The subsequent biological effects of simulated microgravity on endothelial cell growth in HUVECs / 中华创伤杂志(英文版)

<p><b>PURPOSE</b>Microgravity is known to cause endothelium dysfunction in astronauts returning from spaceflight. We aimed to reveal the regulatory mechanism in alterations of human endothelial cells after simulated microgravity (SMG).</p><p><b>METHODS</b>We utilized the rotary cell culture system (RCCS-1) to explore the subsequent effects of SMG on human umbilical vein endothelial cells (HUVECs).</p><p><b>RESULTS</b>SMG-treated HUVECs appeared obvious growth inhibition after return to normal gravity, which might be attributed to a set of responses including alteration of cytoskeleton, decreased cell adhesion capacity and increased apoptosis. Expression levels of mTOR and its downstream Apaf-1 were increased during subsequent culturing after SMG. miR-22 was up-regulated and its target genes SRF and LAMC1 were down-regulated at mRNA levels. LAMC1 siRNAs reduced cell adhesion rate and inhibited stress fiber formation while SRF siRNAs caused apoptosis.</p><p><b>CONCLUSION</b>SMG has the subsequent biological effects on HUVECs, resulting in growth inhibition through mTOR signaling and miR-22-mediated mechanism.</p>

Construction of the full length and N-terminal fragment of serum response factor over-expressing lentiviral plasmid and its impacts on cardiac stem cell differentiation / 临床与实验病理学杂志

Purpose To analyze the effects of full length and N-terminal fragment of serum response factor (SRF-Full and SRF-N) on TGF-β1-induced differentiation in c-Kit + cardiac stem cells (CSC).Methods Rat SRF-Full and SRF-N (1-254 aa) coding sequences were obtained from cDNA library and cloned into the linearized lentviral vector GV358 (Ubi-MCS3FLAG-SV40-EGFP-IRES-puromycin) to generate the recombinant vectors,and then positive clones were selected and sequenced after transducing the competent cells with recombinant vectors.The recombinant lentvirus were packaged through transfecting the HEK293T cells with SRF-Full,SRF-N overexpressing plasmids and viral packaging plasmids.Neonatal SD rat cKit + CSCs were isolated via magnetic activated cell sorting,and TGF-β1-induced differentiation in SRF-Full and SRF-N overexpression virus-infected CSCs was assessed by quantitative PCR.Results SRF-Full and SRF-N coding sequences were successfully obtained and properly cloned into the linearized GV358.The positive clones were selected and further confirmed by sequencing.With the help of packaging plasmids,the SRFFull and SRF-N overexpressing plasmids-transfected HEK293T cells successfully produced the lentiviral particles with the titer of 2 × 108 TU/mL,and the SRF-Full-Flag and SRF-N-Flag fusion protein were detected by Western blot in virus-infected HEK293T cells.Addition of TGF-β1 significantly induced upregulated mRNAs in cardiomyocyte markers (Nkx2.5,Gata4,cTnI) and smooth muscle cell marker (SM22α) but not the epithelia cell marker (vWF) in CSCs.Overexpression of SRF-Full facilitated TGF-β1-triggered cardiomyocyte differentiation.However,SRF-N exerted anti-differentiation effects in TGF-β1-treated cells.Conclusion The SRF-Full and SRF-N overexpressing recombinant lentiviral vectors are successfully constructed.SRF-Full facilitates while SRF-N suppresses TGF-β1-induced cardiomyocyte differentiation in c-Kit + CSCs.

Expression and clinical significance of serum response factor and vascular endothelial growth factor receptor 2 in gastric carcinoma / 肿瘤研究与临床

Objective To investigate the expression and clinical significance of serum response factor (SRF) and vascular endothelial growth factor receptor (VEGFR2) in gastric carcinoma. Methods SABC immunohistochemical method was used to determine the expressions of SRF and VEGFR2 in 50 cases of gastric carcinoma, 50 cases of surgery incisal edges and 29 cases of lymph node metastasis focus. Results The detection positive rates of SRF and VEGFR2 in gastric carcinoma were 52.00 % (26/50) and 60.00 %(30/50), respectively, which were significantly higher than those in the normal gastric mucosa [16.00 % (8/50) and 10.00 % (5/50), respectively] (P0.05), but closely correlated to differentiation degree, invasion depth and lymph node metastasis (P<0.05). The expressions of SRF and VEGFR2 in the gastric carcinoma were positively correlated (r= 0.594, P< 0.05). Conclusion Overexpressions of SRF and VEGFR2 in gastric carcinoma can be regarded as the poorly prognostic markers and play an important role in invasion and metastasis of gastric carcinoma.

Multi-phenotypic Role of Serum Response Factor in the Gastrointestinal System

Serum response factor (SRF) is a master transcription factor of the actin cytoskeleton that binds to highly conserved CArG boxes located within the majority of smooth muscle cell (SMC)-restricted promoters/enhancers. Although most studies of SRF focus on skeletal muscle, cardiac muscle, and vascular SMCs, SRF research has recently expanded into the gastrointestinal (GI) system. Genome scale analyses of GI SMC transcriptome and CArG boxes (CArGome) have identified new SRF target genes. In addition to circular and longitudinal smooth muscle layers, SRF is also expressed in GI mucosa and cancers. In the GI tract, SRF is the central regulator of genes involved in apoptosis, dedifferentiation, proliferation, and migration of cells. Since SRF is the cell phenotypic modulator, it may play an essential role in the development of myopathy, hypertrophy, ulcers, gastric and colon cancers within the GI tract. Given the multi-functional role displayed by SRF in the digestive system, SRF has received more attention emerging as a potential therapeutic target. This review summarizes the findings in SRF research pertaining to the GI tract and provides valuable insight into future directions.

The effect of Y-27632 on invasion and migration of gastric carcinoma cell line SGC-7901 / 基础医学与临床

Objective_To study the effect of Y-27632 on invasion and motility of SGC-7901 gastric carcinoma cells, and to find whether Y-27632 excerts the effect by attenuating SRF expression.Methods_SGC-7901 gastric carcinoma cells were divided into 3 groups:1)blank control group;2)Y-27632 group;3)siRNA-SRF-1107 group. Transfected siRNA-SRF or incubated by Y-27632 48 h.The effect of Y-27632 on proliferation suppressions of SGC-7901 gastric carcinoma cells was detected by CCK-8 assay.Cell invasion was examined by Transwell and wound healing test.The expression of SRF, ROCK1, E-cadherin, β-catenin, F-actin, MRTF-A and Cyclin D1 were detected by Western blot.Results_Y-27632 inhibited invasion (P<0.05)but had no effect on proliferation of SGC-7901 gastric carcinoma cells.Y-27632 reduced ROCK1, MRTF-A, F-actin, SRF protein expressions by 37.0%, 44.3%, 62.7%and 62.7%respectively, and E-cadherin protein expression was up-regulated by 2.64 folds(P<0.05).Conclusions_The inhibition of ROCK and up-regulation of E-cadherin by Y-27632 can inhibit the invasion and migration of SGC-7901 gastric carcinoma cells that is explained at least, in part, by attenuating SRF expression.

Change of serum response factor expression in eyelid of different embryo development stages of B6-Co mice / 中华实验眼科杂志

Background Mutant C57BL/6 mouse with corneal opacity (B6-Co) appears eye open at birth (EOB) phenotype,which is a good animal model in the study of developmental mechanism of eyelid.Investigating the relationship between serum response factor (SRF) and EOB phenotype can provide theoretical support for the research on the mechanism of innate defects in eyelid development in humans.Objective This study was to assess the dynamic expressions of SRF in eyelid of embryonic B6-Co mouse.Methods Total RNA was extracted from B6 and B6-Co mice eyelid tissue at embryonic day 16.5 (E16.5 d),E17.5 d and E18.5 d.The relative expression levels of SRF mRNA and protein in the eyelid tissue of B6 and B6-Co embryonic mice were assayed by real-time quantitative PCR and Western blot,respectively.In situ expressions of SRF protein in eyelid of B6-Co mice and B6 mice were detected using immunofluorescence technique.The use and care of the animals complied with the Regulation for the Administration of Affair Concerning Experimental Animals of Nantong University.Results The relative expression levels ofSRF mRNA in the eyelids were 0.41±0.06 and 0.24±0.17 in E16.5 d and E17.5 d of B6-Co mice,showing a significant decline in comparison with 1.03 ±0.17 and 1.01 ±0.09 in the B6 mice (P =0.025,0.017).The expression levels of SRF protein in the eyelids of E16.5 d and E17.5 d B6-Co mice were 0.08±0.01 and 0.08± 0.01,which were significantly lower than 0.12 ±0.03 and 0.13 ± 0.02 of B6 mice (P =0.036,0.024).However,there were no significant differences in the expression levels of SRF mRNA and protein in E18.5 d between the B6-Co mice and B6 mice (P =0.387,0.774).Immunofluorescence assay displayed that SRF was expressed in the keratinocytes of eyelids in both mice,but the fluorescence intensity was weaker in the B6-Co mice.Conclusions SRF probably interrupts the developing process of eyelid in early embryo of B6-Co mice.

Effect of glycyrrhizin on serum levels of PDGF, IL-17, MCP-1 and SRF in patients with glioma / 中国生化药物杂志

Objective To analyse effect of glycyrrhizin on serum levels of platelet derived growth factor ( PDGF ) , interleukin 17 ( IL-17 ) , monocyte chemotactic protein 1 (MCP-1) and serum response factor (SRF) in patients with glioma.Methods 48 patients who were diagnosed with glioma in our hospital were collected.All patients were randomly divided into experimental group and control group,24 cases in each group.All patients were treated with surgery,control group was treated with temozolomid capsule(150 mg/m2 ) orally, one times per day, 28 days for a treatment period of 5 days continuous administration.On the basis of control group, experimental group was treated with compound ammonium glycyrrhetate injection 40 mL, dissolved in 250 mL 5% glucose solution intravenously,one times per day,four weeks was a period of treatment.After two groups were treated two period of treatment,the serum levels of PDGF, IL-17,MCP-1and SRF were detected in all patients.Results After treatment, compared with control group, the serum PDGF level was lower than that of experimental group (P<0.05);the serum IL-17 level was lower than that of experimental group (P<0.05);the serum MCP-1 level was higher than that of experimental group ( P <0.05 );the serum SRF level was lower than that of experimental group ( P <0.05).Conclusion Glycyrrhizin could significantly reduce the serum PDGF, IL-17 and SRF levels,increase MCP-1 levels, have guidance significance for clinic.

Serum Response Factor Is Essential for Prenatal Gastrointestinal Smooth Muscle Development and Maintenance of Differentiated Phenotype

BACKGROUND/AIMS: Smooth muscle cells (SMCs) characteristically express serum response factor (SRF), which regulates their development. The role of SRF in SMC plasticity in the pathophysiological conditions of gastrointestinal (GI) tract is less characterized. METHODS: We generated SMC-specific Srf knockout mice and characterized the prenatally lethal phenotype using ultrasound biomicroscopy and histological analysis. We used small bowel partial obstruction surgeries and primary cell culture using cell-specific enhanced green fluorescent protein (EGFP) mouse lines to study phenotypic and molecular changes of SMCs by immunofluorescence, Western blotting, and quantitative polymerase chain reaction. Finally we examined SRF change in human rectal prolapse tissue by immunofluorescence. RESULTS: Congenital SMC-specific Srf knockout mice died before birth and displayed severe GI and cardiac defects. Partial obstruction resulted in an overall increase in SRF protein expression. However, individual SMCs appeared to gradually lose SRF in the hypertrophic muscle. Cells expressing low levels of SRF also expressed low levels of platelet-derived growth factor receptor alpha (PDGFRalphalow) and Ki67. SMCs grown in culture recaptured the phenotypic switch from differentiated SMCs to proliferative PDGFRalphalow cells. The immediate and dramatic reduction of Srf and Myh11 mRNA expression confirmed the phenotypic change. Human rectal prolapse tissue also demonstrated significant loss of SRF expression. CONCLUSIONS: SRF expression in SMCs is essential for prenatal development of the GI tract and heart. Following partial obstruction, SMCs down-regulate SRF to transition into proliferative PDGFRalphalow cells that may represent a phenotype responsible for their plasticity. These findings demonstrate that SRF also plays a critical role in the remodeling process following GI injury.

SRF expression and its biological significance in gastric carcinoma cells / 中国肿瘤临床

Objective:This study aims to explore the relationship between serum response factor (SRF) expression level and gas-tric cancer progression by detecting SRF expression level in cancer cells. Methods:The SRF gene in SGC-7901 cells was silenced by RNA interference. Transfection efficiency was detected by fluorescence microscopy, cell proliferation by CCK 8 method, SRF gene and protein expression level by real-time polymerase chain reaction and Western blot, and cell cycle by flow cytometry. Results:Cell treat-ment with siRNA-SRF induced significant reduction in SRF mRNA levels. Western blot analysis showed that SRF protein decreased by 40.1%in the siRNA group compared with that in the control group (P<0.05). Compared with the blank, negative, and mock transfection control groups, cell proliferation of the siRNA-SRF group decreased. The inhibition ratio reached 64.24%, as measured by the CCK-8 assay (P<0.05). Treatment with siRNA could block SGC-7901 cell cycle at G0/G1 phase (P<0.05). Conclusion:SRF expression is close-ly associated with gastric carcinoma cell proliferation. SRF protein level detection can provide a certain reference value in evaluating malignant gastric carcinoma progression. SRF is possibly an important target for the prevention and control of gastric cancers.

Early Growth Response 1— A Transcription Factor in the crossfire of Signal Transduction Cascades.

Early growth response-1 (Egr-1) is a Cys2-His2-type zinc-finger transcription factor. A broad range of extracellular stimuli is capable of activating Egr-1, thus mediating growth, proliferation, differentiation or apoptosis. Egr-1 is, therefore, participating in the progression of a variety of diseases such as atherosclerosis or cancer. Functional response elements connect Egr-1 to signal transduction cascades targeting Egr-1. Five serum response elements (SRE) have been identified in the promoter region of Egr-1, the binding region of serum response factor (SRF). The Rho/Rho-kinase pathway has been shown to regulate actin reorganization via LIM-kinase mediated cofilin phosphorylation. Recent studies have revealed that the actin binding striated muscle activator of Rho signaling (STARS) promotes translocation of myosin related transcription factors (MRTFs) into the nucleus, leading to SRF activation. The ternary complex factor (TCF) Elk-1 eventually bridges the gap between SRF-mediated gene transcription and the Raf/MEK/ERK pathway. Moreover, the Egr-1 promoter owns two cAMP response elements (CREs), whose relevance for gene expression is still unclear. An Egr-1 binding site (EBS) located on the Egr-1 promoter itself is arguing for a negative feedback mechanism. The acquired knowledge on transcriptional regulation of Egr-1 is not entirely understood. In this review, we highlight upstream and downstream signaling in vitro and in vivo associated with Egr-1.

Expression and Prognostic Significance of Serum Response Factor in Cholangiocarcinoma

BACKGROUND: Serum response factor (SRF) is a transcriptional factor that plays an important role in cell growth and differentiation for several types of cells. The expression of SRF in cholangiocarcinoma (CC) and its potential role has not been examined. The aim of this study was to determine the relationship between the expression of SRF in CC and the clinicopathological parameters, as well as patient survival. METHODS: We analyzed the expression of SRF in 84 surgically resected cases of CC (33 cases of intrahepatic CC [ICC] and 51 cases of extrahepatic CC [ECC]) by using immunohistochemistry. RESULTS: The positive expression of SRF was detected in 48.8% of the cases of CC (42.4% in ICC, 52.9% in ECC). SRF was predominantly expressed in the CC cells with intense labeling in the nucleus. A SRF expression was significantly associated with the cell proliferation rate (Ki-67 labeling index, p=0.046) and poor patient survival (p=0.002). The tumor differentiation (p=0.038), the T category (p<0.001), lymph node and distant metastasis (p<0.001, p=0.009) and nerve and vessel invasion (p=0.010, p=0.012) were also found to be significantly associated with a poor CC prognosis. CONCLUSIONS: These results suggest that the SRF may play a role in the tumor cell proliferation of CC, and its expression in tumor cells can provide additional prognostic information.

Serum response factor play a regulative role in the gene expression in heart failure / 老年心脏病学杂志（英文版）

To investigate the relationship between transcription factor and the change of protein expression levels in heart failure. Methods Bioinformatic method was used to analyze the data of binding-sites on the 5 ' flaking regions of four genes whose mRNA level changed in failing heart from three databases about nucleic acid-EMBL, transcriptional regulation factor-TRANSFAC and protein-SWISS-PORT.The expression level of selected transcription factor was determined by immunohischemical method.Results Nine transcription factors were inferred to influence the proteins' levels in occurrence and development of heart failure.Serum response factor (SRF) was selected from the nine factors and assayed. The results showed that there was a higher level of SRF in healthy group than in chronic heart failure (CHF), and the level was associated with the degree of CHF. It was also found that there was a relative higher level of SRF in the acute myocardial infarction (AMI) than that in CHF, but which was lower than the healthy. Conclusion It showed that SRF had a quantitative change in the development of heart failure, and suggested SRF might play an important regulative role in heart failure. The expression changes of proteins related to myocardial function might be regulated by the quantitative change of transcription factor(s).