mechanisms behind their up-regulation are not entirely understood. The cleaved form of the Notch1 protein, intracellular Notch1 (N1IC), is involved in transcriptional regulation of genes. To test whether Notch1 is involved in the expression of multidrug resistance-associated protein 1 (ABCC1/MRP1; herein referred to as ABCC1), we measured N1IC and presenilin 1 (PSEN1), the catalytic subunit of γ-secretase required for Notch activation. We observed higher levels of N1IC and PSEN1 proteins as well as higher activity of N1IC in ABCC1-expressing MDR MCF7/VP cells compared with parental MCF7/WT cells. Reducing N1IC levels in MCF7/VP cells with either a γ-secretase inhibitor or shRNA led to reduction of ABCC1. By contrast, ectopic expression of N1IC in MCF7/WT cells led to increased expression of ABCC1 and associated drug resistance, consistent with expression of this transporter. Inhibition of ABCC1 reversed drug resistance of N1IC-overexpressing stable cells. Using an ABCC1 promoter construct, we observed both its reduced transcriptional activity after blocking the generation of N1IC and its increased transcriptional activity in stable cells overexpressing N1IC. ChIP and gel-shift assays revealed an interaction between a specific promoter region of ABCC1 and the N1IC-activated transcription factor CBF1, suggesting that the regulation of ABCC1 expression by Notch1 is mediated by CBF1. Indeed, deletion or site-directed mutagenesis of these CBF1 binding sites within the ABCC1 promoter region attenuated promoter-reporter activity. Overall, our results reveal a unique regulatory mechanism of ABCC1 expression.

Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice.

Abstract: RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)–expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)—the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.