Abstract. Alternative splicing is a near-ubiquitous phenomenon with important roles in human diseases, including cancers. Splice-switching oligonucleotides (SSOs) have emerged as a class of antisense therapeutics that modulate alternative splicing by hybridizing to the pre-mRNA splice site. The Bcl-x gene is alternatively spliced to express anti-apoptotic Bcl-xL and pro-apoptotic Bcl-xS. Bcl-xL expression is upregulated in many cancers and is considered a general mechanism by which cancer cells evade apoptosis. By redirecting Bcl-x pre-mRNA splicing from Bcl-xL to Bcl-xS, SSO exerted pro-apoptotic and chemosensitizing effects in various cancer cell lines. In this study, we investigated the effects of SSO targeting Bcl-x premRNA in human glioma cell lines. First, we performed reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine the mRNA and protein expression levels of Bcl-xL in glioma cell lines (U87 and U251) and a normal human astrocyte cell line (HA1800). Then, the Bcl-x SSO was designed to bind to the downstream 5' alternative splice site of exon 2 in Bcl-x pre-mRNA and was modified using 2'-O-methoxyethyl-phosphorothioate. An oligonucleotide targeting aberrantly spliced human β-globin intron was used as a negative control. The SSOs were delivered with a cationic lipid into glioma and astrocyte cell lines. The antitumor effects of the SSOs were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and flow cytometry, and the switch in production from Bcl-xL to Bcl-xS was analyzed by RT-PCR and western blotting. Bcl-xL mRNA and protein were highly expressed in both glioma cell lines. The Bcl-x SSO modified Bcl-x pre-mRNA splicing and had pro-apoptotic effects on the glioma cell lines. By contrast, the lipid alone and the control SSO did not affect Bcl-xL expression or induce apoptosis. Our study demonstrated the antitumor activity of an SSO that targets Bcl-x pre-mRNA splicing in glioma cell lines. Bcl-x SSO may be a potential strategy for treating gliomas. IntroductionAlternative splicing is the process by which splice sites in precursor messenger RNA (pre-mRNA) are differentially selected and paired to produce multiple mature mRNAs and protein isoforms with distinct structural and functional properties. Alternative splicing is a very accurate, efficient, and extraordinarily flexible process that regulates many major aspects of eukaryotic cell biology. Approximately 95% of human genes with multiple exons undergo alternative splicing during pre-mRNA maturation (1-3). In addition to its role in human proteome diversity, alternative splicing is now accepted to play important roles in human diseases, including diabetes, neurodegenerative diseases, and cancer (4-6).Aberrant alternative splicing has two major roles in cancer by promoting the emergence of a cancer-specific isoform or disturbing the balanced expression of normally expressed isoforms in cancer cells (7). During tumor growth and development, and during oncogenesis, cells progress through ...
Reducing β amyloid- (Aβ-) induced microglial activation is believed to be effective in treating Alzheimer's disease (AD). Microglia can be activated into classic activated state (M1 state) or alternative activated state (M2 state), and the former is harmful; in contrast, the latter is beneficial. Gypenoside (GP) is the major bioactive constituent of Gynostemma pentaphyllum, a traditional Chinese herb medicine. In this study, we hypothesized that GP attenuates Aβ-induced microglial activation by ameliorating microglial M1/M2 states, and the process may be mediated by suppressor of cell signaling protein 1 (SOCS1). In this study, we found that Aβ exposure increased the levels of microglial M1 markers, including iNOS expression, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 releases, and coadministration of GP reversed the increase of M1 markers and enhanced the levels of M2 markers, including arginase-1 (Arg-1) expression, IL-10, brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF) releases in the Aβ-treated microglial cells. SOCS1-siRNA, however, significantly abolished the GP-induced effects on the levels of microglial M1 and M2 markers. These findings indicated that GP attenuates Aβ-induced microglial activation by ameliorating M1/M2 states, and the process may be mediated by SOCS1.
Objectives Propofol is a popular anesthetic drug that is neuroprotective. However, the mechanisms of propofol for hippocampal neuroprotection remain elusive. This study is aimed at investigating the neuroprotective effect and mechanism of propofol in hippocampal neurons exposed to ischemia-reperfusion (I/R) injury. Methods Hypoxia-reoxygenated (H/R) HT-22 cells were used to mimic I/R injury of the hippocampus in vitro. An MTT assay was used to determine cell viability. Cell apoptosis was detected by a TUNEL assay and a flow cytometry cell apoptosis assay. Expression levels of proteins were measured by Western blotting. Intracellular calcium was assessed by Fura-2/AM staining. Flow cytometry was used to determine the mitochondrial membrane potential (MMP). Coimmunoprecipitation was used to evaluate the stability of the FKBP-RyR complex. Calcineurin enzymatic activity was measured with a colorimetric method. YAP nuclear translocation was tested by immunofluorescence staining. Results H/R induced HT-22 cell viability depression, and apoptosis was reversed by propofol treatment. Propofol could alleviate H/R-induced intracellular calcium accumulation and MMP loss by inhibiting calcineurin activity and FKBP12.6-RyR disassociation in a concentration-dependent manner. In addition, YAP expression was crucial for propofol to protect HT-22 cell apoptosis from H/R injury. Propofol could activate YAP through dephosphorylation. Activated YAP stimulated the transcription of the Bcl2 gene, which promotes cellular survival. Our data also demonstrated that propofol activated YAP through the RhoA-Lats1 pathway without large G proteins or MST involvement. In addition, we showed that there was no interaction between calcineurin signaling and YAP activation in HT-22 cells. Conclusions Propofol protected hippocampal neurons from I/R injury through two independent signaling pathways, including the calcineurin/FKBP12.6-RyR/calcium overload pathway and the RhoA/Lats1/YAP/Bcl-2 pathway.
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