Proteome-Wide Mendelian Randomization Analysis Identified Potential Drug Targets for Atrial Fibrillation.

Background Finding effective and safe therapeutic drugs for atrial fibrillation (AF) is an important concern for clinicians. Proteome-wide Mendelian randomization analysis provides new ideas for finding potential drug targets. Methods and Results Using a proteome-wide Mendelian randomization approach, we assessed the genetic predictive causality between thousands of proteins and AF risk and found that genetically predicted plasma levels of phosphomevalonate kinase, tumor necrosis factor ligand superfamily member 12, sulfhydryl oxidase 2, interleukin-6 receptor subunit alpha, and low-affinity immunoglobulin gamma Fc region receptor II-b might decrease AF risk, while genetically predicted plasma levels of beta-mannosidase, collagen alpha-1(XV) chain, ANXA4 (annexin A4), COF2 (cofilin-2), and RAB1A (Ras-related protein Rab-1A) might increase AF risk (P<3.4×10-5). By using different Mendelian randomization methods and instrumental variable selection thresholds, we performed sensitivity analyses in 30 scenarios to test the robustness of positive findings. Replication analyses were also performed in independent samples to further avoid false-positive findings. Drugs targeting tumor necrosis factor ligand superfamily member 12, interleukin-6 receptor subunit alpha, low-affinity immunoglobulin gamma Fc region receptor II-b, and annexin A4 are approved or in development. The results of the phenome-wide Mendelian randomization analysis showed that changing the plasma levels of phosphomevalonate kinase, cofilin-2, annexin A4, Ras-related protein Rab-1A, sulfhydryl oxidase 2, and collagen alpha-1(XV) chain did not increase the risk of other diseases while decreasing the risk of AF. Conclusions We found a significant causal association between genetically predicted levels of 10 plasma proteins and AF risk. Four of these proteins have drugs targeting them that are approved or in development, and our results suggest the potential for these drugs to treat AF or cause AF. Sulfhydryl oxidase 2, low-affinity immunoglobulin gamma Fc region receptor II-b, and beta-mannosidase have not been suggested by previous laboratory or epidemiological studies to be associated with AF and may reveal new pathophysiological pathways as well as therapeutic targets for AF.

LncRNA TUG1 Exacerbates Myocardial Fibrosis in Diabetic Cardiomyopathy by Modulating the microRNA-145a-5p/Cfl2 Axis.

ABSTRACT: Nowadays, there is limited prevention and treatment for myocardial fibrosis in diabetic cardiomyopathy (DCM). Our study aimed to depict the mechanism of the lncRNA TUG1/miR-145a-5p/Cfl2 axis in DCM and to provide a molecular basis for the study of this disease. Male C57BL/6J mice were intraperitoneally injected with streptozotocin to establish DCM mouse models. The expression levels of lncRNA TUG1, miR-145a-5p, and Cfl2 in myocardial tissues of mice were tested by RT-qPCR or Western blot. Cardiac function was assessed by echocardiography. The contents of Ang-II, TNF-α, and IL-1ß were measured using ELISA. The histopathological observation was performed by HE staining and Masson staining. The expression levels of myocardial fibrosis-related genes COL1A1, MMP2, and FN1 were determined by RT-qPCR. In addition, bioinformatics website, RIP assay, pull-down assay, and luciferase activity assay were conducted to verify the relationships of lncRNA TUG1, miR-145a-5p, and Cfl2. In the DCM mouse model, lncRNA TUG1 and Cfl2 expression levels were upregulated and miR-145a-5p expression was downregulated. Downregulation of lncRNA TUG1 improved cardiac function and myocardial fibrosis; decreased COL1A1, MMP2, and FN1 expression levels; as well as TNF-α, IL-1ß, and Ang-II contents in myocardial tissues of DCM mice. Upregulation of miR-145a-5p showed the same trend as downregulation of lncRNA TUG1. In addition, upregulating miR-145a-5p reversed the promotion roles of lncRNA TUG1 on myocardial fibrosis in DCM mice, and upregulating Cfl2 compromised the improvement effect of downregulated lncRNA TUG1 on myocardial fibrosis in DCM mice. Mechanistically, there was a binding site between lncRNA TUG1 and miR-145a-5p, and miR-145a-5p had a targeting relationship with Cfl2. This study highlights that lncRNA TUG1 sponges miR-145a-5p to aggravate myocardial fibrosis in DCM mice by promoting Cfl2.

Magic angle spinning NMR structure of human cofilin-2 assembled on actin filaments reveals isoform-specific conformation and binding mode.

Actin polymerization dynamics regulated by actin-binding proteins are essential for various cellular functions. The cofilin family of proteins are potent regulators of actin severing and filament disassembly. The structural basis for cofilin-isoform-specific severing activity is poorly understood as their high-resolution structures in complex with filamentous actin (F-actin) are lacking. Here, we present the atomic-resolution structure of the muscle-tissue-specific isoform, cofilin-2 (CFL2), assembled on ADP-F-actin, determined by magic-angle-spinning (MAS) NMR spectroscopy and data-guided molecular dynamics (MD) simulations. We observe an isoform-specific conformation for CFL2. This conformation is the result of a unique network of hydrogen bonding interactions within the α2 helix containing the non-conserved residue, Q26. Our results indicate F-site interactions that are specific between CFL2 and ADP-F-actin, revealing mechanistic insights into isoform-dependent F-actin disassembly.

Circ_0008673 regulates breast cancer malignancy by miR-153-3p/CFL2 axis.

BACKGROUND: Breast cancer is an aggressive tumor, which poses a heavy burden to human health. Circular RNAs have been involved in the pathogenesis of breast cancer. This study aims to investigate whether circ_0008673 mediates breast cancer malignant progression by microRNA-153-3p (miR-153-3p)/cofilin 2 (CFL2) pathway. METHODS: The RNA levels of circ_0008673, miR-153-3p and CFL2 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of CFL2, E-cadherin and N-cadherin was determined by western blot analysis. Cell proliferation was demonstrated through cell counting kit-8 and cell colony-formation assays. Cell apoptosis was detected by flow cytometry analysis. Cell migratory and invasive capacities were determined by transwell assay. The associated relationship between miR-153-3p and circ_0008673 or CFL2 was predicted by online databases, and testified by dual-luciferase reporter and RNA immunoprecipitation assays. In vivo assay was employed to demonstrate the effects of circ_0008673 silencing on tumor formation in vivo. RESULTS: Circ_0008673 and CFL2 expressions were upregulated, while miR-153-3p expression was downregulated in breast cancer tissues and cells compared with adjacent normal breast tissues and cells, respectively. Circ_0008673 overexpression promoted cell proliferation, migration and invasion, and repressed cell apoptosis, while circ_0008673 silencing had opposite effects. Additionally, circ_0008673 served as a sponge of miR-153-3p. And circ_0008673 was proved to regulate breast cancer cell malignancy by sponging miR-153-3p. MiR-153-3p was found to modulate breast cancer cell carcinogenesis via targeting CFL2. Furthermore, circ_0008673 silencing repressed tumor growth in vivo. CONCLUSION: Circ_0008673 promoted breast cancer progression by upregulating CFL2 expression through sponging miR-153-3p. This study provides a theoretical basis for researching circRNA-directed treatment of breast cancer.

Downregulation of Circular RNA PSEN1 ameliorates ferroptosis of the high glucose treated retinal pigment epithelial cells via miR-200b-3p/cofilin-2 axis.

Ferroptosis is a form of programmed cell death that participates in the progression of numerous diseases. Long noncoding RNAs (lncRNAs) are dysregulated in diabetic retinopathy (DR). However, the role of lncRNAs in DR-induced ferroptosis is unclear. Adult retinal pigment epithelial cell line-19 (ARPE19) cells were treated with a high concentration of glucose (high glucose, HG) to mimic DR in vitro. The intracellular contents of glutathione, malondialdehyde, and ferrous ions were analyzed using the corresponding kits. The MTT assay was performed to measure the cell survival rate, and cell death was determined using propidium iodide and terminal deoxynucleotidyl transferase dUTP nick end labeling staining assays. Western blotting was conducted to detect the protein levels of GPX4, SLC7A11, and TFR1. The targeting relationships were verified using luciferase reporter and RNA pull-down assays. circ-PSEN1 was upregulated in HG-treated ARPE19 cells and showed high resistance to RNase R and Act D. Inhibition of circ-PSEN1 in ARPE19 cells ameliorated the ferroptosis induced by HG was ameliorated, as evidenced by changes in the ferroptosis-related biomarkers/genes and decreased cell death. Subsequently, circ-PSEN1 acted as a sponge for miR-200b-3p. Inhibition of miR-200b-3p partially reversed the effects of circ-PSEN1 on ferroptosis. Furthermore, cofilin-2 (CFL2) was the target gene of miR-200b-3p, and it abrogated the inhibitory effect of miR-200b-3p on ferroptosis. Taken together, the findings indicate that knockdown of circ-PSEN1 can mitigate ferroptosis of ARPE19 cells induced by HG via the miR-200b-3p/CFL2 axis.

Platycodin D reverses histone deacetylase inhibitor resistance in hepatocellular carcinoma cells by repressing ERK1/2-mediated cofilin-1 phosphorylation.

BACKGROUND: Chemoresistance remains the main obstacle in hepatocellular carcinoma (HCC) therapy. Despite significant advances in HCC therapy, HCC still has a poor prognosis. Thus, there is an urgent need to identify a treatment target to reverse HCC chemotherapy resistance. Platycodon grandiflorus (PG) is a perennial herb that has been used as food and traditional Chinese medicine for thousands of years in Northeast Asia. Platycodin D (PD), a main active triterpenoid saponin found in the root of PG, has been reported to possess anticancer properties in several cancer cell lines, including HCC; however, the reversal effect of this molecule on HCC chemoresistance remains largely unknown. PURPOSE: This study aimed to investigate the role and the mechanism of PD-mediated reversal of the histone deacetylase inhibitor (HDACi) resistance in HCC cells. METHODS: Human HCC cells (HA22T) and HDACi-resistant (HDACi-R) cells were used. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Combination index was used to calculate the synergism potential. Expression of ERK1/2 (total/phospho), cofilin-1 (total/phospho) and apoptosis-related protein was determined using western blotting. Mitochondrial membrane potential was assessed using the JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide) probe. Apoptosis was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Mitochondrial reactive oxygen species generation was measured using the MitoSOX Red fluorescent probe. RESULTS: We found that PD treatment inhibited cell viability both in HA22T HCC and HDACi-R cells. Inhibition of ERK1/2 by PD98059 could reverse drug resistance in HDACi-R cells treated with PD98059 and PD. Nevertheless, pre-treatment with U46619, an ERK1/2 activator, rescued PD-induced apoptosis by decreasing levels of apoptosis-related proteins in HCC cells. The combined treatment of PD with apicidin a powerful HDACi, dramatically enhanced the apoptotic effect in HDACi-R cells. CONCLUSION: For the first time, we showed that PD reversed HDACi resistance in HCC by repressing ERK1/2-mediated cofilin-1 phosphorylation. Thus, PD can potentially be a treatment target to reverse HCC chemotherapy resistance in future therapeutic trials.

The Cardioprotective PKA-Mediated Hsp20 Phosphorylation Modulates Protein Associations Regulating Cytoskeletal Dynamics.

The cytoskeleton has a primary role in cardiomyocyte function, including the response to mechanical stimuli and injury. The small heat shock protein 20 (Hsp20) conveys protective effects in cardiac muscle that are linked to serine-16 (Ser16) Hsp20 phosphorylation by stress-induced PKA, but the link between Hsp20 and the cytoskeleton remains poorly understood. Herein, we demonstrate a physical and functional interaction of Hsp20 with the cytoskeletal protein 14-3-3. We show that, upon phosphorylation at Ser16, Hsp20 translocates from the cytosol to the cytoskeleton where it binds to 14-3-3. This leads to dissociation of 14-3-3 from the F-actin depolymerization regulator cofilin-2 (CFL2) and enhanced F-actin depolymerization. Importantly, we demonstrate that the P20L Hsp20 mutation associated with dilated cardiomyopathy exhibits reduced physical interaction with 14-3-3 due to diminished Ser16 phosphorylation, with subsequent failure to translocate to the cytoskeleton and inability to disassemble the 14-3-3/CFL2 complex. The topological sequestration of Hsp20 P20L ultimately results in impaired regulation of F-actin dynamics, an effect implicated in loss of cytoskeletal integrity and amelioration of the cardioprotective functions of Hsp20. These findings underscore the significance of Hsp20 phosphorylation in the regulation of actin cytoskeleton dynamics, with important implications in cardiac muscle physiology and pathophysiology.

Phosphorylated cofilin-2 is more prone to oxidative modifications on Cys39 and favors amyloid fibril formation.

Cofilins are small protein of the actin depolymerizing family. Actin polymerization/depolymerization is central to a number of critical cellular physiological tasks making cofilin a key protein for several physiological functions of the cell. Cofilin activity is mainly regulated by phosphorylation on serine residue 3 making this post-translational modification key to the regulation of myofilament integrity. In fact, in this form, the protein segregates in myocardial aggregates in human idiopathic dilated cardiomyopathy. Since myofilament network is an early target of oxidative stress we investigated the molecular changes induced by oxidation on cofilin isoforms and their interplay with the protein phosphorylation state to get insight on whether/how those changes may predispose to early protein aggregation. Using different and complementary approaches we characterized the aggregation properties of cofilin-2 and its phosphomimetic variant (S3D) in response to oxidative stress in silico, in vitro and on isolated cardiomyocytes. We found that the phosphorylated (inactive) form of cofilin-2 is mechanistically linked to the formation of an extended network of fibrillar structures induced by oxidative stress via the formation of a disulfide bond between Cys39 and Cys80. Such phosphorylation-dependent effect is likely controlled by changes in the hydrogen bonding network involving Cys39. We found that the sulfide ion inhibits the formation of such structures. This might represent the mechanism for the protective effect of the therapeutic agent Na2S on ischemic injury.

Knockin mouse model of the human CFL2 p.A35T mutation results in a unique splicing defect and severe myopathy phenotype.

Cofilin-2 is an actin-binding protein that is predominantly expressed in skeletal and cardiac muscles and belongs to the AC group of proteins, which includes cofilin-1 and destrin. In humans, cofilin-2 (CFL2) mutations have been associated with congenital myopathies that include nemaline and myofibrillar myopathy. To understand the pathogenicity of the human CFL2 mutation, p.A35T, that first linked cofilin-2 with the human disease, we created a knock-in mouse model. The Cfl2A35T/A35T (KI) mice were indistinguishable from their wild-type littermates at birth, but they rapidly worsened and died by postnatal day 9. The phenotypic, histopathologic and molecular findings mimicked the constitutive Cfl2-knockout (KO) mice described previously, including sarcomeric disruption and actin accumulations in skeletal muscles and negligible amounts of cofilin-2 protein. In addition, KI mice demonstrated a marked reduction in Cfl2 mRNA levels in various tissues including skeletal muscles. Further investigation revealed evidence of alternative splicing with the presence of two alternate transcripts of smaller size. These alternate transcripts were expressed at very low levels in the wild-type mice and were significantly upregulated in the mutant mice, indicating that pre-translational splicing defects may be a critical component of the disease mechanism associated with the mutation. Evidence of reduced expression of the full-length CFL2 transcript was also observed in the muscle biopsy sample of the patient with p.A35T mutation.

Long noncoding RNA SOX2-OT facilitates prostate cancer cell proliferation and migration via miR-369-3p/CFL2 axis.

Accepted as crucial participators in human malignancies, long noncoding RNAs (lncRNAs) have been proven to exert significant function on the complicated processes of cancer progression. Although existing investigations have revealed the oncogenic role of lncRNA SOX2 overlapping transcript (SOX2-OT) in different kinds of cancers, such as osteosarcoma and cholangiocarcinoma, the potential role of it in prostate cancer (PC) is poorly understood. This study was the first attempt to decipher the underlying regulatory mechanism of SOX2-OT in PC. According to the data from this study, SOX2-OT expression was conspicuously elevated in PC tissues and cells. Silenced SOX2-OT could repress PC cell proliferation and migration. Besides, mechanism assays manifested that SOX2-OT bound with miR-369-3p and negatively correlated with miR-369-3p in PC. Additionally, miR-369-3p was confirmed to elicit suppressive impact on PC progression. What's more, cofilin 2 (CFL2) was testified to be a downstream target gene of miR-369-3p. Final rescue tests uncovered that CFL2 upregulation or miR-369-3p inhibition could largely restore SOX2-OT knockdown-mediated function on PC progression. To sum up, SOX2-OT accelerates cell proliferation and migration by targeting miR-369-3p/CFL2 axis in PC.

Identification of the intermediate filament protein synemin/SYNM as a target of myocardin family coactivators.

Myocardin (MYOCD) is a critical regulator of smooth muscle cell (SMC) differentiation, but its transcriptional targets remain to be exhaustively characterized, especially at the protein level. Here we leveraged human RNA and protein expression data to identify novel potential MYOCD targets. Using correlation analyses we found several targets that we could confirm at the protein level, including SORBS1, SLMAP, SYNM, and MCAM. We focused on SYNM, which encodes the intermediate filament protein synemin. SYNM rivalled smooth muscle myosin (MYH11) for SMC specificity and was controlled at the mRNA and protein levels by all myocardin-related transcription factors (MRTFs: MYOCD, MRTF-A/MKL1, and MRTF-B/MKL2). MRTF activity is regulated by the ratio of filamentous to globular actin, and SYNM was accordingly reduced by interventions that depolymerize actin, such as latrunculin treatment and overexpression of constitutively active cofilin. Many MRTF target genes depend on serum response factor (SRF), but SYNM lacked SRF-binding motifs in its proximal promoter, which was not directly regulated by MYOCD. Furthermore, SYNM resisted SRF silencing, yet the time course of induction closely paralleled that of the SRF-dependent target gene ACTA2. SYNM was repressed by the ternary complex factor (TCF) FLI1 and was increased in mouse embryonic fibroblasts lacking three classical TCFs (ELK1, ELK3, and ELK4). Imaging showed colocalization of SYNM with the intermediate filament proteins desmin and vimentin, and MRTF-A/MKL1 increased SYNM-containing intermediate filaments in SMCs. These studies identify SYNM as a novel SRF-independent target of myocardin that is abundantly expressed in all SMCs.

Codon usage regulates human KRAS expression at both transcriptional and translational levels.

KRAS and HRAS are highly homologous oncogenic Ras GTPase family members that are mutated in a wide spectrum of human cancers. Despite having high amino acid identity, KRAS and HRAS have very different codon usage biases: the HRAS gene contains many common codons, and KRAS is enriched for rare codons. Rare codons in KRAS suppress its protein expression, which has been shown to affect both normal and cancer biology in mammals. Here, using HRAS or KRAS expression in different human cell lines and in vitro transcription and translation assays, we show that KRAS rare codons inhibit both translation efficiency and transcription and that the contribution of these two processes varies among different cell lines. We observed that codon usage regulates mRNA translation efficiency such that WT KRAS mRNA is poorly translated. On the other hand, common codons increased transcriptional rates by promoting activating histone modifications and recruitment of transcriptional coactivators. Finally, we found that codon usage also influences KRAS protein conformation, likely because of its effect on co-translational protein folding. Together, our results reveal that codon usage has multidimensional effects on protein expression, ranging from effects on transcription to protein folding in human cells.

Cofilin-2 Acts as a Marker for Predicting Radiotherapy Response and Is a Potential Therapeutic Target in Nasopharyngeal Carcinoma.

BACKGROUND The purpose of this study was to determine whether cofilin-2 could serve as a protein marker for predicting radiotherapy response and as a potential therapeutic target in nasopharyngeal carcinoma (NPC). MATERIAL AND METHODS Cofilin-2 protein levels in serum and tissue samples from patients with NPC were assessed by sandwich ELISA and IHC. In vitro, cofilin-2 levels in CNE-2R cells were significantly higher than those of CNE-2 cells. Meanwhile, CNE-2R cells were silenced for cofilin-2 to obtain a stable cofilin-2-RNAi-LV3 cell line. Then, cell proliferation, radiosensitivity, invasion and migration abilities, cell cycle, and apoptosis were evaluated by Cell Counting Kit 8 assay (CCK-8), flow cytometry (FCM), clone formation assay, and in vitro. RESULTS The secreted levels of the cofilin-2 protein in radioresistant NPC patients were significantly higher than those of radiosensitive cases. After cofilin-2 knockdown in nasopharyngeal carcinoma CNE-2R cells, proliferation was decreased, while apoptosis and radiosensitivity were enhanced; cell cycle distribution was altered, and the transplanted tumors in nude mice grew significantly less. CONCLUSIONS Overall, our findings suggest that cofilin-2 acts as a marker for predicting radiotherapy response and is a potential therapeutic target in nasopharyngeal carcinoma.

miR-3189-3p Mimics Enhance the Effects of S100A4 siRNA on the Inhibition of Proliferation and Migration of Gastric Cancer Cells by Targeting CFL2.

GDF15 is a downstream gene of S100A4. miR-3189 is embedded in the intron of GDF15-and coexpressed with it. miR-3189-3p functions to inhibit the proliferation and migration of glioblastoma cells. We speculated that S100A4 might regulate miR-3189-3p to affect its function in gastric cancer cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that miR-3189-3p expression was significantly downregulated in MGC803 cells after S100A4 knockdown. Overexpression of miR-3189-3p significantly inhibited the proliferation and migration of the cells. Moreover, miR-3189-3p mimics enhanced the effects of an S100A4 siRNA on the inhibition of cell proliferation and migration. Dual luciferase reporter assays, qRT-PCR, and Western blotting verified that CFL2 is a direct target of miR-3189-3p. CFL2 mediates the regulation of miR-3189-3p on the proliferation and migration of MGC803 cells. Data mining based on Kaplan-Meier plots showed that high CFL2 expression is associated with poor overall survival and first progression in gastric cancer. These data suggested that miR-3189-3p mimics enhanced the effects of the S100A4 siRNA on the inhibition of gastric cancer cell proliferation and migration by targeting CFL2. The findings suggested that when targeting S100A4 to treat gastric cancer, consideration and correction for counteracting factors should obtain a satisfactory effect.

Uncovering the proteome response of murine neuroblastoma cells against low-dose exposure to saxitoxin.

The potent neurotoxin saxitoxin produced by both marine and freshwater phytoplankton causes paralytic shellfish poisoning syndrome. The toxicity and mode of action of the acute exposure of high-dose saxitoxin have been intensively studied for decades; however, the potential risk of exposure of low-dose saxitoxin remained to be uncovered. Here we present a proteomics study of murine neuroblastoma N2A cell with low-dose saxitoxin exposure (1 nM and 10 nM, 24-h intoxication). Differential proteins were profiled by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). A total of 9 proteins, including 14-3-3 beta (1433B), alpha enolase (ENO1) and cofilin 2 (CFL2), were altered by the low-dose saxitoxin exposure. We further validated the expressions of 1433B, ENO1 and CFL2 by Western blot analysis and the enzyme-linked immunosorbent assay. These 9 proteins involve cell apoptotic pathways, cell skeleton maintenance, membrane potentials and mitochondrial functions. Modulation of these 9 proteins by low-dose saxitoxin exposure could correlate to the reports on genotoxicity and neurotoxicity induced by saxitoxin. This study also suggested other potential risks of saxitoxin.

MicroRNA miR-301a is a novel cardiac regulator of Cofilin-2.

Calsarcin-1 deficient mice develop dilated cardiomyopathy (DCM) phenotype in pure C57BL/6 genetic background (Cs1-ko) despite severe contractile dysfunction and robust activation of fetal gene program. Here we performed a microRNA microarray to identify the molecular causes of this cardiac phenotype that revealed the dysregulation of several microRNAs including miR-301a, which was highly downregulated in Cs1-ko mice compared to the wild-type littermates. Cofilin-2 (Cfl2) was identified as one of the potential targets of miR-301a using prediction databases, which we validated by luciferase assay and mutation of predicted binding sites. Furthermore, expression of miR-301a contrastingly regulated Cfl2 expression levels in neonatal rat ventricular cardiomyocytes (NRVCM). Along these lines, Cfl2 was significantly upregulated in Cs1-ko mice, indicating the physiological association between miR-301a and Cfl2 in vivo. Mechanistically, we found that Cfl2 activated serum response factor response element (SRF-RE) driven luciferase activity in neonatal rat cardiomyocytes and in C2C12 cells. Similarly, knockdown of miR301a activated, whereas, its overexpression inhibited the SRF-RE driven luciferase activity, further strengthening physiological interaction between miR-301a and Cfl2. Interestingly, the expression of SRF and its target genes was strikingly increased in Cs1-ko suggesting a possible in vivo correlation between expression levels of Cfl2/miR-301a and SRF activation, which needs to be independently validated. In summary, our data demonstrates that miR-301a regulates Cofilin-2 in vitro in NRVCM, and in vivo in Cs1-ko mice. Our findings provide an additional and important layer of Cfl2 regulation, which we believe has an extended role in cardiac signal transduction and dilated cardiomyopathy presumably due to the reported involvement of Cfl2 in these mechanisms.

Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy.

Cellular actin dynamics is an essential element of numerous cellular processes, such as cell motility, cell division and endocytosis. Actin's involvement in these processes is mediated by many actin-binding proteins, among which the cofilin family plays unique and essential role in accelerating actin treadmilling in filamentous actin (F-actin) in a nucleotide-state dependent manner. Cofilin preferentially interacts with older filaments by recognizing time-dependent changes in F-actin structure associated with the hydrolysis of ATP and release of inorganic phosphate (Pi) from the nucleotide cleft of actin. The structure of cofilin on F-actin and the details of the intermolecular interface remain poorly understood at atomic resolution. Here we report atomic-level characterization by magic angle spinning (MAS) NMR of the muscle isoform of human cofilin 2 (CFL2) bound to F-actin. We demonstrate that resonance assignments for the majority of atoms are readily accomplished and we derive the intermolecular interface between CFL2 and F-actin. The MAS NMR approach reported here establishes the foundation for atomic-resolution characterization of a broad range of actin-associated proteins bound to F-actin.

Changes in the Level of Circulating hsa-miR-297 and hsa-miR-19b-3p miRNA Are Associated with Generalization of Prostate Cancer.

We performed diagnostic classification of plasma specimens from patients with non-metastatic and metastatic prostate cancer based on pairs of miRNA that have no individual diagnostic significance. Of 230 miRNA detected in plasma specimens, 3 pairs were diagnostically significant. The miRNA pair hsa-miR-19b-3p and hsa-miR-297 demonstrated highest sensitivity and specificity. Among common target genes of these miRNA, CFL2 gene associated with cell mobility was detected.

P2Y12 Promotes Migration of Vascular Smooth Muscle Cells Through Cofilin Dephosphorylation During Atherogenesis.

OBJECTIVE: P2Y12 is a well-recognized receptor expressed on platelets and the target of thienopyridine-type antiplatelet drugs. However, recent evidence suggests that P2Y12 expressed in vessel wall plays a role in atherogenesis, but the mechanisms remain elusive. In this study, we examined the molecular mechanisms of how vessel wall P2Y12 mediates vascular smooth muscle cells (VSMCs) migration and promotes the progression of atherosclerosis. APPROACH AND RESULTS: Using a high-fat diet-fed apolipoprotein E-deficient mice model, we found that the expression of P2Y12 in VSMCs increased in a time-dependent manner and had a linear relationship with the plaque area. Moreover, administration of P2Y12 receptor antagonist for 12 weeks caused significant reduction in atheroma and decreased the abundance of VSMCs in plaque. In cultured VSMCs, we found that activation of P2Y12 receptor inhibited cAMP/protein kinase A signaling pathway, which induced cofilin dephosphorylation and filamentous actin disassembly, thereby enhancing VSMCs motility and migration. In addition, the number of P2Y12-positive VSMCs was decreased in the carotid artery plaque from patients receiving clopidogrel. CONCLUSIONS: Vessel wall P2Y12 receptor, which promotes VSMCs migration through cofilin dephosphorylation, plays a critical role in the development of atherosclerotic lesion and may be used as a therapeutic target for atherosclerosis.