Expression of miRNA-210 in human bone marrow-derived mesenchymal stromal cells under oxygen deprivation

A major limitation in the development of efficient clinical protocols for mesenchymal stromal cell (MStroC)-based tissue regeneration therapy is the low retention and survival of MStroC in injured tissue after therapeutic administration. Low oxygen concentration preconditioning (LOP) during ex vivo cultivation of MStroC, as a method for mimicking oxygenation in their physiological microenvironment, has been shown to be beneficial in clinical trials using MStroC. Introducing hypoxia-mimicking molecules into MStroC during cultivation could be an advantageous LOP strategy. MicroRNA (miRNA) drugs are good candidates for this approach. Analysis of the expression of miRNA-210 in human bone marrow-derived MStroC in conditions of acute and extended hypoxia (24 to 72 h) was performed using RT-qPCR methodology. HIF-1 alpha and HIF-2 alpha gene knockdown cell lines were generated using lentiviral transduction of short hairpin RNA (shRNA) in order to examine whether miRNA-210 expression is regulated by transcription factor HIF-1 and/or HIF-2. We detected a significant increase in miRNA-210 expression in hypoxic conditions at time points of 24, 48 and 72 h (p lt 0.05). Knocking down of HIF-1 alpha and HIF-2 alpha genes indicated involvement of both transcription factors in the elevation of miRNA-210 expression. These results point to miRNA-210 as a good candidate for a hypoxia-mimicking molecule in LOP strategy

Molecular basis of differential target regulation by miR-96 and miR-182: the Glypican-3 as a model

Besides the fact that miR-96 and miR-182 belong to the miR-182/183 cluster, their seed region (UUGGCA, nucleotides 2–7) is identical suggesting potential common properties in mRNA target recognition and cellular functions. Here, we used the mRNA encoding Glypican-3, a heparan-sulfate proteoglycan, as a model target as its short 3′ untranslated region is predicted to contain one miR-96/182 site, and assessed whether it is post-transcriptionally regulated by these two microRNAs. We found that miR-96 downregulated GPC3 expression by targeting its mRNA 3′-untranslated region and interacting with the predicted site. This downregulatory effect was due to an increased mRNA degradation and depended on Argonaute-2. Despite its seed similarity with miR-96, miR-182 was unable to regulate GPC3. This differential regulation was confirmed on two other targets, FOXO1 and FN1. By site-directed mutagenesis, we demonstrated that the miRNA nucleotide 8, immediately downstream the UUGGCA seed, plays a critical role in target recognition by miR-96 and miR-182. Our data suggest that because of a base difference at miRNA position 8, these two microRNAs control a completely different set of genes and therefore are functionally independent

Multi-ancestry genome-wide association meta-analysis of Parkinson?s disease

Although over 90 independent risk variants have been identified for Parkinson’s disease using genome-wide association studies, most studies have been performed in just one population at a time. Here we performed a large-scale multi-ancestry meta-analysis of Parkinson’s disease with 49,049 cases, 18,785 proxy cases and 2,458,063 controls including individuals of European, East Asian, Latin American and African ancestry. In a meta-analysis, we identified 78 independent genome-wide significant loci, including 12 potentially novel loci (MTF2, PIK3CA, ADD1, SYBU, IRS2, USP8, PIGL, FASN, MYLK2, USP25, EP300 and PPP6R2) and fine-mapped 6 putative causal variants at 6 known PD loci. By combining our results with publicly available eQTL data, we identified 25 putative risk genes in these novel loci whose expression is associated with PD risk. This work lays the groundwork for future efforts aimed at identifying PD loci in non-European populations

Defining the causes of sporadic Parkinson's disease in the global Parkinson's genetics program (GP2)

The Global Parkinson’s Genetics Program (GP2) will genotype over 150,000 participants from around the world, and integrate genetic and clinical data for use in large-scale analyses to dramatically expand our understanding of the genetic architecture of PD. This report details the workflow for cohort integration into the complex arm of GP2, and together with our outline of the monogenic hub in a companion paper, provides a generalizable blueprint for establishing large scale collaborative research consortia

Unfolded Protein Response Signaling in Liver Disorders: A 2023 Updated Review

Endoplasmic reticulum (ER) is the site for synthesis and folding of secreted and transmembrane proteins. Disturbance in the functioning of ER leads to the accumulation of unfolded and misfolded proteins, which finally activate the unfolded protein response (UPR) signaling. The three branches of UPR—IRE1 (Inositol requiring enzyme 1), PERK (Protein kinase RNA-activated (PKR)-like ER kinase), and ATF6 (Activating transcription factor 6)—modulate the gene expression pattern through increased expression of chaperones and restore ER homeostasis by enhancing ER protein folding capacity. The liver is a central organ which performs a variety of functions which help in maintaining the overall well-being of our body. The liver plays many roles in cellular physiology, blood homeostasis, and detoxification, and is the main site at which protein synthesis occurs. Disturbance in ER homeostasis is triggered by calcium level imbalance, change in redox status, viral infection, and so on. ER dysfunction and subsequent UPR signaling participate in various hepatic disorders like metabolic (dysfunction) associated fatty liver disease, liver cancer, viral hepatitis, and cholestasis. The exact role of ER stress and UPR signaling in various liver diseases is not fully understood and needs further investigation. Targeting UPR signaling with drugs is the subject of intensive research for therapeutic use in liver diseases. The present review summarizes the role of UPR signaling in liver disorders and describes why UPR regulators are promising therapeutic targets

Analysis of post-transcriptional regulation using the FunREG method

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Role of Glycanation and Convertase Maturation of Soluble Glypican-3 in Inhibiting Proliferation of Hepatocellular Carcinoma Cells

Glypican 3 (GPC3) is a complex heparan sulfate proteoglycan associated with the outer surface of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. It is also N-glycosylated and processed by a furin-like convertase. GPC3 has numerous biological functions. Although GPC3 is undetectable in normal liver tissue, it is abnormally and highly overexpressed in hepatocellular carcinoma (HCC). Interestingly, proliferation of HCC cells such as HepG2 and HuH7 is inhibited when they express a soluble form of GPC3 after lentiviral transduction. To obtain more insight into the role of some of its post-translational modifications, we designed a mutant GPC3, sGPC3m, without its GPI anchor, convertase cleavage site, and glycosaminoglycan chains. The highly pure sGPC3m protein strongly inhibited HuH7 and HepG2 cell proliferation in vitro and induced a significant increase in their cell doubling time. It changed the morphology of HuH7 cells but not that of HepG2. It induced the enlargement of HuH7 cell nuclear area and the restructuration of adherent cell junctions. Unexpectedly, for both cell types, the levels of apoptosis, cell division, and beta-catenin were not altered by sGPC3m, although growth inhibition was very efficient. Overall, our data show that glycanation and convertase maturation are not required for sGPC3m to inhibit HCC cell proliferation

New insights into diagnosis and therapeutic options for proliferative hepatoblastoma

International audienceSurgery and cisplatin-based treatment of hepatoblastoma (HB) currently guarantee the survival of 70-80% of patients. However, some important challenges remain in diagnosing high risk tumors and identifying relevant targetable pathways offering new therapeutic avenues. Previously, two molecular subclasses of hepatoblastoma tumors have been described, namely C1 and C2; C2 being the subgroup with the poorest prognosis, a more advanced tumor stage and the worst overall survival rate. An associated 16-gene signature to discriminate the two tumoral subgroups was proposed but it has not been transferred into clinical routine. To address these issues we performed RNA sequencing of 25 tumors and matched normal liver samples from patients. The transcript profiling separated HB into three distinct subgroups named C1, C2A and C2B, identifiable by a concise four-gene signature: HSD17B6, ITGA6, TOP2A and VIM, with TOP2A being characteristic for the proliferative C2A tumors. Differential expression of these genes was confirmed by RT-qPCR on an expanded cohort and by immunohistochemistry. We also revealed significant overexpression of genes involved in Fanconi Anemia (FA) pathway in the C2A subgroup. We then investigated the ability of several described FA inhibitors to block growth of HB cells in vitro and in vivo. We demonstrated that bortezomib, an FDA-approved proteasome inhibitor, strongly impairs the proliferation and survival of HB cell lines in vitro, blocks FA pathway associated double-strand DNA repair and significantly impedes HB growth in vivo. In conclusion, the highly proliferating C2A subtype is characterized by TOP2A gene up-regulation and FA pathway activation and HB therapeutic arsenal could include Bortezomib for the treatment of patients with the most aggressive tumors. This article is protected by copyright. All rights reserved

DLK1/DIO3 locus upregulation by a β-catenin-dependent enhancer drives cell proliferation and liver tumorigenesis

International audienceThe CTNNB1 gene, encoding β-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained β-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (β-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/β-catenin complexes in an open conformation upon sustained β-catenin activation (DLK1-WRE) and increasing DLK1/DIO3 locus transcription in β-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR/Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and β-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case, or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during β-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations