[Zinc finger protein-36 deficiency inhibits osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells and preosteoblasts by activating the ERK/MAPK pathway].

OBJECTIVE: To explore the role of zinc finger protein 36(ZFP36) in regulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and preosteoblasts. METHODS: ZFP36 expression was observed in primary mouse BMSCs and mouse preosteoblasts (MC3T3-E1 cells) during induced osteogenic differentiation. Zfp36-deficient cell models were constructed in the two cells using RNA interference technique and the changes in differentiation capacities of the transfected cells into osteoblasts were observed. Transcriptome sequencing was used to investigate the potential mechanisms of ZFP36 for regulating osteoblast differentiation of the two cells. U0126, a ERK/MAPK signal suppressor, was used to verify the regulatory mechanism of Zfp36 in osteogenic differentiation of Zfp36-deficient cells. RESULTS: During the 14-day induction of osteogenic differentiation, both mouse BMSCs and MC3T3-E1 cells exhibited increased expression of ZFP36, and its mRNA expression reached the peak level on Day 7(P < 0.0001). The Zfp36-deficient cell models showed reduced intensity of alkaline phosphatase (ALP) staining and alizarin red staining with significantly lowered expressions of the osteogenic marker genes including Alpl, Sp7, Bglap and Ibsp (P < 0.01). Transcriptome sequencing verified the reduction of bone mineralization-related gene expressions in Zfp36-deficient cells and indicated the involvement of ERK signaling in the potential regulatory mechanism of Zfp36. Immunoblotting showed that pERK protein expression increased significantly in Zfp36-deficient cells compared with the control cells. In Zfp36-deficient MC3T3-E1 cells, inhibition of activated ERK/MAPK signaling with U0126 resulted in obviously enhanced ALP staining and significantly increased expressions of osteoblast differentiation markers Runx2 and Bglap (P < 0.05). CONCLUSIONS: ZFP36 is involved in the regulation of osteoblast differentiation of mouse BMSCs and preosteoblasts, and ZFP36 deficiency causes inhibition of osteoblast differentiation of the cells by activating the ERK/MAPK signaling pathway.

Distinct microRNA Signature and Suppression of ZFP36L1 Define ASCL1-Positive Lung Adenocarcinoma.

Achaete-scute family bHLH transcription factor 1 (ASCL1) is a master transcription factor involved in neuroendocrine differentiation. ASCL1 is expressed in approximately 10% of lung adenocarcinomas (LUAD) and exerts tumor-promoting effects. Here, we explored miRNA profiles in ASCL1-positive LUADs and identified several miRNAs closely associated with ASCL1 expression, including miR-375, miR-95-3p/miR-95-5p, miR-124-3p, and members of the miR-17∼92 family. Similar to small cell lung cancer, Yes1 associated transcriptional regulator (YAP1), a representative miR-375 target gene, was suppressed in ASCL1-positive LUADs. ASCL1 knockdown followed by miRNA profiling in a cell culture model further revealed that ASCL1 positively regulates miR-124-3p and members of the miR-17∼92 family. Integrative transcriptomic analyses identified ZFP36 ring finger protein like 1 (ZFP36L1) as a target gene of miR-124-3p, and IHC studies demonstrated that ASCL1-positive LUADs are associated with low ZFP36L1 protein levels. Cell culture studies showed that ectopic ZFP36L1 expression inhibits cell proliferation, survival, and cell-cycle progression. Moreover, ZFP36L1 negatively regulated several genes including E2F transcription factor 1 (E2F1) and snail family transcriptional repressor 1 (SNAI1). In conclusion, our study revealed that suppression of ZFP36L1 via ASCL1-regulated miR-124-3p could modulate gene expression, providing evidence that ASCL1-mediated regulation of miRNAs shapes molecular features of ASCL1-positive LUADs. IMPLICATIONS: Our study revealed unique miRNA profiles of ASCL1-positive LUADs and identified ASCL1-regulated miRNAs with functional relevance.

Simplified algorithm for genetic subtyping in diffuse large B-cell lymphoma.

Genetic classification helps to disclose molecular heterogeneity and therapeutic implications in diffuse large B-cell lymphoma (DLBCL). Using whole exome/genome sequencing, RNA-sequencing, and fluorescence in situ hybridization in 337 newly diagnosed DLBCL patients, we established a simplified 38-gene algorithm (termed 'LymphPlex') based on the information on mutations of 35 genes and rearrangements of three genes (BCL2, BCL6, and MYC), identifying seven distinct genetic subtypes: TP53Mut (TP53 mutations), MCD-like (MYD88, CD79B, PIM1, MPEG1, BTG1, TBL1XR1, PRDM1, IRF4 mutations), BN2-like (BCL6 fusion, NOTCH2, CD70, DTX1, BTG2, TNFAIP3, CCND3 mutations), N1-like (NOTCH1 mutations), EZB-like (BCL2 fusion, EZH2, TNFRSF14, KMT2D, B2M, FAS, CREBBP, ARID1A, EP300, CIITA, STAT6, GNA13 mutations, with or without MYC rearrangement), and ST2-like (SGK1, TET2, SOCS1, DDX3X, ZFP36L1, DUSP2, STAT3, IRF8 mutations). Extended validation of 1001 DLBCL patients revealed clinical relevance and biological signature of each genetic subtype. TP53Mut subtype showed poor prognosis, characterized by p53 signaling dysregulation, immune deficiency, and PI3K activation. MCD-like subtype was associated with poor prognosis, activated B-cell (ABC) origin, BCL2/MYC double-expression, and NF-κB activation. BN2-like subtype showed favorable outcome within ABC-DLBCL and featured with NF-κB activation. N1-like and EZB-like subtypes were predominated by ABC-DLBCL and germinal center B-cell (GCB)-DLBCL, respectively. EZB-like-MYC+ subtype was characterized by an immunosuppressive tumor microenvironment, while EZB-like-MYC- subtype by NOTCH activation. ST2-like subtype showed favorable outcome within GCB-DLBCL and featured with stromal-1 modulation. Genetic subtype-guided targeted agents achieved encouraging clinical response when combined with immunochemotherapy. Collectively, LymphPlex provided high efficacy and feasibility, representing a step forward to the mechanism-based targeted therapy in DLBCL.

Time-dependent regulation of cytokine production by RNA binding proteins defines T cell effector function.

Potent T cell responses against infections and malignancies require a rapid yet tightly regulated production of toxic effector molecules. Their production level is defined by post-transcriptional events at 3' untranslated regions (3' UTRs). RNA binding proteins (RBPs) are key regulators in this process. With an RNA aptamer-based capture assay, we identify >130 RBPs interacting with IFNG, TNF, and IL2 3' UTRs in human T cells. RBP-RNA interactions show plasticity upon T cell activation. Furthermore, we uncover the intricate and time-dependent regulation of cytokine production by RBPs: whereas HuR supports early cytokine production, ZFP36L1, ATXN2L, and ZC3HAV1 dampen and shorten the production duration, each at different time points. Strikingly, even though ZFP36L1 deletion does not rescue the dysfunctional phenotype, tumor-infiltrating T cells produce more cytokines and cytotoxic molecules, resulting in superior anti-tumoral T cell responses. Our findings thus show that identifying RBP-RNA interactions reveals key modulators of T cell responses in health and disease.

[ZFP36L1 inhibits proliferation, invasion and migration of human breast cancer cells by inducing epithelial-mesenchymal transition through STAT3/p-STAT3 signaling pathway].

Objective To explore the role and mechanism of zinc finger protein 36 like 1 (ZFP36L1) in breast cancer. Methods Sixty breast cancer patients were enrolled in the study. Immunohistochemistry was performed to evaluate the ZFP36L1 expression. Clinicopathological parameters were observed. MCF-7 cells were transfected with overexpressed ZFP36L1 plasmid. The viability of MCF-7 cells was assayed by the 5-ethynyl-2-deoxyuridine (EdU) and MTS assay. The invasion of MCF-7 cells was assessed by TranswellTM assay. Western blot analysis was used to detect the expression of ß-catenin, vimentin, E-cadherin, signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3 (p-STAT3). Results ZFP36L1-low expression has been found to be associated with poor prognosis in patients with breast cancer. Moreover, ZFP36L1 overexpression inhibited cell proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) in vitro. Accordingly, the expression of STAT3 and p-STAT3 increased significantly. Conclusion ZFP36L1, as a cancer suppressor gene, inhibits cell proliferation, invasion, and migration through EMT and STAT3 signaling pathway.

ZFP36 Family Members Regulate the Proinflammatory Features of Psoriatic Dermal Fibroblasts.

Dermal fibroblasts are strategically positioned underneath the basal epidermis layer to support keratinocyte proliferation and extracellular matrix production. In inflammatory conditions, these fibroblasts produce cytokines and chemokines that promote the chemoattraction of immune cells into the dermis and the hyperplasia of the epidermis, two characteristic hallmarks of psoriasis. However, how dermal fibroblasts specifically contribute to psoriasis development remains largely uncharacterized. In this study, we investigated through which cytokines and signaling pathways dermal fibroblasts contribute to the inflammatory features of psoriatic skin. We show that dermal fibroblasts from lesional psoriatic skin are important producers of inflammatory mediators, including IL-6, CXCL8, and CXCL2. This increased cytokine production was found to be regulated by ZFP36 family members ZFP36, ZFP36L1, and ZFP36L2, RNA-binding proteins with mRNA-degrading properties. In addition, the expression of ZFP36 family proteins was found to be reduced in chronic inflammatory conditions that mimic psoriatic lesional skin. Collectively, these results indicate that dermal fibroblasts are important producers of cytokines in psoriatic skin and that reduced expression of ZFP36 members in psoriasis dermal fibroblasts contributes to their inflammatory phenotype.

Functional screen of inflammatory bowel disease genes reveals key epithelial functions.

BACKGROUND: Genetic studies have been tremendously successful in identifying genomic regions associated with a wide variety of phenotypes, although the success of these studies in identifying causal genes, their variants, and their functional impacts has been more limited. METHODS: We identified 145 genes from IBD-associated genomic loci having endogenous expression within the intestinal epithelial cell compartment. We evaluated the impact of lentiviral transfer of the open reading frame (ORF) of these IBD genes into the HT-29 intestinal epithelial cell line via transcriptomic analyses. By comparing the genes in which expression was modulated by each ORF, as well as the functions enriched within these gene lists, we identified ORFs with shared impacts and their putative disease-relevant biological functions. RESULTS: Analysis of the transcriptomic data for cell lines expressing the ORFs for known causal genes such as HNF4a, IFIH1, and SMAD3 identified functions consistent with what is already known for these genes. These analyses also identified two major clusters of genes: Cluster 1 contained the known IBD causal genes IFIH1, SBNO2, NFKB1, and NOD2, as well as genes from other IBD loci (ZFP36L1, IRF1, GIGYF1, OTUD3, AIRE and PITX1), whereas Cluster 2 contained the known causal gene KSR1 and implicated DUSP16 from another IBD locus. Our analyses highlight how multiple IBD gene candidates can impact on epithelial structure and function, including the protection of the mucosa from intestinal microbiota, and demonstrate that DUSP16 acts a regulator of MAPK activity and contributes to mucosal defense, in part via its regulation of the polymeric immunoglobulin receptor, involved in the protection of the intestinal mucosa from enteric microbiota. CONCLUSIONS: This functional screen, based on expressing IBD genes within an appropriate cellular context, in this instance intestinal epithelial cells, resulted in changes to the cell's transcriptome that are relevant to their endogenous biological function(s). This not only helped in identifying likely causal genes within genetic loci but also provided insight into their biological functions. Furthermore, this work has highlighted the central role of intestinal epithelial cells in IBD pathophysiology, providing a scientific rationale for a drug development strategy that targets epithelial functions in addition to the current therapies targeting immune functions.

ZFP36L1 plays an ambiguous role in the regulation of cell expansion and negatively regulates CDKN1A in chronic myeloid leukemia cells.

The mRNA-destabilizing proteins ZFP36L1 and ZFP36L2 are described as mediators of quiescence and play a pivotal role in hematopoietic malignancies. Both genes are mainly classified as tumor suppressor genes as they posttranscriptionally downregulate the expression of oncogenes and contribute to cellular quiescence. Here, we analyzed the role of ZFP36L1 and ZFP36L2 in chronic myeloid leukemia (CML). We found ZFP36L1 and ZFP36L2 expression to be deregulated in patients with CML. By use of in vitro models of tyrosine kinase inhibitor resistance, an increase in ZFP36L1 and ZFP36L2 expression was detected during the development of imatinib resistance. CRISPR/Cas9-derived knockout of ZFP36L1, but not of ZFP36L2, in imatinib-sensitive cells led to decreased proliferation rates in response to tyrosine kinase inhibitor treatment. This effect was also observed in untreated ZFP36L1 knockout cells, albeit to a lower extent. Genomewide gene expression analyses of ZFP36L1 knockout cells revealed differential expression of cell cycle regulators, in particular upregulation of the cell cycle inhibitor CDKN1A. In addition, the 3' untranslated region of CDKN1A was proven to be a direct target of ZFP36L1. This indicates that tumor suppressor genes can also be targeted by ZFP36L1. Hence, ZFP36L1 cannot unambiguously be regarded as a tumor suppressor gene.

Comprehensive integrative profiling of upper tract urothelial carcinomas.

BACKGROUND: Crosstalk between genetic, epigenetic, and immune alterations in upper tract urothelial carcinomas and their role in shaping muscle invasiveness and patient outcome are poorly understood. RESULTS: We perform an integrative genome- and methylome-wide profiling of diverse non-muscle-invasive and muscle-invasive upper tract urothelial carcinomas. In addition to mutations of FGFR3 and KDM6A, we identify ZFP36L1 as a novel, significantly mutated tumor suppressor gene. Overall, mutations of ZFP36 family genes (ZFP36, ZFP36L1, and ZFP36L2) are identified in 26.7% of cases, which display a high mutational load. Unsupervised DNA methylation subtype classification identifies two epi-clusters associated with distinct muscle-invasive status and patient outcome, namely, EpiC-low and EpiC-high. While the former is hypomethylated, immune-depleted, and enriched for FGFR3-mutated, the latter is hypermethylated, immune-infiltrated, and tightly associated with somatic mutations of SWI/SNF genes. CONCLUSIONS: Our study delineates for the first time the key role for convergence between genetic and epigenetic alterations in shaping clinicopathological and immune upper tract urothelial carcinoma features.

Efficient homing of antibody-secreting cells to the bone marrow requires RNA-binding protein ZFP36L1.

Cell migration relies on coordinated activity of chemotactic and guidance receptors. Here, we report a specific role for the RNA-binding protein ZFP36L1 in limiting the abundance of molecules involved in the homing of antibody-secreting cells (ASCs) to the bone marrow (BM). In the absence of ZFP36L1, ASCs build up in the spleen and the liver and show diminished accumulation in the BM. ZFP36L1 facilitates migration by directly regulating G protein-coupled receptor kinase 2 (GRK2) and the integrin chains α4 and ß1 in splenic ASCs. Expression of CXCR4 and of the integrins α4 and ß1 is differentially regulated on ASCs produced at the early and late stages of the immune response. Consequently, deletion of the Zfp36l1 gene has a stronger effect on BM accumulation of high-affinity ASCs formed late in the response. Thus, ZFP36L1 is an integral part of the regulatory network controlling gene expression during ASC homing.

Mitogen- and Stress-Activated Protein Kinase 1 Mediates Alcohol-Upregulated Transcription of Brf1 and tRNA Genes to Cause Phenotypic Alteration.

Upregulation of Brf1 (TFIIB-related factor 1) and Pol III gene (RNA polymerase III-dependent gene, such as tRNAs and 5S rRNA) activities is associated with cell transformation and tumor development. Alcohol intake causes liver injury, such as steatosis, inflammation, fibrosis, and cirrhosis, which enhances the risk of HCC development. However, the mechanism of alcohol-promoted HCC remains to be explored. We have designed the complementary research system, which is composed of cell lines, an animal model, human samples, and experiments in vivo and in vitro, to carry out this project by using molecular biological, biochemical, and cellular biological approaches. It is a unique system to explore the mechanism of alcohol-associated HCC. Our results indicate that alcohol upregulates Brf1 and Pol III gene (tRNAs and 5S rRNA) transcription in primary mouse hepatocytes, immortalized mouse hepatocyte-AML-12 cells, and engineered human HepG2-ADH cells. Alcohol activates MSK1 to upregulate expression of Brf1 and Pol III genes, while inhibiting MSK1 reduces transcription of Brf1 and Pol III genes in alcohol-treated cells. The inhibitor of MSK1, SB-747651A, decreases the rates of cell proliferation and colony formation. Alcohol feeding promotes liver tumor development of the mouse. These results, for the first time, show the identification of the alcohol-response promoter fragment of the Pol III gene key transcription factor, Brf1. Our studies demonstrate that Brf1 expression is elevated in HCC tumor tissues of mice and humans. Alcohol increases cellular levels of Brf1, resulting in enhancement of Pol III gene transcription in hepatocytes through MSK1. Our mechanism analysis has demonstrated that alcohol-caused high-response fragment of the Brf1 promoter is at p-382/+109bp. The MSK1 inhibitor SB-747651A is an effective reagent to repress alcohol-induced cell proliferation and colony formation, which is a potential pharmaceutical agent. Developing this inhibitor as a therapeutic approach will benefit alcohol-associated HCC patients.

Altered pathways in methylome and transcriptome longitudinal analysis of normal weight and bariatric surgery women.

DNA methylation could provide a link between environmental, genetic factors and weight control and can modify gene expression pattern. This study aimed to identify genes, which are differentially expressed and methylated depending on adiposity state by evaluating normal weight women and obese women before and after bariatric surgery (BS). We enrolled 24 normal weight (BMI: 22.5 ± 1.6 kg/m2) and 24 obese women (BMI: 43.3 ± 5.7 kg/m2) submitted to BS. Genome-wide methylation analysis was conducted using Infinium Human Methylation 450 BeadChip (threshold for significant CpG sites based on delta methylation level with a minimum value of 5%, a false discovery rate correction (FDR) of q < 0.05 was applied). Expression levels were measured using HumanHT-12v4 Expression BeadChip (cutoff of p ≤ 0.05 and fold change ≥2.0 was used to detect differentially expressed probes). The integrative analysis of both array data identified four genes (i.e. TPP2, PSMG6, ARL6IP1 and FAM49B) with higher methylation and lower expression level in pre-surgery women compared to normal weight women: and two genes (i.e. ZFP36L1 and USP32) that were differentially methylated after BS. These methylation changes were in promoter region and gene body. All genes are related to MAPK cascade, NIK/NF-kappaB signaling, cellular response to insulin stimulus, proteolysis and others. Integrating analysis of DNA methylation and gene expression evidenced that there is a set of genes relevant to obesity that changed after BS. A gene ontology analysis showed that these genes were enriched in biological functions related to adipogenesis, orexigenic, oxidative stress and insulin metabolism pathways. Also, our results suggest that although methylation plays a role in gene silencing, the majority of effects were not correlated.

RNA-Binding Protein ZFP36L1 Suppresses Hypoxia and Cell-Cycle Signaling.

ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved adenylate-uridylate-rich elements (ARE) located in 3'untranslated regions (UTR) to mediate mRNA decay. We hypothesized that ZFP36L1 is a negative regulator of a posttranscriptional hub involved in mRNA half-life regulation of cancer-related transcripts. Analysis of in silico data revealed that ZFP36L1 was significantly mutated, epigenetically silenced, and downregulated in a variety of cancers. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo, whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wild-type and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells {plus minus} tet-on ZFP36L1 was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1. These targets included a number of key oncogenic transcripts such as HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3'UTRs of these targets for mRNA degradation, thus suppressing their expression. Dual luciferase reporter assays and RNA electrophoretic mobility shift assays showed that wild-type, but not zinc-finger mutant ZFP36L1, bound to HIF1A 3'UTR and mediated HIF1A mRNA degradation, leading to reduced expression of HIF1A and its downstream targets. Collectively, our findings reveal an indispensable role of ZFP36L1 as a posttranscriptional safeguard against aberrant hypoxic signaling and abnormal cell-cycle progression. SIGNIFICANCE: RNA-binding protein ZFP36L1 functions as a tumor suppressor by regulating the mRNA stability of a number of mRNAs involved in hypoxia and cell-cycle signaling.

Single-Cell Transcriptomics Uncovers Glial Progenitor Diversity and Cell Fate Determinants during Development and Gliomagenesis.

The identity and degree of heterogeneity of glial progenitors and their contributions to brain tumor malignancy remain elusive. By applying lineage-targeted single-cell transcriptomics, we uncover an unanticipated diversity of glial progenitor pools with unique molecular identities in developing brain. Our analysis identifies distinct transitional intermediate states and their divergent developmental trajectories in astroglial and oligodendroglial lineages. Moreover, intersectional analysis uncovers analogous intermediate progenitors during brain tumorigenesis, wherein oligodendrocyte-progenitor intermediates are abundant, hyper-proliferative, and progressively reprogrammed toward a stem-like state susceptible to further malignant transformation. Similar actively cycling intermediate progenitors are prominent components in human gliomas with distinct driver mutations. We further unveil lineage-driving networks underlying glial fate specification and identify Zfp36l1 as necessary for oligodendrocyte-astrocyte lineage transition and glioma growth. Together, our results resolve the dynamic repertoire of common and divergent glial progenitors during development and tumorigenesis and highlight Zfp36l1 as a molecular nexus for balancing glial cell-fate decision and controlling gliomagenesis.

Brf1 loss and not overexpression disrupts tissues homeostasis in the intestine, liver and pancreas.

RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut.

Epigenomic profiling of myelofibrosis reveals widespread DNA methylation changes in enhancer elements and ZFP36L1 as a potential tumor suppressor gene that is epigenetically regulated.

In this study we interrogated the DNA methylome of myelofibrosis patients using high-density DNA methylation arrays. We detected 35,215 differentially methylated CpG, corresponding to 10,253 genes, between myelofibrosis patients and healthy controls. These changes were present both in primary and secondary myelofibrosis, which showed no differences between them. Remarkably, most differentially methylated CpG were located outside gene promoter regions and showed significant association with enhancer regions. This aberrant enhancer hypermethylation was negatively correlated with the expression of 27 genes in the myelofibrosis cohort. Of these, we focused on the ZFP36L1 gene and validated its decreased expression and enhancer DNA hypermethylation in an independent cohort of patients and myeloid cell-lines. In vitro reporter assay and 5'-azacitidine treatment confirmed the functional relevance of hyper-methylation of ZFP36L1 enhancer. Furthermore, in vitro rescue of ZFP36L1 expression had an impact on cell proliferation and induced apoptosis in SET-2 cell line indicating a possible role of ZFP36L1 as a tumor suppressor gene in myelofibrosis. Collectively, we describe the DNA methylation profile of myelofibrosis, identifying extensive changes in enhancer elements and revealing ZFP36L1 as a novel candidate tumor suppressor gene.

RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family.

Osteoarthritis (OA) is a whole-joint disease characterized by cartilage destruction and other whole-joint pathological changes. There is currently no effective disease-modifying therapy. Here we investigate the post-transcriptional mRNA regulation of OA-modulating proteins in chondrocytes and show that the ZFP36 family member, ZFP36L1, is specifically upregulated in OA chondrocytes and OA cartilage of humans and mice. Adenovirus-mediated overexpression of ZFP36L1 alone in mouse knee-joint tissue does not modulate OA pathogenesis. However, genetic ablation or silencing of Zfp36l1 significantly abrogates experimental OA in mice. Knockdown of Zfp36l1 increases the mRNA expression of two heat shock protein 70 (HSP70) family members, which act as its direct targets. Furthermore, overexpression of HSPA1A in joint tissues protects mice against experimental OA by inhibiting chondrocyte apoptosis. Our results indicate that the RNA-binding protein, ZFP36L1, regulates HSP70 family members that appear to protect against OA pathogenesis by inhibiting chondrocyte apoptosis.

Levamisole suppresses adipogenesis of aplastic anaemia-derived bone marrow mesenchymal stem cells through ZFP36L1-PPARGC1B axis.

Aplastic anaemia (AA) is a life-threatening hematopoietic disorder characterized by hypoplasia and pancytopenia with increasing fat cells in the bone marrow (BM). The BM-derived mesenchymal stem cells (MSCs) from AA are more susceptible to be induced into adipogenic differentiation compared with that from control, which may be causatively associated with the fatty BM and defective hematopoiesis of AA. Here in this study, we first demonstrated that levamisole displayed a significant suppressive effect on the in vitro adipogenic differentiation of AA BM-MSCs. Mechanistic investigation revealed that levamisole could increase the expression of ZFP36L1 which was subsequently demonstrated to function as a negative regulator of adipogenic differentiation of AA BM-MSCs through lentivirus-mediated ZFP36L1 knock-down and overexpression assay. Peroxisome proliferator-activated receptor gamma coactivator 1 beta (PPARGC1B) whose 3'-untranslated region bears adenine-uridine-rich elements was verified as a direct downstream target of ZFP36L1, and knock-down of PPARGC1B impaired the adipogenesis of AA BM-MSCs. Collectively, our work demonstrated that ZFP36L1-mediated post-transcriptional control of PPARGC1B expression underlies the suppressive effect of levamisole on the adipogenic differentiation of AA BM-MSCs, which not only provides novel therapeutic targets for alleviating the BM fatty phenomenon of AA patients, but also lays the theoretical and experimental foundation for the clinical application of levamisole in AA therapy.

Systematic-analysis of mRNA expression profiles in skeletal muscle of patients with type II diabetes: The glucocorticoid was central in pathogenesis.

Since the past 30 years, the prevalence of diabetes has more than doubled, making it an urgent challenge globally. We carried out systematic analysis with the public data of mRNA expression profiles in skeletal muscle to study the pathogenesis, since insulin resistance in the skeletal muscle is an early feature. We utilized three GEO datasets, containing total 60 cases and 63 normal samples. After the background removal, R package QC was utilized to finish the preprocessing of datasets. We obtained a dataset containing 2481 genes and 123 samples after the preprocessing. Quantitative quality control measures were calculated to represent the quality of these datasets. MetaDE package provides functions for conducting different systematic analysis methods for differential expression analysis. The GO term enrichment was carried out using PANTHER. Protein-protein interactions, drug-gene interactions, and genetic association of the identified differentially expressed genes were analyzed using STRING v10.0 online tool, DGIdb, and the Genetic Association Database, respectively. The datasets had good performances on IQC and EQC, which suggested that the datasets had good internal and external quality. Totally 96 differentially expressed genes were detected using 0.01 as cutoff of AW. The enriched GO terms were mainly associated with the response to glucocorticoid. There were seven genes involving in the gluconeogenesis were differentially expressed, which might be the potential treatment target for this disease. The closely connected networks and potential targets of existed drugs suggested that some of the drugs might be applied to the treatment of diabetes as well.

Post-transcriptional regulation of BCL2 mRNA by the RNA-binding protein ZFP36L1 in malignant B cells.

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3' untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the 'maximum information coefficient' (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3' untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3' untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.