Molecular mechanism of actin filament elongation by formins.

Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action remain unclear. In this work, we present high-resolution cryo-electron microscopy structures of F-actin barbed ends bound by three distinct formins, revealing a common asymmetric formin conformation imposed by the filament. Formation of new intersubunit contacts during actin polymerization sterically displaces formin and triggers its translocation. This "undock-and-lock" mechanism explains how actin-filament growth is coordinated with formin movement. Filament elongation speeds are controlled by the positioning and stability of actin-formin interfaces, which distinguish fast and slow formins. Furthermore, we provide a structure of the actin-formin-profilin ring complex, which resolves how profilin is rapidly released from the barbed end during filament elongation.

Altered Fhod3 expression involved in progressive high-frequency hearing loss via dysregulation of actin polymerization stoichiometry in the cuticular plate.

Age-related hearing loss (ARHL) is a common sensory impairment with complex underlying mechanisms. In our previous study, we performed a meta-analysis of genome-wide association studies (GWAS) in mice and identified a novel locus on chromosome 18 associated with ARHL specifically linked to a 32 kHz tone burst stimulus. Consequently, we investigated the role of Formin Homology 2 Domain Containing 3 (Fhod3), a newly discovered candidate gene for ARHL based on the GWAS results. We observed Fhod3 expression in auditory hair cells (HCs) primarily localized at the cuticular plate (CP). To understand the functional implications of Fhod3 in the cochlea, we generated Fhod3 overexpression mice (Pax2-Cre+/-; Fhod3Tg/+) (TG) and HC-specific conditional knockout mice (Atoh1-Cre+/-; Fhod3fl/fl) (KO). Audiological assessments in TG mice demonstrated progressive high-frequency hearing loss, characterized by predominant loss of outer hair cells, and a decreased phalloidin intensities of CP. Ultrastructural analysis revealed loss of the shortest row of stereocilia in the basal turn of the cochlea, and alterations in the cuticular plate surrounding stereocilia rootlets. Importantly, the hearing and HC phenotype in TG mice phenocopied that of the KO mice. These findings suggest that balanced expression of Fhod3 is critical for proper CP and stereocilia structure and function. Further investigation of Fhod3 related hearing impairment mechanisms may lend new insight towards the myriad mechanisms underlying ARHL, which in turn could facilitate the development of therapeutic strategies for ARHL.

Integrated analysis of transcriptome and genome variations in pediatric T cell acute lymphoblastic leukemia: data from north Indian tertiary care center.

INTRODUCTION: T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with poor prognosis and inferior outcome. Although multiple studies have been perform on genomics of T-ALL, data from Indian sub-continent is scarce. METHODS: In the current study we aimed to identify the genetic variability of T-ALL in an Indian cohort of pediatric (age ≤ 12 years) T-ALL patients (n = 25) by whole transcriptome sequencing along with whole exome sequencing and correlated the findings with clinical characteristics and disease outcome. RESULTS: The median age was 7 years (range 3 -12 years). RNA sequencing revealed a definitive fusion event in 14 cases (56%) (including a novel fusions) with STIL::TAL1 in 4 (16%), followed by NUP21::ABL1, TCF7::SPI1, ETV6::HDAC8, LMO1::RIC3, DIAPH1::JAK2, SETD2::CCDC12 and RCBTB2::LPAR6 in 1 (4%) case each. Significant aberrant expression was noted in RAG1 (64%), RAG2 (80%), MYCN (52%), NKX3-1 (52%), NKX3-2 (32%), TLX3 (28%), LMO1 (20%) and MYB (16%) genes. WES data showed frequent mutations in NOTCH1 (35%) followed by WT1 (23%), FBXW7 (12%), KRAS (12%), PHF6 (12%) and JAK3 (12%). Nearly 88.2% of cases showed a deletion of CDKN2A/CDKN2B/MTAP genes. Clinically significant association of a better EFS and OS (p=0.01) was noted with RAG2 over-expression at a median follow up of 22 months, while a poor EFS (p=0.041) and high relapse rate (p=0.045) was observed with MYB over-expression. CONCLUSION: Overall, the present study demonstrates the frequencies of transcriptomic and genetic alterations from Indian cohort of pediatric T-ALL and is a salient addition to current genomics data sets available in T-ALL.

Global analysis of urinary extracellular vesicle small RNAs in autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent monogenic renal disease progressing to end-stage renal disease. There is a pressing need for the identification of early ADPKD biomarkers to enable timely intervention and the development of effective therapeutic approaches. Here, we profiled human urinary extracellular vesicles small RNAs by small RNA sequencing in patients with ADPKD and compared their differential expression considering healthy control individuals to identify dysregulated small RNAs and analyze downstream interaction to gain insight about molecular pathophysiology. METHODS: This is a cross-sectional study where urine samples were collected from a total of 23 PKD1-ADPKD patients and 28 healthy individuals. Urinary extracellular vesicles were purified, and small RNA was isolated and sequenced. Differentially expressed Small RNA were identified and functional enrichment analysis of the critical miRNAs was performed to identify driver genes and affected pathways. RESULTS: miR-320b, miR-320c, miR-146a-5p, miR-199b-3p, miR-671-5p, miR-1246, miR-8485, miR-3656, has_piR_020497, has_piR_020496 and has_piR_016271 were significantly upregulated in ADPKD patient urine extracellular vesicles and miRNA-29c was significantly downregulated. Five 'driver' target genes (FBRS, EDC3, FMNL3, CTNNBIP1 and KMT2A) were identified. CONCLUSIONS: The findings of the present study make significant contributions to the understanding of ADPKD pathogenesis and to the identification of novel biomarkers and potential drug targets aimed at slowing disease progression in ADPKD.

Establishing a human-induced pluripotent stem cell line (SMUSHi003-A) from a patient with Charcot-Marie-Tooth disease and focal segmental glomerulosclerosis.

INF2 mutations cause Charcot-Marie-Tooth disease (CMT), and /or focal segmental glomerulosclerosis (FSGS) in an autosomal dominant inheritance mode, whose underlying mechanism remainsunclear. Here, we report the generation of an iPSC line from a female patient with CMT and FSGS. The iPSC line from the patient's PBMCscarried aheterozygous INF2 deletion mutation (c.315_323delGCGCGCCGT) within the conserved E2. This line exhibited a normal karyotype, high expression of pluripotency markers, and trilineage differentiation potential. This line can be used to dissect the complex pathomechanism through further induction of differentiation into related cells and as a drug screening tool for INF2-associated diseases.

Filopodial protrusion driven by density-dependent Ena-TOCA-1 interactions.

Filopodia are narrow actin-rich protrusions with important roles in neuronal development where membrane-binding adaptor proteins, such as I-BAR- and F-BAR-domain-containing proteins, have emerged as upstream regulators that link membrane interactions to actin regulators such as formins and proteins of the Ena/VASP family. Both the adaptors and their binding partners are part of diverse and redundant protein networks that can functionally compensate for each other. To explore the significance of the F-BAR domain-containing neuronal membrane adaptor TOCA-1 (also known as FNBP1L) in filopodia we performed a quantitative analysis of TOCA-1 and filopodial dynamics in Xenopus retinal ganglion cells, where Ena/VASP proteins have a native role in filopodial extension. Increasing the density of TOCA-1 enhances Ena/VASP protein binding in vitro, and an accumulation of TOCA-1, as well as its coincidence with Ena, correlates with filopodial protrusion in vivo. Two-colour single-molecule localisation microscopy of TOCA-1 and Ena supports their nanoscale association. TOCA-1 clusters promote filopodial protrusion and this depends on a functional TOCA-1 SH3 domain and activation of Cdc42, which we perturbed using the small-molecule inhibitor CASIN. We propose that TOCA-1 clusters act independently of membrane curvature to recruit and promote Ena activity for filopodial protrusion.

KIF20B and MET, hub genes of DIAPHs, predict poor prognosis and promote pancreatic cancer progression.

BACKGROUND: The DIAPHs (DIAPH1, DIAPH2, and DIAPH3) are members of the diaphanous subfamily of the formin family. KIF20B and MET, hub genes of DIAPHs, play crucial roles in cytoskeletal remodeling, cell migration, and adhesion. However, their combined prognostic and treatment value in pancreatic adenocarcinoma (PC) warrants further investigation. METHODS: Multiomics analysis tools were used to comprehensively assess the genomic expression and prognostic value of KIF20B and MET in PC. Immune cell infiltration, functional enrichment, single-cell RNA-seq (scRNA) analysis, potential therapeutic drugs, and nomograms were established and analyzed. CCK-8 levels, transwell assay, Co-IP assay, mass spectrometry, and western blotting were performed to assess the role of KIF20B and MET as modulators of ß-catenin and Lactate Dehydrogenase A (LDHA) in vitro. Xenograft tumor models were used to evaluate the anti-tumor effects in vivo. RESULTS: DIAPHs, KIF20B, and MET were overexpressed and functioned as poor prognostic markers of PC. Immunoinfiltration analysis revealed that pDC and NK cells were enriched with low expression levels of KIF20B and MET, whereas Th2 cells were enriched with high expression levels of these two genes. The copy number variations (CNVs) in KIF20B and MET were positively correlated with B cell and CD4 + T cell infiltration. Immunological checkpoints NT5E and CD44 were positively correlated with KIF20B and MET expression. Moreover, the nomogram constructed based on KIF20B and MET demonstrated predictive value for overall survival. scRNA-Seq analysis indicated that KIF20B and MET were enriched in endothelial, malignant, B, T, and CD8 + T cells, which correlated with glycolysis and the epithelial-mesenchymal transition (EMT). The interactions of KIF20B and MET with ß-catenin and LDHA were verified by Co-IP assay and mass spectrometry. Knockdown of KIF20B and MET downregulates ß-catenin and LDHA in vitro. Furthermore, dual knockdown of KIF20B and MET exhibited a synergistic suppressive effect on PC progression in vitro and in vivo. CONCLUSION: DIAPHs, KIF20B, and MET are promising candidates for the prognosis and treatment of PC. More importantly, downregulation of KIF20B and MET inhibited pancreatic cancer progression by regulating LDHA and EMT.

Functional genetic variants of the disulfidptosis-related INF2 gene predict survival of hepatitis B virus-related hepatocellular carcinoma.

Disulfidptosis is a novel form of programmed cell death involved in migration and invasion of cancer cells, but few studies investigated the roles of genetic variants in disulfidptosis-related genes in survival of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We used Cox proportional hazards regression analyses, Kaplan-Meier curves and receiver operating characteristic curves to assess effects of genetic variants in 14 disulfidptosis-related genes on overall survival of 866 HBV-HCC patients. The Bayesian false discovery probability was used for multiple testing corrections. We also investigated biological mechanisms of the significant variants through expression quantitative trait loci analyses using the data from publicly available databases, luciferase reporter assays and differential expression analyses. As a result, we identified two independently functional single nucleotide polymorphisms (SNPs) (INF2 rs4072285 G > A and INF2 rs4444271 A > T) that predicted overall survival of HBV-HCC patients, with adjusted hazard ratios of 1.60 (95% CI = 1.22-2.11, P = 0.001) and 1.50 (95% CI = 1.80-1.90, P < 0.001), respectively, after multiple testing correction. Luciferase reporter assays indicated that both INF2 rs4072285 A and INF2 rs4444271 T alleles increased INF2 mRNA expression levels (P < 0.001) that were also higher in HCC tumor tissues than in adjacent normal tissues (P < 0.001); such elevated INF2 expression levels were associated with a poorer survival of HBV-HCC patients (P < 0.001) in the TCGA database. In summary, this study supported that INF2 rs4072285 and INF2 rs4444271 may be novel biomarkers for survival of HBV-HCC patients.

Peripheral thickening of the sarcomeres and pointed end elongation of the thin filaments are both promoted by SALS and its formin interaction partners.

During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-of-function studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.

Using meta-analysis and machine learning to investigate the transcriptional response of immune cells to Leishmania infection.

BACKGROUND: Leishmaniasis is a parasitic disease caused by the Leishmania protozoan affecting millions of people worldwide, especially in tropical and subtropical regions. The immune response involves the activation of various cells to eliminate the infection. Understanding the complex interplay between Leishmania and the host immune system is crucial for developing effective treatments against this disease. METHODS: This study collected extensive transcriptomic data from macrophages, dendritic, and NK cells exposed to Leishmania spp. Our objective was to determine the Leishmania-responsive genes in immune system cells by applying meta-analysis and feature selection algorithms, followed by co-expression analysis. RESULTS: As a result of meta-analysis, we discovered 703 differentially expressed genes (DEGs), primarily associated with the immune system and cellular metabolic processes. In addition, we have substantiated the significance of transcription factor families, such as bZIP and C2H2 ZF, in response to Leishmania infection. Furthermore, the feature selection techniques revealed the potential of two genes, namely G0S2 and CXCL8, as biomarkers and therapeutic targets for Leishmania infection. Lastly, our co-expression analysis has unveiled seven hub genes, including PFKFB3, DIAPH1, BSG, BIRC3, GOT2, EIF3H, and ATF3, chiefly related to signaling pathways. CONCLUSIONS: These findings provide valuable insights into the molecular mechanisms underlying the response of immune system cells to Leishmania infection and offer novel potential targets for the therapeutic goals.

Helical motors and formins synergize to compact chiral filopodial bundles: A theoretical perspective.

Chiral actin bundles have been shown to play an important role in cell dynamics, but our understanding of the molecular mechanisms which combine to generate chirality remains incomplete. To address this, we numerically simulate a crosslinked filopodial bundle under the actions of helical myosin motors and/or formins and examine the collective buckling and twisting of the actin bundle. We first show that a number of proposed mechanisms to buckle polymerizing actin bundles without motor activity fail under biologically-realistic parameters. We then demonstrate that a simplified model of myosin spinning action at the bundle base effectively "braids" the bundle, but cannot control compaction at the fiber tips. Finally, we show that formin-mediated polymerization and motor activity can act synergitically to compact filopodium bundles, as motor activity bends filaments into shapes that activate twist forces induced by formins. Stochastic fluctuations of actin polymerization rates and slower cross linking dynamics both increase buckling and decrease compaction. We discuss implications of our findings for mechanisms of cytoskeletal chirality.

Phosphorylation of INF2 by AMPK promotes mitochondrial fission and oncogenic function in endometrial cancer.

Mitochondria are highly dynamic organelles capable of altering their sizes and shapes to maintain metabolic balance through coordinated fission and fusion processes. In various cancer types, mitochondrial hyperfragmentation has been frequently observed, contributing to the progression of cancer toward metastasis. Inverted formin 2 (INF2), which resides in the endoplasmic reticulum (ER), has been found to accelerate actin polymerization and drive mitochondrial fission. In this study, we demonstrate that INF2 expression is significantly upregulated in endometrial cancer (EC) and is associated with a poor prognosis in EC patients. INF2 promotes anchorage-dependent and independent EC cell growth in part by facilitating mitochondrial fission. Furthermore, in conditions of energy stress, AMP-activated protein kinase (AMPK) phosphorylates INF2 at Ser1077, leading to increased localization of INF2 to the ER and enhanced recruitment of the dynamin-related protein 1 (DRP1) to mitochondria. This AMPK-mediated phosphorylation of INF2 at Ser1077 facilitates mitochondrial division and promotes EC cell growth. Pathological examination using immunohistochemical analyses revealed a positive correlation between AMPK activity and phosphorylated INF2 (Ser1077) in EC specimens. Collectively, our findings uncover novel molecular mechanisms involving the AMPK-INF2 axis, which regulates mitochondrial dynamics and malignant cell growth in EC.

Cooperative actin filament nucleation by the Arp2/3 complex and formins maintains the homeostatic cortical array in Arabidopsis epidermal cells.

Precise control over how and where actin filaments are created leads to the construction of unique cytoskeletal arrays within a common cytoplasm. Actin filament nucleators are key players in this activity and include the conserved actin-related protein 2/3 (Arp2/3) complex as well as a large family of formins. In some eukaryotic cells, these nucleators compete for a common pool of actin monomers and loss of one favors the activity of the other. To test whether this mechanism is conserved, we combined the ability to image single filament dynamics in the homeostatic cortical actin array of living Arabidopsis (Arabidopsis thaliana) epidermal cells with genetic and/or small molecule inhibitor approaches to stably or acutely disrupt nucleator activity. We found that Arp2/3 mutants or acute CK-666 treatment markedly reduced the frequency of side-branched nucleation events as well as overall actin filament abundance. We also confirmed that plant formins contribute to side-branched filament nucleation in vivo. Surprisingly, simultaneous inhibition of both classes of nucleator increased overall actin filament abundance and enhanced the frequency of de novo nucleation events by an unknown mechanism. Collectively, our findings suggest that multiple actin nucleation mechanisms cooperate to generate and maintain the homeostatic cortical array of plant epidermal cells.

Intermediate-effect size p.Arg637Gln in FHOD3 increases risk of HCM and is associated with an aggressive phenotype in homozygous carriers.

Formin homology 2 domain-containing 3 (FHOD3) gene has emerged as one of the main non-sarcomeric genes associated with hypertrophic cardiomyopathy (HCM), but no cases of biallelic variants associated with disease have been described to date. From 2014 until 2021, FHOD3 was evaluated in our center by next-generation sequencing in 22 806 consecutive unrelated probands. The p.Arg637Gln variant in FHOD3 was enriched in our HCM cohort (284 of 9668 probands; 2.94%) compared with internal controls (64 of 11 480; 0.59%) and gnomAD controls (373 of 64 409; 0.58%), with ORs of 5.40 (95% CI: 4.11 to 7.09) and 5.19 (95% CI: 4.44 to 6.07). The variant affects a highly conserved residue localised in a supercoiled alpha helix considered a clustering site for HCM variants, and in heterozygosis can act as a predisposing factor (intermediate-effect variant) for HCM, with an estimated penetrance of around 1%. Additionally, seven homozygous carriers of p.Arg637Gln in FHOD3 were identified. All but one (unaffected) showed an early presentation and a severe HCM phenotype. All this information suggest that p.Arg637Gln variant in FHOD3 is a low-penetrant variant, with an intermediate effect, that contributes to the development of HCM in simple heterozygosis, being associated with a more severe phenotype in homozygous carriers.

INF2 and ROBO2 gene mutation in an Indian family with end stage renal failure and follow-up of renal transplantation.

BACKGROUND: Accurate genetic diagnosis of end-stage renal disease patients with a family history of renal dysfunction is very essential. It not only helps in proper prognosis, but becomes crucial in designating donor for live related renal transplant. We here present a case of family with deleterious mutations in INF2 and ROBO2 and its importance of genetic testing before preparing for kidney transplantation. CASE PRESENTATION: We report the case of a 29-year-female with end-stage renal disease and rapidly progressive renal failure. Mutational analysis revealed an Autosomal Dominant inheritance pattern and mutation in exon 4 of the INF2 gene (p. Thr215Ser) and exon 26 of the ROBO2 gene (p. Arg1371Cys). Her mother was diagnosed for CKD stage 4 with creatinine level of 4.3 mg/dL. Genetic variants (INF2 and ROBO2) identified in proband were tested in her sisters and mother. Her elder sister was positive for both heterozygous variants (INF2 and ROBO2). Her mother was positive for mutation in INF2 gene, and her donor elder sister did not showed mutation in INF2 gene and had mutation in ROBO2 gene without any clinical symptoms. CONCLUSION: This case report emphasize that familial genetic screening has allowed us in allocating the donor selection in family where family member had history of genetic defect of Chronic Kidney Disease. Information of the causative renal disorder is extremely valuable for risk-assessment and planning of kidney transplantation.

Drebrin Protects Assembled Actin from INF2-FFC-mediated Severing and Stabilizes Cell Protrusions.

Highly specialized cells, such as neurons and podocytes, have arborized morphologies that serve their specific functions. Actin cytoskeleton and its associated proteins are responsible for the distinctive shapes of cells. The mechanism of their cytoskeleton regulation - contributing to cell shape maintenance - is yet to be fully clarified. Inverted formin 2 (INF2), one of the modulators of the cytoskeleton, is an atypical formin that can both polymerize and depolymerize actin filaments depending on its molar ratio to actin. Prior work has established that INF2 binds to the sides of actin filaments and severs them. Drebrin is another actin-binding protein that also binds filaments laterally and stabilizes them, but the interplay between drebrin and INF2 on actin filament stabilization is not well understood. Here, we have used biochemical assays, electron microscopy, and total internal reflection fluorescence microscopy imaging to show that drebrin protects actin filaments from severing by INF2 without inhibiting its polymerization activity. Notably, truncated drebrin - DrbA1-300 - is sufficient for this protection, though not as effective as the full-length protein. INF2 and drebrin are abundantly expressed in highly specialized cells and are crucial for the temporal regulation of their actin cytoskeleton, consistent with their involvement in peripheral neuropathy.

A novel splice-site FHOD3 founder variant is a common cause of hypertrophic cardiomyopathy in the population of the Balkans-A cohort study.

Founder variants in sarcomere protein genes account for a significant proportion of disease-causing variants in patients with hypertrophic cardiomyopathy (HCM). However, information on founder variants in non-sarcomeric protein genes, such as FHOD3, which have only recently been associated with HCM, remains scarce. In this study, we conducted a retrospective analysis of exome sequencing data of 134 probands with HCM for recurrent pathogenic variants. We discovered a novel likely pathogenic variant c.1646+2T>C in FHOD3 in heterozygous state in eight probands with HCM and confirmed its presence in seven additional relatives. Individuals with this variant had a wide range of ages at onset of the disease (4-63 years). No adverse cardiac events were observed. Haplotype analysis revealed that the individuals with this variant shared a genomic region of approximately 5 Mbp surrounding the variant, confirming the founder effect of the variant. FHOD3 c.1646+2T>C is estimated to have arisen 58 generations ago (95% CI: 45-81) in a common ancestor living on the Balkans. A founder FHOD3 c.1646+2T>C variant is the second most common genetic variant in our cohort of patients with HCM, occurring in 16% of probands with a known genetic cause of HCM, which represents a substantially higher proportion than the currently estimated 0.5-2% for causal FHOD3 variants. Our study broadens the understanding of the genetic causes of HCM and may improve the diagnosis of this condition, particularly in patients from the Balkans.

Genome-wide study reveals novel roles for formin-2 in axon regeneration as a microtubule dynamics regulator and therapeutic target for nerve repair.

Peripheral nerves regenerate successfully; however, clinical outcome after injury is poor. We demonstrated that low-dose ionizing radiation (LDIR) promoted axon regeneration and function recovery after peripheral nerve injury (PNI). Genome-wide CpG methylation profiling identified LDIR-induced hypermethylation of the Fmn2 promoter, exhibiting injury-induced Fmn2 downregulation in dorsal root ganglia (DRGs). Constitutive knockout or neuronal Fmn2 knockdown accelerated nerve repair and function recovery. Mechanistically, increased microtubule dynamics at growth cones was observed in time-lapse imaging of Fmn2-deficient DRG neurons. Increased HDAC5 phosphorylation and rapid tubulin deacetylation were found in regenerating axons of neuronal Fmn2-knockdown mice after injury. Growth-promoting effect of neuronal Fmn2 knockdown was eliminated by pharmaceutical blockade of HDAC5 or neuronal Hdac5 knockdown, suggesting that Fmn2deletion promotes axon regeneration via microtubule post-translational modification. In silico screening of FDA-approved drugs identified metaxalone, administered either immediately or 24-h post-injury, accelerating function recovery. This work uncovers a novel axon regeneration function of Fmn2 and a small-molecule strategy for PNI.

[Effect of miR-22 Targeting FMNL2 on Cell Migration and Apoptosis in Childhood Acute Myeloid Leukemia].

OBJECTIVE: To investigate the effect of miR-22 targeting formin-like protein 2 (FMNL2) on the migration and apoptosis of childhood acute myeloid leukemia (AML) cells. METHOD: Peripheral blood samples from 11 children with AML, 10 children with immune thrombocytopenia, human AML cell lines TF-1a, HL-60, THP-1 and human bone marrow stromal cells HS-5 were used as the research objects. UniCel DxH 800 automatic hematology analyzer detected platelet count, hemoglobin, and white blood cell count in peripheral blood samples, and RT-qPCR detected miR-22 expression in peripheral blood samples and AML cells. HL-60 cells were transfected with LipofectamineTM 2000 kit, the experiments were divided into seven groups: blank (no cells transfected), miR-NC, miR-22 mimics, si-NC, si-FMNL2 , miR-22 mimics+OE-NC and miR-22 mimics+OE-FMNL2 . RT-qPCR was used to detect the expression of miR-22 in each group. Transwell was used to detect cell migration. Flow cytometry was used to detect cell apoptosis. Dual-luciferase reporter gene detection experiments verified the targeting relationship between miR-22 and FMNL2 . Western blot was used to detect the expression of FMNL2 protein. RESULTS: Compared with the control group, the number of leukocytes in the peripheral blood of children with AML was significantly increased (P <0.001), while the concentration of hemoglobin and the number of platelets were significantly decreased P <0.001). The expression level of miR-22 in peripheral blood of children with AML was significantly lower than that in control group (P <0.001). Compared with HS-5 cells, the expression levels of miR-22 in TF-1a, HL-60, and THP-1 cells were significantly decreased (P <0.05), and in HL-60 cells was the lowest. Therefore, HL-60 cells were selected for subsequent experiments. Up-regulation of miR-22 or silencing of FMNL2 could reduce the number of migrating cells and increase apoptosis rate (P <0.05). MiR-22 targeted and negatively regulated the expression of FMNL2 . FMNL2 overexpression reversed the effects of up-regulated miR-22 on migration and apoptosis of HL-60 cells. CONCLUSION: MiR-22 can inhibit the migration and promote apoptosis of HL-60 cells by down regulating the expression of FMNL2 .