PLEKHA4 is a novel prognostic biomarker that reshapes the tumor microenvironment in lower-grade glioma.

Background: Lower-grade glioma (LGG) is a primary intracranial tumor that carry a high risk of malignant transformation and limited therapeutic options. Emerging evidence indicates that the tumor microenvironment (TME) is a superior predictor for tumor progression and therapy response. PLEKHA4 has been demonstrated to be a biomarker for LGG that correlate with immune infiltration. However, the fundamental mechanism by which PLEKHA4 contributes to LGG is still poorly understood. Methods: Multiple bioinformatic tools, including Tumor Immune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA2), Shiny Methylation Analysis Resource Tool (SMART), etc., were incorporated to analyze the PLEKHA4. ESTIMATE, ssGSEA, CIBERSORT, TIDE and CellMiner algorithms were employed to determine the association of PLEKHA4 with TME, immunotherapy response and drug sensitivities. Immunohistochemistry (IHC)-based tissue microarrays and M2 macrophage infiltration assay were conducted to verify their associations. Results: PLEKHA4 expression was found to be dramatically upregulated and strongly associated with unfavorable overall survival (OS) and disease-specific survival (DSS) in LGG patients, as well as their poor clinicopathological characteristics. Cox regression analysis identified that PLEKHA4 was an independent prognostic factor. Methylation analysis revealed that DNA methylation correlates with PLEKHA4 expression and indicates a better outcome in LGG. Moreover, PLEKHA4 was remarkably correlated with immune responses and TME remodeling, as evidenced by its positive correlation with particular immune marker subsets and the putative infiltration of immune cells. Surprisingly, the proportion of M2 macrophages in TME was strikingly higher than others, inferring that PLEKHA4 may regulate the infiltration and polarization of M2 macrophages. Evidence provided by IHC-based tissue microarrays and M2 macrophage infiltration assay further validated our findings. Moreover, PLEKHA4 expression was found to be significantly correlated with chemokines, interleukins, and their receptors, further supporting the critical role of PLEKHA4 in reshaping the TME. Additionally, we found that PLEKHA4 expression was closely associated with drug sensitivities and immunotherapy responses, indicating that PLEKHA4 expression also had potential clinical significance in guiding immunotherapy and chemotherapy in LGG. Conclusion: PLEKHA4 plays a pivotal role in reshaping the TME of LGG patients, and may serve as a potential predictor for LGG prognosis and therapy.

LncRNA Pnky Positively Regulates Neural Stem Cell Migration by Modulating mRNA Splicing and Export of Target Genes.

Directed migration of neural stem cells (NSCs) is critical for embryonic neurogenesis and the healing of neurological injuries. The long noncoding RNA (lncRNA) Pnky has been reported to regulate neuronal differentiation of NSCs by interacting with PTBP1. However, its regulatory effect on NSC migration remains to be determined. Herein, we identified that Pnky is also a key regulator of NSC migration in mice, as underscored by the finding that Pnky silencing suppressed but Pnky overexpression promoted the in vitro migration of both C17.2 and NE4C murine NSCs. Additionally, in vivo cell tracking demonstrated that Pnky depletion attenuated but Pnky overexpression facilitated the migration of NE4C cells in the spinal canal after transplantation via injection into the spinal canal. Mechanistically, Pnky regulated the expression of a core set of critical regulators that direct NSC migration, including MMP2, MMP9, Connexin43, Paxillin, AKT, ERK, and P38MAPK. Using catRAPID, a web server for large-scale prediction of protein-RNA interactions, the splicing factors U2AF1 and U2AF1L4, as well as the mRNA export adaptors SARNP, Aly/Ref, and THOC7, were predicted to interact strongly with Pnky. Further investigations using colocalization and RNA immunoprecipitation (RIP) assays confirmed the direct binding of Pnky to U2AF1, SARNP, Aly/Ref, and THOC7. Transcriptomic profiling revealed that as many as 5319 differential splicing events of 3848 genes, which were highly enriched in focal adhesion, PI3K-Akt and MAPK signaling pathways, were affected by Pnky depletion. The predominant subtype of differential splicing by Pnky depletion is intron retention, followed by alternative 5' and 3' splice sites and mutually exclusive exons. Moreover, Pnky knockdown substantially blocked but Pnky overexpression facilitated the export of MMP2, Paxillin, AKT, p38MAPK, and other mRNAs to the cytosol. Collectively, our data showed that through interacting with U2AF1, SARNP, Aly/Ref, and THOC7, Pnky couples and modulates the splicing and export of target mRNAs, which consequently controlling NSC migration. These findings provide a possible theoretical basis of NSC migration regulation.

Inhibition of Rat CYP1A2 and CYP2C11 by Honokiol, a Component of Traditional Chinese Medicine.

BACKGROUND AND OBJECTIVES: Honokiol, a major constituent isolated from Magnolia officinalis, is regarded as a phytochemical marker and bioactive substance present in many traditional Chinese medicines. However, the effect of honokiol on cytochrome P450 (CYP) has not been thoroughly investigated. The aim of this study was to investigate the effect of honokiol on CYP1A2 and CYP2C11 in vitro and in vivo. METHODS: The effect of honokiol on CYP1A2 and CYP2C11 was investigated with rat liver microsomes (RLMs) by measuring phenacetin and tolbutamide metabolism (probe drugs for CYP1A2 and CYP2C11, respectively), and then explored in vivo by measuring the effect of honokiol (2.5 and 5 mg/kg, intravenous injection) on the pharmacokinetics of theophylline and tolbutamide (probe drugs for CYP1A2 and CYP2C11, respectively) in rats in vivo. RESULTS: Honokiol inhibited the formation of acetaminophen from phenacetin and 4-hydroxytolbutamide from tolbutamide in RLMs, with inhibition constant (Ki) values of 1.6 µM and 16.5 µM, respectively. In vivo, honokiol (2.5 or 5.0 mg/kg) increased the half-life (t1/2) of theophylline by 40.9% and 119.9%, decreased the clearance (CL) by 23.8% and 42.9%, and increased the area under the curve (AUC) by 41.3% and 83.4%, respectively. Similarly, the t1/2 of tolbutamide increased by 25.5% and 33.8%, the CL decreased by 14.3% and 19.1%, and the AUC increased by 19.2% and 25.7%, respectively. CONCLUSION: The inhibition of CYP1A2 by honokiol is greater than the inhibition of CYP2C11. The changes in the pharmacokinetics of theophylline and tolbutamide in rats treated with honokiol are due to the inhibition of CYP1A2 and CYP2C11 activity in a dose-dependent manner.