A clinical prognostic model related to T cells based on machine learning for predicting the prognosis and immune response of ovarian cancer.

Background: Ovarian cancer (OV) is regarded as one of the most lethal malignancies affecting the female reproductive system, with individuals diagnosed with OV often facing a dismal prognosis due to resistance to chemotherapy and the presence of an immunosuppressive environment. T cells serve as a crucial mediator for immune surveillance and cancer elimination. This study aims to analyze the mechanism of T cell-associated markers in OV and create a prognostic model for clinical use in enhancing outcomes for OV patients. Methods: Based on the single-cell dataset GSE184880, this study used single-cell data analysis to identify characteristic T cell subsets. Analysis of high dimensional weighted gene co-expression network analysis (hdWGCNA) is utilized to identify crucial gene modules along with their corresponding hub genes. A grand total of 113 predictive models were formed utilizing ten distinct machine learning algorithms along with the combination of the cancer genome atlas (TCGA)-OV dataset and the GSE140082 dataset. The most dependable clinical prognostic model was created utilizing the leave one out cross validation (LOOCV) framework. The validation process for the models was achieved by conducting survival curve analysis and receiver operating characteristic (ROC) analysis. The relationship between risk scores and immune cells was explored through the utilization of the Cibersort algorithm. Additionally, an analysis of drug sensitivity was carried out to anticipate chemotherapy responses across various risk groups. The genes implicated in the model were authenticated utilizing qRT-PCR, cell viability experiments, and EdU assay. Results: This study developed a clinical prognostic model that includes ten risk genes. The results obtained from the training set of the study indicate that patients classified in the low-risk group experience a significant survival advantage compared to those in the high-risk group. The ROC analysis demonstrates that the model holds significant clinical utility. These results were verified using an independent dataset, strengthening the model's precision and dependability. The risk assessment provided by the model also serves as an independent prognostic factor for OV patients. The study also unveiled a noteworthy relationship between the risk scores calculated by the model and various immune cells, suggesting that the model may potentially serve as a valuable tool in forecasting responses to both immune therapy and chemotherapy in ovarian cancer patients. Notably, experimental evidence suggests that PFN1, one of the genes included in the model, is upregulated in human OV cell lines and has the capacity to promote cancer progression in in vitro models. Conclusion: We have created an accurate and dependable clinical prognostic model for OV capable of predicting clinical outcomes and categorizing patients. This model effectively forecasts responses to both immune therapy and chemotherapy. By regulating the immune microenvironment and targeting the key gene PFN1, it may improve the prognosis for high-risk patients.

PFN1 Knockdown Aggravates Mitophagy to Retard Lung Adenocarcinoma Initiation and M2 Macrophage Polarization.

Tumor-associated macrophages (TAM) are considered as crucial influencing factors of lung adenocarcinoma (LUAD) carcinogenesis and metastasis. Profilin 1 (PFN1) has been proposed as a potent driver of migration and drug resistance in LUAD. The focus of this work was to figure out the functional mechanism of PFN1 in macrophage polarization in LUAD. PFN1 expression and its significance in patients' survival were detected by ENCORI and Kaplan-Meier Plotter. RT-qPCR and western blotting examined PFN1 expression in LUAD cells. CCK-8 assay and colony formation assay detected cell proliferation. Flow cytometry detected cell apoptosis. Relevant assay kit tested caspase3 concentration. Western blotting analyzed the expression of proliferation- and apoptosis-related proteins. RT-qPCR and immunofluorescence staining measured M1 and M2 macrophages markers. Mitophagy was assessed by MitoTracker Red staining, immunofluorescence staining, and western blotting. PFN1 expression was increased in LUAD tissues and cells and correlated with the poor survival rate of LUAD patients. Deficiency of PFN1 hindered the proliferation, whereas facilitated the apoptosis of LUAD cells. Additionally, PFN1 interference impaired M2 macrophage polarization. Moreover, PFN1 knockdown exacerbated the mitophagy in LUAD cells and mitophagy inhibitor mitochondrial division inhibitor 1 (Mdivi-1) notably reversed the effects of PFN1 down-regulation on the proliferation, apoptosis as well as macrophage polarization in LUAD cells. To sum up, activation of mitophagy initiated by PFN1 depletion might obstruct the occurrence and M2 macrophage polarization in LUAD.

Identification of a novel lactylation-related gene signature predicts the prognosis of multiple myeloma and experiment verification.

Multiple myeloma (MM) is an incurable hematological malignancy with poor survival. Accumulating evidence reveals that lactylation modification plays a vital role in tumorigenesis. However, research on lactylation-related genes (LRGs) in predicting the prognosis of MM remains limited. Differentially expressed LRGs (DELRGs) between MM and normal samples were investigated from the Gene Expression Omnibus database. Univariate Cox regression and LASSO Cox regression analysis were applied to construct gene signature associated with overall survival. The signature was validated in two external datasets. A nomogram was further constructed and evaluated. Additionally, Enrichment analysis, immune analysis, and drug chemosensitivity analysis between the two groups were investigated. qPCR and immunofluorescence staining were performed to validate the expression and localization of PFN1. CCK-8 and flow cytometry were performed to validate biological function. A total of 9 LRGs (TRIM28, PPIA, SOD1, RRP1B, IARS2, RB1, PFN1, PRCC, and FABP5) were selected to establish the prognostic signature. Kaplan-Meier survival curves showed that high-risk group patients had a remarkably worse prognosis in the training and validation cohorts. A nomogram was constructed based on LRGs signature and clinical characteristics, and showed excellent predictive power by calibration curve and C-index. Moreover, biological pathways, immunologic status, as well as sensitivity to chemotherapy drugs were different between high- and low-risk groups. Additionally, the hub gene PFN1 is highly expressed in MM, knocking down PFN1 induces cell cycle arrest, suppresses cell proliferation and promotes cell apoptosis. In conclusion, our study revealed that LRGs signature is a promising biomarker for MM that can effectively early distinguish high-risk patients and predict prognosis.

Detergent-insoluble PFN1 inoculation expedites disease onset and progression in PFN1 transgenic rats.

Accumulating evidence suggests a gain of elusive toxicity in pathogenically mutated PFN1. The prominence of PFN1 aggregates as a pivotal pathological hallmark in PFN1 transgenic rats underscores the crucial involvement of protein aggregation in the initiation and progression of neurodegeneration. Detergent-insoluble materials were extracted from the spinal cords of paralyzed rats afflicted with ALS and were intramuscularly administered to asymptomatic recipient rats expressing mutant PFN1, resulting in an accelerated development of PFN1 inclusions and ALS-like phenotypes. This effect diminished when the extracts derived from wildtype PFN1 transgenic rats were employed, as detergent-insoluble PFN1 was detected exclusively in mutant PFN1 transgenic rats. Consequently, the factor influencing the progression of ALS pathology in recipient rats is likely associated with the presence of detergent-insoluble PFN1 within the extracted materials. Noteworthy is the absence of disease course modification upon administering detergent-insoluble extracts to rats that already displayed PFN1 inclusions, suggesting a seeding rather than augmenting role of such extracts in initiating neuropathological changes. Remarkably, pathogenic PFN1 exhibited an enhanced affinity for the molecular chaperone DNAJB6, leading to the sequestration of DNAJB6 within protein inclusions, thereby depleting its availability for cellular functions. These findings shed light on a novel mechanism that underscores the prion-like characteristics of pathogenic PFN1 in driving neurodegeneration in the context of PFN1-related ALS.

Long noncoding RNA RMRP ameliorates doxorubicin-induced apoptosis by interacting with PFN1 in a P53-Dependent manner.

Doxorubicin (DOX) often causes acute or chronic cardiotoxicity during its application. LncRNA RMRP has been reported to be associated with several biological processes, such as cartilage-hair hypoplasia, but the relationship between RMRP and DOX-induced cardiotoxicity and chronic heart failure remains obscure. To test this hypothesis, GSE124401 and GSE149870 were processed for bioinformatics, and differentially expressed RMRP was then verified in the peripheral blood of 21 patients with heart failure compared with 7 controls. For in vitro validation, we used AC16 and HEK-293T cells. qPCR was used to detect the mRNA expression levels. The degree of apoptosis was detected by Western blot and TUNEL staining. Furthermore, the interaction between RMRP and PFN1 mRNA was verified by dual-luciferase reporter assays. In bioinformatics, RMRP showed significant downregulation, which was verified in clinical samples (p < 0.001) and DOX-treated AC16 models (p < 0.0001). Next, overexpression of RMRP could significantly alleviate DOX-induced apoptosis, and a potential downstream molecule of RMRP, PFN1, was also negatively associated with this change. RESCUE experiments further confirmed that PFN1 could be regulated by RMRP at both the RNA and protein levels, serving as a downstream mediator of RMRP's cardioprotective effects. This interaction was then confirmed to be a direct combination (p < 0.0001). Finally, we found that overexpression of RMRP could inhibit the expression of p53 and its phosphorylation level by suppressing PFN1. In summary, RMRP could exert cardioprotective effects via the PFN1/p53 axis, holding great promise for serving as a therapeutic target and potential biomarker.

Genetic Screening of ZNF687 and PFN1 in a Paget's Disease of Bone Cohort Indicates an Important Role for the Nuclear Localization Signal of ZNF687.

Paget's disease of bone (PDB) is a common, late-onset bone disorder, characterized by focal increases of bone turnover that can result in bone lesions. Heterozygous pathogenic variants in the Sequestosome 1 (SQSTM1) gene are found to be the main genetic cause of PDB. More recently, PFN1 and ZNF687 have been identified as causal genes in patients with a severe, early-onset, polyostotic form of PDB, and an increased likelihood to develop giant cell tumors. In our study, we screened the coding regions of PFN1 and ZNF687 in a Belgian PDB cohort (n = 188). In the PFN1 gene, no variants could be identified, supporting the observation that variants in this gene are extremely rare in PDB. However, we identified 3 non-synonymous coding variants in ZNF687. Interestingly, two of these rare variants (p.Pro937His and p.Arg939Cys) were clustering in the nuclear localization signal of the encoded ZNF687 protein, also harboring the p.Pro937Arg variant, a previously reported disease-causing variant. In conclusion, our findings support the involvement of genetic variation in ZNF687 in the pathogenesis of classical PDB, thereby expanding its mutational spectrum.

The identification of high-performing antibodies for Profilin-1 for use in Western blot, immunoprecipitation and immunofluorescence.

Profilin-1, a member of the Profilin family, is a ubiquitously expressed protein that controls actin polymerization in a concentration-dependent manner. As mutations in the Profilin-1 gene have potential implications in neurodegenerative disease progression, well-characterized anti-Profilin-1 antibodies would be beneficial to the scientific community. In this study, we characterized sixteen Profilin-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence applications, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Association of PFN1 Gene Polymorphisms with Bone Mineral Density, Bone Turnover Markers, and Osteoporotic Fractures in Chinese Population.

Recent studies have discovered an association between the PFN1 gene and Paget's disease. However, it is currently unknown whether the PFN1 gene is related to osteoporosis. This study was performed to investigate the association of Single-Nucleotide Polymorphisms (SNPs) in the PFN1 gene with Bone Mineral Density (BMD) as well as bone turnover markers and osteoporotic fractures in Chinese subjects. A total of 2836 unrelated Chinese subjects comprising 1247 healthy subjects and 1589 osteoporotic fractures patients (Fracture group) were enrolled in this study. Seven tagSNPs (rs117337116, rs238243, rs6559, rs238242, rs78224458, rs4790714, and rs13204) of the PFN1 gene were genotyped. The BMD of the lumbar spine 1-4 (L1-4), femoral neck, and total hip as well as bone turnover markers, such as ß-C-Terminal telopeptide of type 1 collagen (ß-CTX) and Procollagen type 1 N-terminal Propeptide (P1NP), were measured. The association between 7 tagSNPs and BMD and bone turnover markers was analyzed in 1247 healthy subjects only. After age matching, we selected 1589 osteoporotic fracture patients (Fracture group) and 756 nonfracture controls (Control group, selected from 1247 healthy subjects) for a case-control study, respectively. For the case-control study, we used logistic regression to investigate the relationship between 7 tagSNPs and osteoporotic fractures risk. In the All group, the PFN1 haplotype GAT was associated with the ß-CTX (P = 0.007). In the Female group, the PFN1 haplotype GAT was associated with the ß-CTX (P = 0.005). In the Male group, the rs13204, the rs78224458, and the PFN1 haplotype GAC were associated with the BMD of the L1-4 (all P = 0.012); the rs13204, the rs78224458, and the PFN1 haplotype GAC were associated with the BMD of the femoral neck (all P = 0.012); the rs13204 and rs78224458 were associated with the BMD of the total hip (both P = 0.015); and the PFN1 haplotype GAT was associated with the ß-CTX (P = 0.013). In the subsequent case-control study, the rs13204 and rs78224458 in the male group were associated with the risk of L1-4 fracture (P = 0.016 and 0.010, respectively) and total hip fracture (P = 0.013 and 0.016, respectively). Our study reveals that PFN1 gene polymorphisms are associated with BMD in Chinese males and ß-CTX in Chinese people and confirmed the relationship between PFN1 gene polymorphisms and Chinese male osteoporotic fractures in a case-control study.

Profilin1 Promotes Renal Tubular Epithelial Cell Apoptosis in Diabetic Nephropathy Through the Hedgehog Signaling Pathway.

Background: Profilin-1 (PFN1) regulates the dynamic balance of actin and plays an important role in cell functions as a hub protein in signaling molecule interaction networks. Dysregulation of PFN1 is related to pathologic kidney diseases. Diabetic nephropathy (DN) was recently reported as an inflammatory disorder, however, the molecular mechanisms of PFN1 in DN remain unclear. Therefore, the present study was conducted to explore the molecular and bioinformatic characteristics of PFN1 in DN. Methods: Bioinformatics analyses were performed on the chip of database in DN kidney tissues. A cellular model of DN was established in human renal tubular epithelial cells (HK-2) induced by high glucose. The PFN1 gene was overexpressed or knocked-down to investigate its function in DN. Flow cytometry was used to detect cell proliferation and apoptosis. PFN1 and proteins in the related signaling pathways were evaluated by Western blotting. Results: The expression of PFN1 was significantly increased in DN kidney tissues (P < 0.001) and was correlated with a high apoptosis-associated score (Pearson's correlation = 0.664) and cellular senescence-associated score (Pearson's correlation = 0.703). PFN1 protein was mainly located in cytoplasm. Overexpression of PFN1 promoted apoptosis and blocked the proliferation of HK-2 cells treated with high levels of glucose. Knockdown of PFN1 led to the opposite effects. Additionally, we found that PFN1 was correlated with the inactivation of the Hedgehog signaling pathway in HK-2 cells treated with high levels of glucose. Conclusion: PFN1 might play an integral role in the regulation of cell proliferation and apoptosis during DN development by activating the Hedgehog signaling pathway. This study provided molecular and bioinformatic characterizations of PFN1, and contributed to the understanding of the molecular mechanisms leading to DN.

Novel variant c.92T > G (p.Val31Gly) in the PFN1 gene (ALS18) responsible for a specific phenotype in a large Bulgarian amyotrophic lateral sclerosis pedigree.

Objectives: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive deterioration of motor function, disability, and death. Variants in the PFN1 gene, encoding the Profilin-1 protein, are related to ALS18. Methods: We present a pedigree consisting of 3 generations and 4 affected individuals, 3 of which carry a novel heterozygous variant: c.92T > G (p.Val31Gly) in the PFN1 gene. This variant was discovered through means of whole exome sequencing (WES) and targeted analysis of ALS-related genes. Results: The mean age of onset in our pedigree was 59.75 (±10.11 SD) years with a significant difference between the first two generations (females) and the third (male) of 22.33 (±3.4 SD) years. For this ALS form, we observed a longer disease progression of 4 (±1.87 SD) years (three of four affected are still alive). Clinical manifestations displayed predominant impairment of the lower motor neuron (LMN) in one limb, with gradual involvement of other limbs. A novel heterozygous missense variant c.92T > G, p. Val31Gly (NM_005022.4) in exon 1 in the PFN1 gene was discovered through means of whole exome sequencing (WES). Segregation analysis in the family showed that the detected variant was inherited from the affected mother, and the affected aunt also turned out to be a variant carrier. Conclusions: ALS18 is a very rare form of the disease. We report here a relatively large pedigree with a novel variant, leading to late onset (after 50 years), initial involvement of the lower limbs and relatively slow progression.

LncRNA PEG11as aggravates cerebral ischemia/reperfusion injury after ischemic stroke through miR-342-5p/PFN1 axis.

AIM: LncRNAs are highly expressed in the CNS and regulate pathophysiological processes. However, the potential role of lncRNAs inischemic stroke (IS) remains unknown. In this study, we investigated the functions and possible molecular mechanism of lncRNA paternal expressed gene 11 antisense (PEG11as) in this process. METHODS: Middle cerebral artery occlusion/reperfusion (MCAO/R) mice model and N2a cells model from oxygen-glucose deprivation/reoxygenation (OGD/R) were used to simulate cerebral I/R in vivo and in vitro. High-throughput sequencing (RNA-Seq) was used todetect differential expression of lncRNAs in cerebral I/R. QRT-PCR was used to detect the expression of PEG11as and miR-342-5p. Bioinformatics analysis, FISH, luciferase reporter assay, RIP, Western blot, and immunofluorescence were used to detect the interaction between PEG11as, miR-342-5p and PFN1. The effect on neuronal apoptosis was analyzed using loss-of-function combined with TUNEL, Hoechst, and caspase3 activity assays. KEY FINDINGS: 254 lncRNAs were differentially expressed in MCAO1h/R6h mice. Among them, PEG11as was significantly up-regulated. PEG11as down-regulated could markedly attenuate the brain infarct volume, alleviate neurological deficit in vivo, and effectively promote neuron survival, attenuate neuronal apoptosis both in vivo and in vitro. FISH assay discovered that PEG11as was mainly located in the cytoplasm. Furthermore, we demonstrated that PEG11as was able to bind miR-342-5p to inhibit miR-342-5p activity, whereas the down-regulated of miR-342-5p resulted in profilin 1 (PFN1) overexpression and thus promoting apoptosis. SIGNIFICANCE: This study suggests that PEG11as regulates neuronal apoptosis by miR-342-5p/PFN1 axis, which may contribute to our understanding of pathogenesis and provide a potential therapeutic option for cerebral I/R.

PFN1 Inhibits Myogenesis of Bovine Myoblast Cells via Cdc42-PAK/JNK.

Myoblast differentiation is essential for the formation of skeletal muscle myofibers. Profilin1 (Pfn1) has been identified as an actin-associated protein, and has been shown to be critically important to cellular function. Our previous study found that PFN1 may inhibit the differentiation of bovine skeletal muscle satellite cells, but the underlying mechanism is not known. Here, we confirmed that PFN1 negatively regulated the myogenic differentiation of bovine skeletal muscle satellite cells. Immunoprecipitation assay combined with mass spectrometry showed that Cdc42 was a binding protein of PFN1. Cdc42 could be activated by PFN1 and could inhibit the myogenic differentiation like PFN1. Mechanistically, activated Cdc42 increased the phosphorylation level of p2l-activated kinase (PAK), which further activated the phosphorylation activity of c-Jun N-terminal kinase (JNK), whereas PAK and JNK are inhibitors of myogenic differentiation. Taken together, our results reveal that PFN1 is a repressor of bovine myogenic differentiation, and provide the regulatory mechanism.

Decrease in LINC00963 attenuates the progression of pulmonary arterial hypertension via microRNA-328-3p/profilin 1 axis.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a severe cardiopulmonary disease characterized by vascular hyperplasia and remodeling. Long noncoding RNA LINC00963 can regulate cell proliferation and metastasis in nonsmall cell lung cancer. However, the function of LINC00963 on PAH progression is rarely reported. METHODS: Quantitative real-time PCR was used to determine the expression levels of LINC00963, microRNA (miRNA)-328-3p, and profilin 1 (PFN1), as well as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), and hypoxia-inducible factor (HIF)-α. The protein level of PFN1 was measured by western blotting. The viability and migration of hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs) were assessed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide, and transwell assays, respectively. The target relationships between miR-328-3p and LINC00963/PFN1 were confirmed by dual-luciferase reporter assay. A PAH mouse model was conducted to explore the effects of hypoxia on cardiopulmonary functions. RESULTS: In hypoxia-induced PASMCs and PAH mouse model, high expression levels of LINC00963 and PFN1, and low expression of miR-328-3p, were determined. The viability, migration of hypoxia-induced PASMCs, the expression of VEGF, FGF-2, and HIF-α were significantly repressed by transfection of si-LINC00963 or miR-328-3p mimics. The inhibitory effects of LINC00963 silencing on cell viability, migration, and the levels of VEGF, FGF-2, and HIF-α were partly eliminated by miR-328-3p inhibitor or increasing the expression of PFN1. Hypoxia treatment increased the levels of RVSP, mPAP, and RV/(LV+S), as well as the thickness of pulmonary artery wall. CONCLUSIONS: Silencing of LINC00963 ameliorates PAH via modulating miR-328-3p/PFN1.

Detection of structural and conformational changes in ALS-causing mutant profilin-1 with hydrogen/deuterium exchange mass spectrometry and bioinformatics techniques.

The hydrogen/deuterium exchange (HDX) is a reliable method to survey the dynamic behavior of proteins and epitope mapping. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a quantifying tool to assay for HDX in the protein of interest. We combined HDX-MALDI-TOF MS and molecular docking/MD simulation to identify accessible amino acids and analyze their contribution into the structural changes of profilin-1 (PFN-1). The molecular docking/MD simulations are computational tools for enabling the analysis of the type of amino acids that may be involved via HDX identified under the lowest binding energy condition. Glycine to valine amino acid (G117V) substitution mutation is linked to amyotrophic lateral sclerosis (ALS). This mutation is found to be in the actin-binding site of PFN-1 and prevents the dimerization/polymerization of actin and invokes a pathologic toxicity that leads to ALS. In this study, we sought to understand the PFN-1 protein dynamic behavior using purified wild type and mutant PFN-1 proteins. The data obtained from HDX-MALDI-TOF MS for PFN-1WT and PFN-1G117V at various time intervals, from seconds to hours, revealed multiple peaks corresponding to molecular weights from monomers to multimers. PFN-1/Benzaldehyde complexes identified 20 accessible amino acids to HDX that participate in the docking simulation in the surface of WT and mutant PFN-1. Consistent results from HDX-MALDI-TOF MS and docking simulation predict candidate amino acid(s) involved in the dimerization/polymerization of PFNG117V. This information may shed critical light on the structural and conformational changes with details of amino acid epitopes for mutant PFN-1s' dimerization, oligomerization, and aggregation.

Mutant Profilin1 Aggregation in Amyotrophic Lateral Sclerosis: An in Vivo Biochemical Analysis.

INTRODUCTION: Profilin1 (PFN1) is a ubiquitously expressed protein known for its function as a regulator of actin polymerization and dynamics. A recent discovery linked mutant PFN1 to Amyotrophic Lateral Sclerosis (ALS), which is a fatal and progressive motor neuron disease. We have also demonstrated that Gly118Val mutation in PFN1 is a cause of ALS, and the formation of aggregates containing mutant PFN1 may be a mechanism for motor neuron death. Hence, we were interested in investigating the aggregation of PFN1 further and searching for co-aggregated proteins in our mouse model overexpressing mutant PFN1. METHODS: We investigated protein aggregation in several tissues of transgenic and notransgenic mice using western blotting. To further understand the neurotoxicity of mutant PFN1, we conducted a pull-down assay using an insoluble fraction of spinal cord lysates from hPFN1G118V transgenic mice. For this assay, we expressed His6-tagged PFN1WT and PFN1G118V in E. coli and purified these proteins using the Ni-NTA column. RESULTS: In this study, we demonstrated that mutant PFN1 forms aggregate in the brain and spinal cord of hPFN1G118V mice, while WT-PFN1 remains soluble. Among these tissues, spinal cord lysates were found to have PFN1 bands at higher molecular weights recognized with anti-PFN1. Moreover, the pull-down assay using His6-PFN1G118V showed that Myelin Binding Protein (MBP) was present in the insoluble fraction. CONCLUSION: Our analysis of PFN1 aggregation in vivo revealed further details of mutant PFN1 aggregation and its possible complex formation with other proteins, providing new insights into the ALS mechanism.

A novel lncRNA promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1.

Myogenesis, the process of skeletal muscle formation, is a highly coordinated multistep biological process. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are emerging as a gatekeeper in myogenesis. Up to now, most studies on muscle development-related lncRNAs are mainly focussed on humans and mice. In this study, a novel muscle highly expressed lncRNA, named lnc23, localized in nucleus, was found differentially expressed in different stages of embryonic development and myogenic differentiation. The knockdown and over-expression experiments showed that lnc23 positively regulated the myogenic differentiation of bovine skeletal muscle satellite cells. Then, TMT 10-plex labelling quantitative proteomics was performed to screen the potentially regulatory proteins of lnc23. Results indicated that lnc23 was involved in the key processes of myogenic differentiation such as cell fusion, further demonstrated that down-regulation of lnc23 may inhibit myogenic differentiation by reducing signal transduction and cell fusion among cells. Furthermore, RNA pulldown/LC-MS and RIP experiment illustrated that PFN1 was a binding protein of lnc23. Further, we also found that lnc23 positively regulated the protein expression of RhoA and Rac1, and PFN1 may negatively regulate myogenic differentiation and the expression of its interacting proteins RhoA and Rac1. Hence, we support that lnc23 may reduce the inhibiting effect of PFN1 on RhoA and Rac1 by binding to PFN1, thereby promoting myogenic differentiation. In short, the novel identified lnc23 promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1.

Mutations in Profilin 1 Cause Early-Onset Paget's Disease of Bone With Giant Cell Tumors.

Paget's disease of bone (PDB) is a late-onset chronic progressive bone disease characterized by abnormal activation of osteoclasts that results in bone pain, deformities, and fractures. PDB is very rare in Asia. A subset of PDB patients have early onset and can develop malignant giant cell tumors (GCTs) of the bone (PDB/GCTs), which arise within Paget bone lesions; the result is a significantly higher mortality rate. SQSTM1, TNFRSF11A, OPG, VCP, and HNRNPA2B1 have been identified as pathogenic genes of PDB, and ZNF687 is the only confirmed gene to date known to cause PDB/GCT. However, the molecular mechanism underlying PDB/GCT has not been fully elucidated. Here, we investigate an extended Chinese pedigree with eight individuals affected by early-onset and polyostotic PDB, two of whom developed GCTs. We identified a heterozygous 4-bp deletion in the Profilin 1 (PFN1) gene (c.318_321delTGAC) by genetic linkage analysis and exome sequencing for the family. Sanger sequencing revealed another heterozygous 1-bp deletion in PFN1 (c.324_324delG) in a sporadic early-onset PDB/GCT patient, further proving its causative role. Interestingly, a heterozygous missense mutation of PFN1 (c.335 T > C) was identified in another PDB/GCT family, revealing that not only deletion but also missense mutations in PFN1 can cause PDB/GCT. Furthermore, we established a Pfn1-mutated mouse model (C57BL/6J mice) and successfully obtained Pagetic phenotypes in heterozygous mice, verifying loss of function of PFN1 as the cause of PDB/GCT development. In conclusion, our findings reveal mutations in PFN1 as the pathological mechanism in PDB/GCT, and we successfully established Pfn1-mutated mice as a suitable animal model for studying PDB-associated pathological mechanisms. The identification of PFN1 mutations has great diagnostic value for identifying PDB individuals predisposed toward developing GCTs. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

PFN1 Gene Polymorphisms and the Bone Mineral Density Response to Alendronate Therapy in Postmenopausal Chinese Women with Low Bone Mass.

PURPOSE: Alendronate is a widely used anti-osteoporotic drug. PFN1 gene is a newly identified early-onset Paget's disease pathogenic gene. The purpose of this study is to study whether the genetic variations in this gene affect the clinical efficacy of alendronate in postmenopausal Chinese women with low bone mass. PATIENTS AND METHODS: Seven single nucleotide polymorphisms in PFN1 gene were genotyped. A total of 500 postmenopausal women with osteoporosis or osteopenia were included. All participants were treated with weekly alendronate 70 mg for 12 months. A total of 466 subjects completed the follow-up. Bone mineral density (BMD) of lumbar spine, femoral neck and total hip were measured at baseline and after treatment. RESULTS: After 12 months of treatment, the BMD of lumbar spine, femoral neck and total hip all increased significantly (all P < 0.001), with an average increase of 4.72 ± 5.31%, 2.08 ± 4.45%, and 2.42 ± 3.46%, respectively. At baseline, there were no significant differences in BMD at lumbar spine, femoral neck and total hip between different genotype groups (P > 0.05). We failed to identify any significant association between the genotypes or haplotypes of PFN1 and the BMD response to alendronate therapy. CONCLUSION: Genetic polymorphisms of PFN1 may not be a major contributor to the therapeutic response to alendronate treatment in Chinese women with low bone mass.

Detergent-insoluble inclusion constitutes the first pathology in PFN1 transgenic rats.

Mutation of profilin 1 (PFN1) can cause amyotrophic lateral sclerosis (ALS). To assess how PFN1 mutation causes the disease, we created transgenic rats with human genomic DNA that harbors both the coding and the regulatory sequences of the human PFN1 gene. Selected transgenic lines expressed human PFN1 with or without the pathogenic mutation C71G at a moderate and a comparable level and in the similar pattern of spatial and temporal expression to rat endogenous PFN1. The artificial effects of arbitrary transgene expression commonly observed in cDNA transgenic animals were minimized in PFN1 transgenic rats. Expression of the mutant, but not the wild type, human PFN1 in rats recapitulated the cardinal features of ALS including the progressive loss of motor neurons and the subsequent denervation atrophy of skeletal muscles. Detergent-insoluble PFN1 inclusions were detected as the first pathology in otherwise asymptomatic transgenic rats expressing mutant human PFN1. The findings suggest that protein aggregation is involved in the neurodegeneration of ALS associated with PFN1 mutation. The resulting rat model is useful to mechanistic study on the ALS.

The Neglected Genes of ALS: Cytoskeletal Dynamics Impact Synaptic Degeneration in ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that selectively affects motor neurons (MNs) of the cortex, brainstem, and spinal cord. Several genes have been linked to both familial (fALS) and sporadic (sALS) cases of ALS. Among all the ALS-related genes, a group of genes known to directly affect cytoskeletal dynamics (ALS2, DCTN1, PFN1, KIF5A, NF-L, NF-H, PRPH, SPAST, and TUBA4A) is of high importance for MN health and survival, considering that MNs are large polarized cells with axons that can reach up to 1 m in length. In particular, cytoskeletal dynamics facilitate the transport of organelles and molecules across the long axonal distances within the cell, playing a key role in synapse maintenance. The majority of ALS-related genes affecting cytoskeletal dynamics were identified within the past two decades, making it a new area to explore for ALS. The purpose of this review is to provide insights into ALS-associated cytoskeletal genes and outline how recent studies have pointed towards novel pathways that might be impacted in ALS. Further studies making use of extensive analysis models to look for true hits, the newest technologies such as CRIPSR/Cas9, human induced pluripotent stem cells (iPSCs) and axon sequencing, as well as the development of more transgenic animal models could potentially help to: differentiate the variants that truly act as a primary cause of the disease from the ones that act as risk factors or disease modifiers, identify potential interactions between two or more ALS-related genes in disease onset and progression and increase our understanding of the molecular mechanisms leading to cytoskeletal defects. Altogether, this information will give us a hint on the real contribution of the cytoskeletal ALS-related genes during this lethal disease.