Alcohol-related liver disease (ALD) is a common chronic liver disease caused by long-term excessive alcohol consumption and responsible for more than half of all liver-related deaths worldwide. The molecular mechanisms associated with ALD were not fully understood. In this study, we performed single-cell RNA sequencing on liver tissues obtained from ALD patients and healthy liver donors. We identified an ALB+KRT7+ epithelial population that expressed both hepatocyte and biliary markers significantly expanded in ALD livers. The ALB+KRT7+ epithelial cells were demonstrated to have stem cell properties and malignant transformation potentials. Moreover, ALB+KRT7+ epithelium-derived ALD organoids promote the tumour growth by activating Wnt/β-catenin signalling of liver cancer cells. Most importantly, blocking the Wnt protein secretion or knockdown the Wnt receptor suppressed the tumour promoting effect of ALD organoids. Our study provides important insights that Wnt signalling can be targeted in patients with advanced alcohol-related cirrhosis to prevent malignant transformation. In addition, our results also uncovered the important alterations of nonparenchymal cells, especially macrophages and T/NK populations that responsible for active inflammation responses in alcohol-related hepatitis and immunosuppressive microenvironment in advanced cirrhosis livers, which likely facilitated the malignant progression of ALD.
RNA pseudouridylation, a dynamic and reversible post-transcriptional modification found in diverse RNA species, is crucial for various biological processes, including tRNA homeostasis, tRNA transport, translation initiation regulation, pre-mRNA splicing, enhancement of mRNA translation, and translational fidelity. Disruption of pseudouridylation impairs cellular homeostasis, contributing to pathological alterations. Recent studies have highlighted its regulatory role in human diseases, particularly in tumourigenesis. Cellular stresses trigger RNA pseudouridylation in organisms, suggesting that pseudouridylation-mediated epigenetic reprogramming is essential for maintaining cellular viability and responding to stress. This review examines the regulatory mechanisms and pathological implications of pseudouridylation in human diseases, with a focus on its involvement in tumourigenesis. Additionally, it explores the therapeutic potential of targeting pseudouridylation, presenting novel strategies for disease treatment.
Numerous pathogenic variants causing human oocyte maturation arrest have been reported on the primate-specific TUBB8 gene. The main etiology is the dramatic reduction of tubulin α/β dimer, but still large numbers of variants remain unexplained.
Using microinjection mRNA and genome engineering to reintroduce the conserved pathogenic missense variants into oocytes or in generating TUBB8 variant knock-in mouse models, we investigated that the human deleterious variants alter microtubule nucleation and spindle assembly during meiosis. Live-cell imaging and immunofluorescence were utilised to track the dynamic expression of microtubule plus end-tracking proteins in vivo and analysed microtubule nucleation or spindle assembly in vitro, respectively. Immunoprecipitation-mass spectrometry and ultramicro-quantitative proteomics were performed to identify the differential abundance proteins and affected interactome of TUBB8 protein.
First, we observed a significant depletion of the EB1 signal upon microinjection of mutated TUBB8 mRNA (including R262Q, M300I, and D417N missense variants), indicating disruption of microtubule nucleation caused by these introduced TUBB8 missense variants. Mechanically, we demonstrated that the in vivo TUBB8-D417N missense variant diminished the affinity of EB1 and microtubules. It also harmed the interaction between microtubules and CKAP5/TACC3, which are crucial for initiating microtubule nucleation. Attenuated Ran-GTP pathway was also found in TUBB8-D417N oocytes, leading to disrupted spindle assembly. Stable microtubule was largely abolished on the spindle of TUBB8-D417N oocytes, reflected by reduced tubulin acetylation and accumulated HDAC6. More importantly, selective inhibition of HDAC6 by culturing TUBB8-D417N oocytes with Tubacin or Tubastatin A showed morphologically normal spindle and drastically recovered polar-body extrusion rate. These rescue results shed light on the strategy to treat meiotic defects in a certain group of TUBB8 mutated patients.
Our study provides a comprehensive mechanism elucidating how TUBB8 missense variants cause oocyte maturation arrest and offers new therapeutic avenues for treating female infertility in the clinic.
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