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.

Matrix Metalloproteinase-9 Regulates Tumor Cell Invasion through Cleavage of Protease Nexin-1

Abstract: Matrix metalloproteinase-9 (MMP-9) expression is known to enhance the invasion and metastasis of tumor cells. In previous work based on a proteomic screen, we identified the serpin protease nexin-1 (PN-1) as a potential target of MMP-9. Here, we show that PN-1 is a substrate for MMP-9 and establish a link between PN-1 degradation by MMP-9 and regulation of invasion. PN-1 levels increased in prostate carcinoma cells after downregulation of MMP-9 and in tissues of MMP-9-deficient mice, consistent with PN-1 degradation by MMP-9. We identified three MMP-9 cleavage sites in PN-1 and showed that mutations in those sites made PN-1 more resistant to MMP-9. Urokinase plasminogen activator (uPA) is inhibited by PN-1. MMP-9 augmented uPA activity in the medium of PC3-ML cells by degrading PN-1. Prostate cancer cells, overexpressing PN-1 or treated with MMP-9 shRNA, had reduced cell invasion in Matrigel. PN-1 siRNA restored uPA activity and the invasive capacity. PN-1 mutated in the serpin inhibitory domain, the reactive center loop, failed to inhibit uPA and to reduce Matrigel invasion. This study shows a novel molecular pathway in which MMP-9 regulates uPA activity and tumor cell invasion through cleavage of PN-1.

Effective Suppression of Human Immunodeficiency Virus Type 1 through a Combination of Short- or Long-Hairpin RNAs Targeting Essential Sequences for Retroviral Integration

Abstract: Gene targeting in mammalian cells through the use of short-hairpin RNAs (shRNAs) has been advanced by the development of vector systems for efficient delivery and stable expression of shRNA sequences (4, 7, 22, 30). Upon delivery into cells, shRNAs are converted into short double-stranded RNAs, termed small interfering RNAs (siRNAs), that mediate a sequence-specific RNA degradation process termed RNA interference (RNAi) (12, 14, 42). Antiviral therapy based on siRNA has been proposed as a new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) (16, 31, 32) and hepatitis C virus (HCV) (34). When viral genes are targeted, viruses can escape from RNAi-mediated inhibition due to their high mutation rate (6, 11, 39). An alternative approach that shows promise is the use of siRNAs targeting cellular genes essential for virus replication. In the case of HIV-1, siRNAs against the cell surface CD4 receptor (31) or CXCR4 and CCR5 coreceptors for HIV-1 entry conferred viral resistance (2, 3, 33). However, CD4 and CXCR4 are essential for T-cell development and proper immunologic function. In addition, although CCR5 might be nonessential for normal function (23), not all HIV-1 strains require CCR5. Downregulation of an essential cellular coreceptor could potentially result in the emergence of HIV-1 variants that use another coreceptor(s) for viral entry into the cell. To achieve long-term control of viral replication by siRNA and prevent the emergence of escape variants, it is important to target highly conserved and/or essential HIV-1 sequences. For example, many sites in the cis-regulatory regions, as well as the protein-coding regions, of HIV-1 have been examined as potential targets for siRNA. These regions include the primer-binding site, the polypurine tract, the long terminal repeat, and the gag, pol, env, tat, rev, vif, and nef genes (6, 10, 11, 16, 18, 21, 30, 39). The degree to which siRNAs inhibited HIV-1 replication and the underlying mechanisms varied considerably, depending on the target sequence (10, 11). For example, RNAi-resistant HIV-1 variants can emerge not only through mutations in the siRNA target sequence but also through mutations that alter the local RNA structure (39). These results emphasize the need for empirical studies to determine effective siRNA target sites within the HIV-1 genome. In the present study, we selected several sequences for lentivirus-mediated shRNA expression based on a preliminary screening of HIV-1 RNAi target sites using synthetic siRNA duplexes. These sequences mapped within the integrase (IN) gene (int) and the attachment site (att), which are essential for HIV-1 integration. We evaluated the anti-HIV-1 activity of these expressed shRNAs using a highly susceptible CD4+ T-cell line. Genetic analysis of HIV-1 escape mutants that emerged after treatment with combinations of shRNAs revealed that two or more shRNAs targeting different essential sequences had the strongest impact on antiviral activity. The results also suggest that shRNAs or long-hairpin RNA (lhRNA) that targets incoming viral RNA before proviral DNA formation is more efficient at mediating RNAi antiviral therapy.

METTL3 Promotes the Proliferation and Mobility of Gastric Cancer Cells.

Abstract: Methyltransferase-like 3 (METTL3) was originally known to be responsible for N6-methyladenosine (m6A) modification of mRNA. Recent studies have found that METTL3 plays important roles in a variety of tumors by regulating the translation of oncogenes. However, the functional and regulating mechanisms of METTL3 in human gastric cancer have not yet been understood. Here we knocked down METTL3 in human gastric cancer cell lines, AGS and MKN45, by using shRNA transfection. RT-qPCR assay and western blotting verified the effectiveness of RNA interference on mRNA and protein levels, respectively. Then we found that METTL3 knockdown inhibited cell proliferation, migration and invasion in AGS and MKN45 cells. Moreover, METTL3 knockdown decreased Bcl2 and increased Bax and active Caspase-3 in gastric cancer cells, which suggested the apoptotic pathway was activated. Mechanistic investigation suggested that METTL3 led to inactivation of the AKT signaling pathway in human gastric cancer cells, including decreased phosphorylation levels of AKT and expression of down-stream effectors p70S6K and Cyclin D1. In conclusion, our study reveals that down-regulation of METTL3 inhibits the proliferation and mobility of human gastric cancer cells and leads to inactivation of the AKT signaling pathway, suggesting that METTL3 may be a potential target for the treatment of human gastric cancer.