Cofilin Research Articles

Effect of PGK1 on radiosensitivity through regulation of CIN and CFL1 in human glioma cells.

e14631 Background: Phosphoglycerate kinase 1 (PGK1) has been detected overexpressed in many malignancies and usually correlated with increased tumorigenesis and poor survival. Our previous study revealed that PGK1 significantly up-regulated and positively correlated with cofilin-1 (CFL1) expression in radioresistant astrocytomas samples. However, the further mechanism remains unknown. Methods: In the present study, functional interactions among PGK1, chronophin (CIN) and CFL1 were analyzed by co-immunoprecipitation and immunofluorescence experiments. Transfection of shRNA and pcDNA3.1-plasmid were respectively utilized to silence and overexpress PGK1 or CIN in both normal glioma cells (U251 and U373) and estabilshed radioresistant (RR-U251 and RR-U373) cells. The effect of PGK1 on CIN and CFL1 expression levels was investigated with Western-blot method; while the effect of PGK1 or CIN on radiosensitivity of glioma cells was evaluated using cell viability, migration and invasion assays after irradiation. Results: Cell proliferation, migration and invasion abilities significantly increased in RR-U251 and RR-U373 cells compared with those of normal cells, indicating the successful establishment of RR-glioma cell models. Laser confocal experiment in vitro showed the colocalization of PGK1 and CIN. Meanwhile, PGK1 specifically and directly combined with CIN, while did not interplay with CFL1. Elevated PGK1 expression level was observed in radioresistant cells compared with that of normal cells. The western blot analysis showed that CFL1 and CIN levels were positively correlated with the PGK1 expression in both normal and radioresistant cells. The proliferation, migration and invasion capabilities significantly decreased in PGK1-silenced and CIN-silenced glioma cells, indicating the improvement of the radiosensitivity of radioresistant cells. Conclusions: Our research implied the effect of CIN-mediated PGK1 regulation on CFL1 level, thereby altering the migration and invasion abilities in nomal and notably radioresistant glioma cells. These findings clarify a novel underlying mechanism of radioresistance in glioma cells and provide valid basis for further explorations concerning radiotherapy.

Hypoxia aggravates Aβ1-42-induced toxicity in human neuroblastoma cells through PAK1/LIMK1/cofilin pathway

Objective To investigate the pathways by which hypoxia aggravates the neurotoxic effects of amyloid-beta protein(Aβ)on neurons. Methods Human neuroblastoma cells(SH-SY5Y cells)were cultured in vitro, and were divided into four groups: the control group, Aβ intervention group, hypoxia group, Aβ intervention plus hypoxia group.Quantitative real time polymerase chain reaction(qRT-PCR)was adopted to detect the mRNA expression levels of p21-activated kinase 1(PAK1), LIM kinase 1 protein(LIMK1)and cofilin.Western blot was used to measure the protein levels of PAK1, LIMK1, phosphate-LIMK1(P-LIMK1), cofilin and phosphate-cofilin(P-cofilin). Results After Aβ treatment, the activity of SH-SY5Y cells was decreased.Compared with the control group, the protein levels of PAK1, LIMK1, P-LIMK1, P-cofilin, and the mRNA expression levels of PAK1 and LIMK1 were decreased(all P<0.05), but the protein and mRNA expression of cofilin had no significant changes after 24 h of treatment with 10 μmol/L Aβ.Compared with the Aβ intervention group, the protein levels of PAK1, LIMK1, P-LIMK1 and P-cofilin were decreased(all P<0.05), and the mRNA expression levels of PAK1 and LIMK1 were decreased(both P<0.05), but the protein and mRNA expression of cofilin had no significant changes after 24 h of treatment of SH-SY5Y cells with 10 μmol/L Aβ plus 2% oxygen. Conclusions Aβ may reduce P-LIMK1 expression by inhibiting the activity of PAK1, thereby reducing the P-cofilin, increasing the formation of dephosphorylated cofilin, leading to neural cells damage, and hypoxia aggravates the neurotoxicity of Aβ through this pathway. Key words: Alzheimer disease; Amyloid beta-protein; Anoxia

Identify Cross Talk Between Circadian Rhythm and Coronary Heart Disease by Multiple Correlation Analysis.

Disorder in circadian rhythm has been revealed as a risk factor for coronary heart disease. Several studies in molecular biology established a gene interaction network using coronary heart susceptibility genes and the circadian rhythm pathway. However, cross talk between genes was mostly discovered in single gene pairs. There might be combination sets of genes intergraded as a unit to regulate the network. To resolve multiple variables in coronary heart susceptibility genes controlling circadian rhythm pathways, a multiple correlation analysis was applied to the transcriptome. Nine genes, including CUGBP, Elav-like family member (CELF); sodium leak channel, nonselective (NALCN); protein phosphatase 2 regulatory subunit B gamma (PPP2R2C); tubulin alpha 1c (TUBA1C); microtubule-associated protein 4 (MAP4); cofilin 1 (CFL1); myosin heavy chain 7 (MYH7); QKI, KH domain containing RNA binding (QKI); and maternal embryonic leucine zipper kinase (MELK), from coronary heart susceptibility were identified to predict the outcome of a linear combination of circadian rhythm pathway genes with R factor more than 0.7. G protein subunit alpha o1 (GNAO1), protein kinase C gamma (PRKCG), RBX, and G protein subunit beta 1 (GNB1) in the circadian rhythm pathway are characterized as combination variables to coexpress with coronary heart susceptibility genes.

Regulation of cofilin phosphorylation in glomerular podocytes by testis specific kinase 1 (TESK1)

Expression of a constitutively active Rho A (V14Rho) in podocytes in vivo induces albuminuria and foot process (FP) effacement. These effects may be mediated by the Rho A effector Rho kinase (ROK); but inhibition of ROK with Y27632 failed to attenuate albuminuria or FP effacement in V14Rho mice. ROK activates LIM kinases (LIMKs), which phosphorylate and inhibit the actin depolymerizing factor cofilin 1 (CFL1). Sustained phosphorylation of CFL1 is implicated in human nephrotic diseases, but Y27632 did not inhibit phosphorylation of CFL1 in vivo, despite effective ROK inhibition. CFL1 is also phosphorylated by testis-specific kinase 1 (TESK1) on the same serine residue. TESK1 was expressed in podocytes, and, similar to the in vivo situation, Y27632 had little effect on phospho-CFL1 (pCFL1) levels in cultured podocytes. In contrast, Y27632 reduced pCFL1 levels in TESK1 knockout (KO) cells. ROK inhibition enhanced podocyte motility but, the motility promoting effect of Y27632 was absent in TESK1 KO podocytes. Thus, TESK1 regulates podocyte cytoskeletal dynamics in glomerular podocytes and may play an important role in regulating glomerular filtration barrier integrity in glomerular disease processes.

Immunopeptidomic Profiling of HLA-A2-Positive Triple Negative Breast Cancer Identifies Potential Immunotherapy Target Antigens.

The recent development in immune checkpoint inhibitors and chimeric antigen receptor (CAR) T‐cells in the treatment of cancer has not only demonstrated the potency of utilizing T‐cell reactivity for cancer therapy, but has also highlighted the need for developing new approaches to discover targets suitable for such novel therapeutics. Here we analyzed the immunopeptidomes of six HLA‐A2‐positive triple negative breast cancer (TNBC) samples by nano‐ultra performance liquid chromatography tandem mass spectrometry (nUPLC‐MS2). Immunopeptidomic profiling identified a total of 19 675 peptides from tumor and adjacent normal tissue and 130 of the peptides were found to have higher abundance in tumor than in normal tissues. To determine potential therapeutic target proteins, we calculated the average tumor‐associated cohort coverage (aTaCC) that represents the percentage coverage of each protein in this cohort by peptides that had higher tumoral abundance. Cofilin‐1 (CFL‐1), interleukin‐32 (IL‐32), proliferating cell nuclear antigen (PCNA), syntenin‐1 (SDCBP), and ribophorin‐2 (RPN‐2) were found to have the highest aTaCC scores. We propose that these antigens could be evaluated further for their potential as targets in breast cancer immunotherapy and the small cohort immunopeptidomics analysis technique could be used in a wide spectrum of target discovery. Data are available via ProteomeXchange with identifier PXD009738.

Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation.

Expression of UGP2 and CFL1 expression levels in benign and malignant pancreatic lesions and their clinicopathological significance

BackgroundThis study investigated UGP2 (uridine diphosphate-glucose pyrophosphorylase-2) and CFL1 (cofilin-1) expression in pancreatic ductal carcinoma (PDC), paracancerous tissue (PT), benign lesions (BL), and normal tissue (NT) and their clinicopathological significance.MethodsSurgical specimens, which were collected from 106 cases of pancreatic ductal carcinoma, 35 cases of paracancerous tissues, 55 cases of benign lesions and 13 cases of normal pancreatic tissues, were fixed with 4% formaldehyde to prepare conventional paraffin-embedded sections. EnVision immunohistochemical was used to stain for UGP2 and CFL1. Kaplan-Meier survival analysis was performed to assess the correlation of expression pattern with survival.ResultsWe found that positive UGP2 and CFL1 expression in PDC were significantly higher than those in PT, BL, and NT. In PT and BL with positive UGP2 and CFL1 expression, mild to severe atypical hyperplasia or intraepithelial neoplasia of grades II–III was observed in ductal epithelium. Positive UGP2 and CFL1 expression in cases with high differentiation, no lymph node metastasis, no surrounding invasion, and TNM (tumor-node-metastasis) staging I or/and II were significantly lower than those in cases with poor differentiation, lymph node metastasis, surrounding invasion, and TNM stage III and/or IV. Positive UGP2 expression in male patients was significantly lower than that in female patients. UGP2 and CFL1 expression in PDC were positively correlated. Kaplan-Meier survival analysis showed the degree of differentiation, tumor maximal diameter, TNM stage, lymph node metastasis, and surrounding invasion, and UGP2 and CFL1 expression were closely related to the average survival time of patients with PDC. The survival time of patients with positive UGP2 and CFL1 expression was significantly shorter than that of patients with negative expression. Cox multivariate analysis showed that poor differentiation, tumor maximal diameter ≥ 3 cm, TNM stage III or IV, lymph node metastasis, surrounding invasion, and positive UGP2 and CFL1 expression was negatively correlated with the postoperative survival rate and positively correlated with the mortality of patients with PDC.ConclusionPositive expression of UGP2 and CFL1 can serve a valuable prognostic factor in pancreatic cancer.

Detection of sputum cofilin-1 as indicator of malignancy.

Cofilin-1 (CFL1), a small protein of 18 kDa, has been studied as a biomarker due to its involvement in tumor cell migration and invasion. Our aim was to evaluate CFL1 as an indicator of malignancy and aggressiveness in sputum samples. CFL1 was analyzed by ELISA immunoassay in the sputum of 73 lung cancer patients, 13 cancer-free patients, and 6 healthy volunteers. Statistical analyses included ANOVA, ROC curves, Spearman correlation, and logistic regression. Sputum CFL1 levels were increased in cancer patients compared to cancer-free patients and volunteers (P<0.05). High expression of sputum CFL1 was correlated to T4 stage (P=0.01) and N stage (P=0.03), tobacco history (P=0.01), and squamous cell carcinoma histologic type (P=0.04). The accuracy of sputum CFL1 in discriminating cancer patients from cancer-free patients and healthy volunteers were 0.78 and 0.69, respectively. CFL1 at a cut-off value of 415.25 pg/mL showed sensitivity/specificity of 0.80/0.70 in differentiating between healthy volunteers and cancer patients. Sputum CFL1 was also able to identify cancer-free patients from patients with lung cancer. The AUC was 0.70 and, at a cut-off point ≥662.63 pg/mL, we obtained 60% sensitivity and 54% specificity. Logistic regression analysis controlled for tobacco history, histologic types, and N stage showed that cancer cell-associated CFL1 was an independent predictor of death. Smoker patients with squamous cell carcinoma, lymph node metastasis and sputum CFL1>1.475 pg/mL showed augmented chance of death, suggesting lung cancer aggressiveness. CFL1 presented diagnostic value in detecting lung cancer and was associated to tumor aggressiveness.

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.

MiR-519d-3p suppresses breast cancer cell growth and motility via targeting LIM domain kinase 1.

Breast cancer is the most common female cancer in women, and its estrogen receptor (ER)-negative subtype (ENBC) and triple-negative subtype (TNBC) have unfavorable prognosis in comparison with ER-positive subtype. MiRNAs are small noncoding RNAs that bind to the 3'-UTR region of targeting mRNAs to regulate gene expression. Mir-519d-3p was found to be associated with breast cancer for its potential role in proliferation and metastasis. To explore its potential role and mechanism of miR-519d-3p in breast carcinogenesis, we determined whether miR-519d-3p regulates breast cancer cell proliferation and motility by performing wound-healing assays and migration-invasion assays. We found that miR-519d-3p significantly inhibits proliferation and motility of ENBC and TNBC cells. Overexpression of miR-519d-3p arrested breast cancer cells in the G0/G1 phase and reduced the expression of CDK4, 6/Cyclin D1, and CDK2/Cyclin E1. It was reported that miR-519d-3p or miR-519d-3p expression was associated with cancer metastasis and clinical staging. Since LIM domain kinase 1 (LIMK1) was highly expressed in breast cancer and a major regulator of breast cancer growth and metastasis, we further demonstrated that LIMK1 is a potential target of miR-519d-3p by dual-luciferase report assay. Mir-519d-3p decreases LIMK1 expression at mRNA and protein levels, and the protein level and phosphorylation of cofilin 1 (CFL1), one of the key downstream targets of LIMK1. Our findings suggest that miR-519d-3p regulates the LIMK1/CFL1 pathway in breast cancer and this new venue could be targeted for future breast cancer therapy.

Role of the cofilin 2 gene in regulating the myosin heavy chain genes in mouse myoblast C2C12 cells.

The cofilin2 (CFL2) and myosin heavy chain(MyHC) genes play a key role in muscle development and myofibrillar formation. The aim of the present study was to investigate the effect of CFL2 on genes involved in fiber formation and the mechanisms underlying this process. Undifferentiated and differentiated C2C12 cells (UDT and DT, respectively) were transfected with CFL2 small interfering RNA (siRNA). CFL2 mRNA and protein levels were assessed using reverse transcription polymerase chain reaction (RT-PCR) and western blotting, respectively. MyHC gene expression in UDT and signaling pathway-related factors were observed with quantitative PCR (RT‑qPCR) and western blotting. Fluorescence microscopy was used to analyze the cytoskeletal effects of CFL2. The mRNA and protein expressions of CFL2, four MyHC isoforms (MyHC-I, MyHC-IIa, MyHC-IIb and MyHC-IIx), p38 mitogen-activated protein kinase, cAMP-response element-binding protein, AMP-activated protein kinaseα1, and myocyte enhancer factor2C,were significantly decreased in UDT. However, extracellular signal-regulated kinase2expression was significantly increased. Slightly decreased CFL2 protein and mRNA expression was observed in DT C2C12 cells transfected with CFL2 siRNA. Fluorescence microscopy revealed a significant decrease of CFL2 in the cytoplasm, but not the nucleus, of UDT, compared with normal cells. These results indicated that the mouse CFL2 gene may be involved in the regulation of MyHC via the key signaling molecules of CFL2-related signaling pathways.

The microRNAs miR-200b-3p and miR-429-5p target the LIMK1/CFL1 pathway to inhibit growth and motility of breast cancer cells

Triple-negative breast cancer (TNBC) has the worst prognosis of all subtypes of breast cancer (BC), with limited options for conventional therapy and no targeted therapies. MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression. In this study, we aimed to determine whether two members of the miR-200 family, miR-200b-3p and miR-429-5p, are involved in BC cell proliferation and motility and to elucidate their target genes and pathways. We performed a meta-analysis that reveals down-regulated expression of miR-200b-3p and miR-429-5p in BC tissues and cell lines, consistent with a lower expression of miR-200b-3p and miR-429-5p in MDA-MB-231 and HCC1937 cells than in MCF-7 and MCF-10 cells. Overexpression of miR-200b-3p and miR-429-5p significantly inhibited the proliferation, migration, and invasion of TNBC cells; suppressed the expression of markers for proliferation and metastasis in TNBC cells. We next demonstrated that LIM domain kinase 1 (LIMK1) is a direct target gene of miR-200b-3p and miR-429-5p. Inhibition of LIMK1 reduced the expression and phosphorylation of cofilin 1 (CFL1), which polymerizes and depolymerizes F-actin and G-actin to reorganize cellular actin cytoskeleton. In addition, transfection with mimics for miR-200b-3p and miR-429-5p arrested G2/M and G0/G1 cell cycles respectively, suppressed the expression of the cell cycle–related complexes, cyclin D1/CDK4/CDK6 and cyclin E1/CDK2, in TNBC cells. In conclusion, miR-200b-3p and miR-429-5p suppress proliferation, migration, and invasion in TNBC cells, via the LIMK1/CFL1 pathway. These results provide insight into how specific miRNAs regulate TNBC progression and suggest that the LIMK1/CFL1 pathway is a therapeutic target for treating TNBC.

Altered protein expression pattern in colon tissue of mice upon supplementation with distinct selenium compounds.

The essential trace element selenium (Se) is controversially discussed concerning its role in health and disease. Its various physiological functions are largely mediated by Se incorporation in the catalytic center of selenoproteins. In order to gain insights into the impact of Se deficiency and of supplementation with different Se compounds (selenite, selenate, selenomethionine) at defined concentrations (recommended, 150μg/kg diet; excessive, 750μg/kg diet) in murine colon tissues, a 20-week feeding experiment was performed followed by analysis of the protein expression pattern of colon tissue specimens by 2D-DIGE and MALDI-TOF MS. Using this approach, 24 protein spots were identified to be significantly regulated by the different Se compounds. These included the antioxidant enzyme peroxiredoxin-5 (PRDX5), proteins with binding capabilities, such as cofilin-1 (COF1), calmodulin, and annexin A2 (ANXA2), and proteins involved in catalytic processes, such as 6-phosphogluconate dehydrogenase (6PGD). Furthermore, the Se compounds demonstrated a differential impact on the expression of the identified proteins. Selected target structures were validated by qPCR and Western blot which mainly confirmed the proteomic profiling data. Thus, novel Se-regulated proteins in colon tissues have been identified, which expand our understanding of the physiologic role of Se in colon tissue.

Cofilin 1 induces the epithelial-mesenchymal transition of gastric cancer cells by promoting cytoskeletal rearrangement.

Epithelial-mesenchymal transition (EMT) is an important biological process whereby malignant tumor cells obtain the ability to migrate, invade, resist apoptosis and degrade the extracellular matrix. We found that Cofilin1 (CFL1) expression was elevated in clinical gastric cancer specimens and correlated with biomarkers of EMT in BGC-823 gastric cancer cells. BGC-823 cells exhibited EMT phenotypes and increased metastatic ability when induced by TGF-β1. By contrast, BGC-823 cells transfected with Lv-siRNA-CFL1 did not exhibit EMT phenotypes under the same inducing conditions. As CFL1 expression increased, EMT cell filopodia stretched out. In addition, the ultrastructures observed using transmission electron microscopy indicated that silencing of CFL1 markedly inhibited depolymerization of fibrous actin and cytoskeletal reorganization during EMT. Similar results were obtained in vivo. These findings demonstrate that CFL1 induces EMT by promoting cytoskeletal rearrangement. Our results may provide the basis for developing new anticancer drugs to inhibit CFL1.

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.

Targeted proteomics driven verification of biomarker candidates associated with breast cancer aggressiveness

Breast cancer is the most common and molecularly relatively well characterized malignant disease in women, however, its progression to metastatic cancer remains lethal for 78% of patients 5years after diagnosis. Novel markers could identify the high risk patients and their verification using quantitative methods is essential to overcome genetic, inter-tumor and intra-tumor variability and translate novel findings into cancer diagnosis and treatment. We recently identified 13 proteins associated with estrogen receptor, tumor grade and lymph node status, the key factors of breast cancer aggressiveness, using untargeted proteomics. Here we verified these findings in the same set of 96 tumors using targeted proteomics based on selected reaction monitoring with mTRAQ labeling (mTRAQ-SRM), transcriptomics and immunohistochemistry and validated in 5 independent sets of 715 patients using transcriptomics. We confirmed: (i) positive association of anterior gradient protein 2 homolog (AGR2) and periostin (POSTN) and negative association of annexin A1 (ANXA1) with estrogen receptor status; (ii) positive association of stathmin (STMN1), cofilin-1 (COF1), plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1) and negative associations of thrombospondin-2 (TSP2) and POSTN levels with tumor grade; and (iii) positive association of POSTN, alpha-actinin-4 (ACTN4) and STMN1 with lymph node status. This study highlights a panel of gene products that can contribute to breast cancer aggressiveness and metastasis, the understanding of which is important for development of more precise breast cancer treatment.

The relationship between phosphorylation at Ser3 of cofilin1 and Taxol resistance of ovarian cancer in elderly female patients

Objectives To analyze the relationship between phosphorylation of serine-3 of cofilin1 and Taxol resistance of ovarian cancer and to evaluate the prognostic value of cofilin1 phosphorylation in estimating ovarian cancer survival using clinical samples. Methods Wild type(WT)plasmids with over-expression of wild cofilin1, S3L plasmids with over-expression of cofilin1 and inhibited phosphorylation at serine-3, and siRNA-C plasmids with down-regulation of cofilin1 were constructed using molecular biology techniques.After the SKOV3 and SK-TR30 cell-lines were transfected with these plasmids, changes in biological characteristics and drug resistance after instant transfection were compared.Fifty-one elderly female patients with microscopically confirmed ovarian cancer, who had received chemotherapy with Taxol and cis-platinum(TC)after surgery, including 30 chemo-sensitive and 21 chemo-resistant cases, were recruited in this study.Immunohistochemical methods were used to detect the expression of cofilinl and phosphorylated cofilin in tissue sections from the two groups.Progression free survival(PFS)was analyzed. Results Compared with the control group, the proportion of cells increased at the G0/G1 phase(P=0.034)and decreased at the S phase(P=0.031), and the apoptosis rate decreased(P=0.020), in SKOV3 cells with high expression of cofilin1.However, these characteristics disappeared when the wild type was replaced with the inactive mutant S3L.When cofilin1 expression was down-regulated in SK-TR30 cells, the proportion of cells at the G0/G1 phase decreased(P=0.020). The expression of cofilin1 was detected in 56.7%(17/30)and 57.1%(12/21), respectively, sections of ovarian tumor tissues of the chemo-sensitive and chemo-resistant groups, and there was no statistic difference(χ2=0.332, P=0.943)between the two groups.The expression of phosphorylated cofilin was much higher in the chemo-resistant group(85.7% or 18/21)than in the chemo-sensitive group(53.3% or 16/30), and the difference was statistically significant(χ2=5.829, P=0.016). The higher expression of phosphorylated cofilin was also correlated with shorter PFS(χ2=21.440, P<0.01, 95% CI: 0.068-0.883), especially in the chemo-sensitive group. Conclusions Serine-3 phosphorylation status of cofilin 1 is associated with paclitaxel resistance in ovarian cancer, but the underlying mechanisms of regulation need further investigation. Key words: Ovarian neoplasms; Drug resistance; Cofilin 1; Phosphorylases

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.

Effects of RhoD overexpression on the cytoskeleton, migration and invasion of a human melanoma cell line A375

Objective To evaluate effects of RhoD overexpression on the cytoskeleton, migration and invasion of a human melanoma cell line A375, and to explore their mechanisms. Methods Cultured A375 cells were divided into 2 groups to be transfected with a lentiviral vector carrying the RhoD gene (A375-RhoD group) and a negative lentiviral vector expressing enhanced green fluorescent protein (EGFP) (A375-EGFP group) respectively. After additional culture for 24 hours, rhodamine-phalloidin was used to stain cytoskeletal proteins, Transwell assay was conducted to evaluate migratory and invasive activities of cells, flow cytometry to estimate cell cycle distribution, and Western-blot analysis to measure protein expressions of cofilin, phosphorylated cofilin (p-cofilin), Diaph2 and RhoD. Results Compared with the A375-EGFP group, the A375-RhoD group showed larger cells with thinner, softer and weaker stress fibers, more focal adhesions and filopodias, but no obvious changes in lamellipodias or ruffled borders. Transwell assay showed that the number of A375 cells crossing the polycarbonate membrane as well as that crossing the artificial basement membrane (matrigel) per high-power field (× 200) were significantly smaller in the A375-RhoD group than in the A375-EGFP group (migration assay: 72.67 ± 5.03 vs. 152.67 ± 11.23, t = 11.25, P 0.05). Western-blot analysis showed that RhoD overexpression activated and promoted the expression of the downstream signaling protein Diaph2 in the A375-RhoD group, which was not observed in the A375-EGFP group. In addition, there were no significant differences in expressions of cofilin or p-cofilin between the two groups. Conclusion Overexpression of RhoD may regulate the cytoskeleton and suppress migratory and invasive activities of A375 cells by activating the downstream effector molecule Diaph2. Key words: Melanoma, experimental; GTP phosphohydrolases; Cytoskeleton; Cell migration assays; A375 cells; RhoD

Identification of functional pathways associated with the conditional ablation of serum response factor in Dstncorn1 mice.

The aim of the present study was to investigate the signaling pathways associated with functional alterations in corneal tissues following the conditional ablation of serum response factor (Srf) in Dstncorn1 mice. The gene expression profiling array GSE49688, which includes 3 samples each from the wild-type (WT), Dstncorn1 mutant (corn1) and corn1 mice following the conditional ablation of Srf from the corneal epithelium [namely rescued (res)] mouse groups, was downloaded from the Gene Expression Omnibus database. The limma package was used to identify differentially expressed genes (DEGs) among the three mouse groups. DEGs were subsequently analyzed by dynamic comparison, hierarchical clustering and pathway enrichment analysis. Pathway alteration scores were also calculated in order to study the dynamic metergasis of each identified pathway. A total of 788 DEGs were identified between the corn1 and res groups, 1,365 DEGs were identified between the corn1 and WT groups, and 852 DEGs were identified between the res and WT groups. Among these DEGs, 228 genes were differentially expressed across all three groups, and were mainly enriched in signaling pathways involved in the regulation of the actin cytoskeleton, including the cofilin 1 (CFL1), the mitogen-activated protein kinase (MAPK) signaling pathway and focal adhesion. The dilated cardiomyopathy signaling pathway displayed the highest alteration score, and was enriched with integrin and integrin β-6 (ITGB6). In conclusion, the actin cytoskeleton regulatory pathway, MAPK and dilated cardiomyopathy signaling pathways, as well as CFL1 and ITGB6 genes, may be regulated by Srf to serve important roles in the progression of corneal disease.