Smad3 Research Articles

The effect of lipopolysaccharide cooperating with transforming growth factor on MCF-7 breast cancer cells in the aspects of epithelial-mesenchymal transition,migration and invasion

Objective To investigate into the biological effect of lipopolysaccharide(LPS) combined with transforming growth factor-β1(TGF-β1) on MCF-7 breast cancer cells and discuss the molecular mechanism underneath the phenomena. Methods The morphology of MCF-7 cells was observed under the inverted microscope.Rhodamine-phalloidine was used for staining the actin cytoskeleton.Real-time quantitative polymerase chain reaction(Real-time PCR) was used to detect genetic expression of E-cadherin, Vimentin,Snail-2, Twist and matrix metalloproteinase-9(MMP-9).Transwell was used to examine cell migration and invasion.Zymography experiment was used to test active protein MMP-9.Western blotting was used to detect signal pathways of nuclear factor-κB(NF-κB), extracellular signal-regulated kinase(ERK) and Smad. Results Almost all the cells in the group of LPS combined with TGF-β1(L+ T group)acquired fibroblastoid properties along with obvious cytoskeleton changes.L+ T group had E-cadherin down-regulated obviously while Snail-2, Twist and MMP-9 up-regulated sharply.The number of migrated cells in control group, LPS group, TGF-β1 group, L+ T group were 6. 8±4. 1, 10. 2±4. 3, 39. 5± 3. 8, 69. 7±4. 4 respectively.The number of invaded cells in control group, LPS group, TGF-β1 group, L+ T group were 4. 4±1. 1,6. 8±2. 4,32. 1±2. 3, 57. 9±4. 5 respectively.The active protein of MMP-9 was the most in L+ T group.L+ T group has much higher phosphorylated ERK and phosphorylated Smad-2 than TGF-β1 group's. Conclusion LPS cooperates with TGF-β1 to promote the epithelial to mesenchymal transition of MCF-7 breast cancer cells, and stimulation of LPS and TGF-β1 on MCF-7 make those cells have stronger capabilities of migration and invasion. Key words: Breast cancer; Transforming growth factor-β1; Lipopolysaccharide; Tumor metastasis; Epithelial-mesenchymal transition

Role of transforming growth factor-β1 in epithelial-mesenchymal transition of mesothelial cells and its effect on peritoneal metastasis of gastric cancer

To elucidate the role of transforming growth factor-beta1(TGF-β1) in epithelial-mesenchymal transition of mesothelial cells and peritoneal metastasis of gastric cancer. HMrSV5 cells, a human peritoneal mesothelial cell line, were incubated with TGF-β1, and their morphological changes were observed by phase contrast microscopy. Expressions of α-smooth muscle actin (α-SMA), vimentin, cytokeratin, E-cadherin, phosphorylated-Smad2 and Smad2 were examined by Western blotting. After fibroblastic-like mesothelial cells were co-incubate with HSC-39 cells(gastric cancer cell line), the adhesion and invasion potential of HSC-39 were evaluated by adhesion and invasion assay in vitro. Few mesothelial cells converted to spindle fibroblast-like morphology for 24 h, and remarkable phenotypic changes were observed at 72 h of TGF-β1 activation. TGF-β1 could induce α-SMA and vimentin expression, and down-regulate cytokeratin and E-cadherin expression in mesothelial cells (P<0.05). TGF-β1 induced phosphorylation of Smad2 within 15 min of stimulation, reached a maximum at 30 min after treatment and remained high level during the experiment without affecting total Smad2 expression(P>0.05). The percentage of HSC-39 gastric cancer cells adhered were significantly increased as compared to the control. When the mesothelial cells were treated by TGF-β1 for 72 h, the increased adhesion percentage was(146±17)%(P<0.05). After fibroblastic-like mesothelial cells co-incubated with HSC-39 cells for 48 h, more cancer cells [(61.1±11.4) cells/view field] invaded the coated membrane as compared to the control group [(31.9±8.1) cells/view field] (P<0.05). TGF-β1 can induce the transition of mesothelial cells into myofibroblasts and Smad2 signal pathway may play a role in this transition, which is associated with increased adhesion and invasiveness of gastric cancer cells, and provides favorable environment for the dissemination of gastric cancer.

Suppression of transforming growth factor β receptor 2 and Smad5 is associated with high levels of microRNA miR-155 in the oral mucosa during chronic simian immunodeficiency virus infection.

Chronic human immunodeficiency virus and simian immunodeficiency virus (HIV and SIV) infections are characterized by mucosal inflammation in the presence of anti-inflammatory cytokines such as transforming growth factor β (TGFβ). The mechanisms for refractiveness to TGFβ are not clear. Here we show that the expression of microRNA miR-155 was significantly upregulated in the oropharyngeal mucosa during chronic SIV infection and was coincident with downregulation of TGFβ receptor 2 (TGFβ-R2) and SMAD5, key TGFβ signaling genes that harbor putative target sites for miR-155. Ectopic expression of miR-155 in vitro was found to significantly downregulate TGFβ-R2 and Smad5 expression, suggesting a role for miR-155 in the suppression of TGFβ-R2 and SMAD5 genes in vivo. The downregulation of TGFβ signaling genes by miR-155 likely contributes to the nonresponsiveness to TGFβ during SIV infection and may inadvertently aid in increased immune activation during HIV and SIV infections.

Effect of antisense oligodeoxynucleotide of basic fibroblast growth factor on rat model of pulmonary fibrosis

Objective To observe the effect of antisense oligodeoxynucleotide (AODN) of basic fibroblast growth factor (bFGF) on the rat models of pulmonary fibrosis.Methods Thirty rats were divided into five groups (6 rats of each group):control group,pulmonary fibrosis group,empty vector transfection group,AODN group and RODN group.Lung coefficient was counted,pathologic changes in the lung tissue were observated to determine degree of pulmonary fibrosis,enzyme linked immunosorbent assay (ELISA) was used to determine the concentrations of hydroxyproline in lung tissue homogenate and the expression of bFGF in the serum and bronchoalveolar lavage fluid,and reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect the mRNA expression of bFGF,α-smooth muscle actin (α-SMA),type Ⅰ collagen,transforming growth factor-β1 (TGF-β1),connective tissue growth factor (CTGF),Smad3 and Smad7 in lung tissue homogenate.Results In control group,pulmonary fibrosis gToup,empty vector transfection group,AODN group and RODN group,the lung coefficient was 7.5 ± 1.5,10.8 ± 1.4,11.0 ± 1.4,8.8 ± 1.7 and 12.5 ± 1.6 respectively,the degree of pulmonary fibrosis was 0,2.38 ± 0.52,2.41 ± 0.48,1.62 ± 0.45 and 2.82 ±0.54 respectively,the synthesis of bFGF was significantly decreased in AODN group and significantly increased in RODN group (P ＜ 0.05),the concentration of HYP was markedly decreased in AODN group and markedly increased in RODN group (P ＜ 0.05),the mRNA expression of bFGF,α-SMA,type Ⅰ collagen,TGF-β1,CTGF and Smad3 was markedly decreased in AODN group and markedly increased RODN group (P ＜ 0.05),and that of Smad7 was markedly increased in AODN group and markedly decreased in RODN group.Conclusion:These results demonstrate that AODN of bFGF can inhibit the development of pulmonary fibrosis induced by Bleomycyn in rats possibly by down-regulating the TGF-β1-Smad signaling pathway. Key words: Basic fibroblast growth factor; Antisense oligodeoxynucleotide; Pulmonary fibrosis; Transforming growth factor-β1-Smad

Prognostic significance of p16 and Smad4 deleted expression in patients with lung adenocarcinoma

Objective To investigate the loss of p16 and Smad4 expression in human lung adenocarcinoma and their relationship with the clinicopathological characteristics and prognosis.Methods The protein expression of p16 and Smad4 in 120 cases of lung adenocarcinoma tissues was detected.The relationship between the expression of these proteins and clinicopathological features was analyzed,and the application of p16 and Smad4 in disease prognosis was evaluated.Results The loss frequency of p16 and Smad4 in lung adenocarcinoma tissues was 45.0％ and 62.5％,respectively.Loss of p16 expression was associated with lymph node metastasis (P ＜ 0.01) and clinicopathological stage (P ＜ 0.01).Loss of Smad4 expression was associated with tumor size (P ＜ 0.05),lymph node metastasis (P ＜ 0.05),and tumor differentiation (P ＜ 0.05).Loss of p16 and Smad4 expression was negatively correlated with overall survival,and the overall survival time in patients with two genetic loss was shortest (P ＜ 0.01).The Spearman correlation analysis showed that the expression of p16 had a positive correlation with the expression of Smad4 (r =0.182,P ＜ 0.05).Conclusion The loss of p1 6 and Smad4 expression in lung adenocarcinoma was associated with malignant biological behaviors and poor prognosis. Key words: p16 gene ; Smad4 gene ; Lung neoplasms ; Prognosis

MicroRNA-140 suppresses the migration and invasion of colorectal cancer cells through targeting Smad3

To investigate the effect of microRNA-140 (miR-140) on the migration and invasion of colorectal cancer (CRC) cells and the possible mechanism. miR-140 mimics, miR-140 specific inhibitor or small interfering RNA (siRNA) against Smad3 were transfected into human CRC cell line RKO cells respectively, using Oligofectamine or Lipofectamine2000. Quantitative real-time PCR (real-time PCR) was used to measure the expression levels of miR-140 and Smad3 mRNA. Smad3 protein was analyzed by Western blot. The in vitro cell migrating and invasive abilities were determined by wound-healing and Transwell chamber assay after up-regulating or down-regulating miR-140 or knocking down Smad3. The Western blot assays showed that the Smad3 protein level was significantly reduced after up-regulating miR-140 (0.04 ± 0.01), compared with that of (0.47 ± 0.02, P < 0.05) in the control group and that of (0.52 ± 0.06) in the negative control group (P < 0.05 for both). The results of real-time PCR indicated that no significant difference was found in the levels of Smad3 mRNA between miR-140 transfection and NC groups (1.11 ± 0.13 vs. 1.00 ± 0.06, P > 0.05). The wound-healing assay showed that the migrating ability was dramatically attenuated by miR-140 compared with that in the control and NC groups, whereas no significance was found when compared with that of the Smad3 siRNA transfected cells. The number of cells migrating through Transwell chamber without matrigel in the miR-140 group was (76.2 ± 4.4), remarkably lowered than that in the control (267.1 ± 4.9) and NC (336.1 ± 5.7) groups (P < 0.05 for both), but no significant difference between the miR-140 (76.2 ± 4.4) and Smad3 siRNA (83.5 ± 7.3) groups. Transwell chamber with matrigel assay showed that number of cells penetrating through the membrane was (109.5 ± 7.4) in the miR-140 group, significantly lower than that in the control (403.1 ± 5.1) and NC (392.6 ± 8.4) groups (P < 0.05 for both), while Smad3 siRNA transfection had a similar effect (138.8 ± 3.6)(P > 0.05). Down-regulation of miR-140 increased the level of smad3 protein expression, and partially reversed the inhibition of the cell migration and invasion mediated by miR-140. Co-transfection of miR-140 inhibitor and Smad3 siRNA had no significant effect on the Smad3 protein expression and the abilities of cell migration and invasion. miR-140 regulates the Smad3 expression at the post-transcriptional level. miR-140 suppresses the migrating and invasive abilities of CRC cells, possibly through down-regulation of Smad3. The findings of this study suggest that miR-140 may have a unique potential as a possible biomarker candidate for diagnosis and therapy of tumor metastasis.

Crosstalk of TGF[beta] superfamily with Smad1 in testis

Crosstalk of TGF[beta] superfamily with Smad1 in testis

Effect of bilirubin on proliferation of pulmonary microvascular endothelial cells of rats

Objective To evaluate the effect of bilirubin on proliferation of pulmonary microvascular endothelial cells (PMVECs) of rats.Methods Primary PMVECs harvested from 10 healthy male Sprague-Dawleyrats,weighing 120-150 g,aged 2-3 months,were cultured,purified and identified.PMVECs were seeded in low-glucose DMEM culture medium or 96-well culture plates,and divided into 5 groups according to the random number table:control group (group C) and different concentrations of bilirubin groups (B1-4 groups) with 30 wells or48 flasks in each group.In group C,low-glucose DMEM 1 ml was added.In B1-4 groups,5,10,20 and 50 μmol/L bilirubin 1 ml were added,respectively.At 24,48 and 72 h of incubation,proliferation of PMVECs was measured using CCK-8 assay and 3 H-TdR incorporation assay,Akt1 mRNA and Smad3 mRNA expression was measured by RT-PCR,and phosphor-Akt1 (p-Akt1) protein and Smad3 protein expression was detected using Western blot.Results Compared with group C,no significant changes were found in each parameter mentioned above at each time point in B1 and B2 groups,the proliferation of PMVECs was weakened,and Akt1 mRNA,p-Akt1 protein and Smad3 protein and mRNA expression was down-regulated at 48 h of incubation in B3 group,and the proliferation of PMVECs was weakened,and the expression of Akt1 mRNA,p-Akt1 protein and Smad3 mRNA and protein was down-regulated at 48 and 72 h of incubation in B4 group.Compared with group B3,the proliferation of PMVECs was weakened,and the expression of Akt1 mRNA,p-Akt1 protein and Smad3 mRNA and protein was down-regulated at 72 h of incubation in B4 group.Conclusion High concentration of bilirubin can inhibit proliferation of PMVECs and down-regulated expression of Akt1 and Smad3 is involved in the mechanism. Key words: Bilirubin; Cell proliferation; Endothelial cells

Effects of transforming growth factor-β1 and Smad-3 on chronic cystitis and fibrosis

Objective To evaluate the effects of transforming growth factor-β1 (TGF-β1) and Smad-3 on chronic cystitis and fibrosis.Methods Forty Sprague-Dawley rats were randomized into control group,model group,low dose group and large dose group.Urodynamic detection,real time reverse transcription polymerase chain reaction,hematoxylin and eosin staining and Masson staining were done to determine the difference among the groups.Results As compared with control group,chronic cystitis with diffuse fibrosis in model group was aggravated,bladder compliance was increased [(0.99 ± 0.11) ml/mmHg to (0.29 ± 0.05) m]/mmHg],and the mRNA levels of TGF-β1,Smad-3,Collagen Ⅰ,Collagen Ⅲ increased were significantly (0.79 ±0.07,0.73 ±0.06,1.09 ±0.09,1.32 ±0.05,P ＜0.05).After administration of two doses of SB431542,bladder compliance was increased significantly [(0.64 ± 0.07) and (0.71 ± 0.05) m]/mmHg],the mRNA levels of Smad-3 decreased significantly (0.30 ± 0.04 and 0.25 ± 0.05),while the inflammation and fibrosis reduced (P ＜ 0.05).Conclusion TGF-β1 promotes the bladder inflammatory fibrosis probably through the Smad-3 pathway. Key words: Bladder ; Fibrosis ; Transforming growth factor-β ; Inflammation

Excess SMAD signaling contributes to heart and muscle dysfunction in muscular dystrophy.

Disruption of the dystrophin complex causes muscle injury, dysfunction, cell death and fibrosis. Excess transforming growth factor (TGF) β signaling has been described in human muscular dystrophy and animal models, where it is thought to relate to the progressive fibrosis that characterizes dystrophic muscle. We now found that canonical TGFβ signaling acutely increases when dystrophic muscle is stimulated to contract. Muscle lacking the dystrophin-associated protein γ-sarcoglycan (Sgcg null) was subjected to a lengthening protocol to produce maximal muscle injury, which produced rapid accumulation of nuclear phosphorylated SMAD2/3. To test whether reducing SMAD signaling improves muscular dystrophy in mice, we introduced a heterozygous mutation of SMAD4 (S4) into Sgcg mice to reduce but not ablate SMAD4. Sgcg/S4 mice had improved body mass compared with Sgcg mice, which normally show a wasting phenotype similar to human muscular dystrophy patients. Sgcg/S4 mice had improved cardiac function as well as improved twitch and tetanic force in skeletal muscle. Functional enhancement in Sgcg/S4 muscle occurred without a reduction in fibrosis, suggesting that intracellular SMAD4 targets may be important. An assessment of genes differentially expressed in Sgcg muscle focused on those encoding calcium-handling proteins and responsive to TGFβ since this pathway is a target for mediating improvement in muscular dystrophy. These data demonstrate that excessive TGFβ signaling alters cardiac and muscle performance through the intracellular SMAD pathway.

An experimental study of the signal pathway of transforming growth factor-β1/Smad in rat dorsal root ganglia neurons

Objective To explore whether the TGF-β1/Smad signaling pathway exists in rat dorsal root ganglia neurons.Methods Rat dorsal root ganglia neurons were cultured ex vivo.Immunofluorescence histochemistry and real time fluorescence quantitative PCR were employed to detect the expression of Smad molecules in the rat DRG neurons and to test the influence of TGF-β1 on gene transcription of Smads.Results TβR1,Smad2 and Smad4 were expressed in DRG neurons of rats.The gene expression of these molecules in DRG neurons was regulated by TGF-β1.Conclusion The TGF β1/ Smad signaling pathway exists in rat dorsal root ganglia neurons. Key words: Rats; Dorsal root ganglia; Neurons; TGF beta 1/Smad signal pathway

Polymorphism of SMAD7 and susceptibility to non-small cell lung cancer

Objective To explore the relationship between single nucleotide polymorphisms (rs12953-717) of SMAD7 and susceptibility of non-small cell lung cancer (NSCLC) in Chinese Han population.Methods A single nucleotide polymorphisms (rs12953717) from SMAD7 was detected via Sequenom system in 528 NSCLC cases and 762 healthy controls.Data was statistically analyzed by unconditional Logistic regression method.Results rs12953717 had significant differences between non-small cell lung cancer patients and the controls.Compared with CC/TT (CC combined with TT) genotype,the adjusted odds ratio for the CT genotype was 4.107 (95％ CI:3.206 ～ 5.260,P =0.000 1).Smokers had a 2.004 odds ratio (95 ％ CI =1.583 ～ 2.537,P =0.000 1) of NSCLC compared with the controls.There was a 10.074-fold increased risk of NSCLC among the subjects with CT genotype and smokers.Conclusion The polymorphism of rs12953717 may have relation with risk of NSCLC.Heterozygote (CT) is a susceptibility genotype of NSCLC.Smoking is one of the risk factors of NSCLC.Smoking and CT genotype have synergistic effects on NSCLC susceptibility. Key words: Polymorphism, single nucleotide; Smoking; Carcinoma, non-small-cell lung; Disease susceptibility; SMAD7

ShRNA interference on TGF-β2/Smads signal pathway in human glioma cell

Objective Using shot hairpin RNA (shRNA) interference to specifically deplete cells expression of Smad2/3 gene of TGF-β2/Smads signal pathway and to investigate their effects on the proliferation of hunman glioblastoma mutiforme (GBM) cells.Methods GBM cell line U251 was transfected with plasmids taking psh-Smad2 or psh-Smad3 that expressed shRNA targeting Smad2 or Smad3 gene,and the negative control plasmid.The expression levels of Smad2/3 mRNA and protein were measured by Real-time fluorescence Quantitative PCR (Real-time PCR) and Western blot.Cells were treated by TGF-β2 (+ /-) after being transfected with plasmids.The proliferation rate was evaluated by CCK-8 assay.Results (1) Real-time PCR and Western blot illustrated that the Smad2/3 expression levels were dramatically down-regulated in U251 cells by shRNA interference (P =0.01).(2)The proliferation of cells transfected with psh-Smad2 and psh-Smad3 group was obviously higher than that of negative control group(P =0.018,P =0.008).(3)The rate of cell proliferation was similar between the cells treated with and without TGF-β2 when Smad3 was knocked down (P =0.001).(4)Additionally,the expression level of Smad3 in psh-Smad2 group was significantly higher than that of the psh-NC group and the mRNA expression of Smad2 in psh-Smad3 group also was significantly higher than that of the psh-NC group(P =0.258).Conclusions Knockdown of Smad2/3 both enhanced the cellular proliferation.U251 cell proliferation inducted by TGFβ2 was dependent on Smad3 but not Smad2.And depletion of Smad2 enhanced Smad3 expression,meanwhile depletion of Smad3 also enhanced Smad2 expression. Key words: shRNA interference; Glioma; Transforming growth factor beta2; Genes

Effect of integrin-linked kinase on the transforming growth factor-β1/Smad3 signaling pathway in hypertrophic scar

Objective To explore the role of integrin-linked kinase (ILK) in the transforming growth factor-β1 (TGF-β1)/Smad3 signal pathway,and the action mechanism of ILK on the differentiation of fibroblasts in hypertrophic scar.Methods The fibroblasts of human hypertrophic scar were isolated and cultured in vitro.The cells were divided into 4 groups:control group,ILK small interfering RNA (siRNA) group,TGF-β1 group,and ILK siRNA + TGF-β1 group.The ILK expression levels were detected by using immunofiuorescence.The mRNA expression levels of ILK,Smad3 and α-smooth muscle actin (α-SMA) were detected by using real-time fluorescent quantitative polyrnerase chain reaction (FQ-PCR).Results The immunofluorescence showed that ILK expression was decreased after transfection of siRNA,and increased again by stimulation with TGF-β1 (5 μg/L).FQ-PCR revealed that the mRNA expression of Smad3 (0.222 ± 0.089) and α-SMA (0.118 ± 0.080) was decreased with ILK (0.522 ± 0.113) after transfection of ILK siRNA,and then the mRNA expression of Smad3 (2.891 ± 0.475) and α-SMA (2.581 ± 0.826) was decreased with ILK (2.233 ± 0.511) increased significantly after stimulation of TGF-β1 as compared with that in control group and ILK siRNA group (P ＜ 0.05).Conclusion The mRNA level of Smad3 and α-SMA could be up-regulated by ILK,which could be stimulated by TGF-β1.ILK may be an important upstream gene for Smad proteins to affect the differentiation of fibroblasts in hypertrophic scar via TGF-β1/Smad3 signal pathway. Key words: Integrin-linked kinase; Cicatrix; Fibroblast ; Transforming growth factor-β1

Involvement of Smad pathway in proteoglycan 4 expression induced by hydrostatic pressure in temporomandibular synovial fibroblasts

To examine the expression of proteoglycan 4 (PRG-4) induced by hydrostatic pressure in rat temporomandibular synovial fibroblasts and investigate the possible mechanism. The cultured rat temporomandibular synovial fibroblasts were subjected to 100 kPa magnitude intermittent hydrostatic pressure (IHP) at frequency of 4 h/day, and the static group served as control. The expressions of Smad pathway proteins and p38MAPK pathway proteins were analyzed by Western blot and immunofluorescence staining. Then the cells were incubated with SB431542, the inhibitor of transforming growth factor (TGF)-β receptor. Western blot and reverse transcription PCR were used to detect the PRG-4 expression after 72 h. The expression of phosphorylated Smad-2 and phosphorylated Smad-3 were increased after 1 h of IHP, reaching a maximum after 2 h and 4 h of IHP, respectively.However, the protein content of phosphorylated p38 did not vary significantly. In addition, IHP induced nuclear translocation of Smad-2/-3, and the immunofluorescence staining signal intensity markedly increased (24.11 ± 4.70)(P < 0.05). The levels of PRG-4 mRNA were significantly increased by IHP (1.48 ± 0.08)(P < 0.05). Treatment of cells with SB431542 could decrease the expression of PRG-4 mRNA significantly after IHP (0.47 ± 0.05)(P < 0.05). In addition, SB431542 inhibited the expression of PRG-4 protein induced by IHP. Smad signal acts as an essential signal pathway to regulate PRG-4 expression induced by IHP.

ZFP423, a transcription factor implicated in Joubert Syndrome and Cerebellar Vermis Hypoplasia, orchestrates the pace and mode of cerebellar neurogenesis

Neurogenesis is a tightly regulated process, both in the embryonic and in the adult brain. Its success depends on the ability of a germinative epithelium to establish the appropriate balance between maintaining an undifferentiated progenitor pool and giving birth to sequential generations of neurons and glia. The Zfp423 gene encodes a 30 Zn-finger transcription factor (TF) which interacts with the SMAD1- SMAD4 complex (BMP signaling), Notch intracellular domain, retinoic acid receptors and Collier/Olf-1/EBF TFs. This gene has been previosly implicated in cerebellar development. Mutations in the human ortholog ZNF423 have been identified in patients carrying cerebellar vermis hypoplasia (CVH) or Joubert Syndrome (JS), and/ or exhibiting other signs of ciliopathy outside the central nervous system. We have been analyzing two mouse mutant lines carrying allelic in-frame deletions of Zfp423. One of them lacks Zn-finger domains 9-20 (Δ9-20), implicated in BMP and Notch signal transduction, while the other lacks a C-terminal domain (Δ28-30). Both mutants exhibit cerebellar malformations and severe ataxia. However, our results indicate that the two protein domains play sharply distinct roles in the context of cerebellar neurogenesis. In Zfp423Δ9-20/Δ9-20 mutants, GABAergic Purkinje cell (PC) neurogenesis is impaired and the PC progenitor pool in the ventricular zone is precociously depleted. Conversely, Zfp423Δ28-30/Δ28-30 mutants display a selective impairment in the development of glutamatergic cerebellar neurons.

The effects of helium based atmospheric pressure cold plasma (APCP) on wound healing

Cold plasmas have been proposed for many applications in medicine, such as tissue disinfection and wound healing. Their antibacterial effects have been well demonstrated and depend mainly to the action of reactive oxygen species or ROS, but the molecular mechanisms promoting tissue regeneration in wound healing are not yet fully understood. We previously reported that the applications of 2-5 min of atmospheric pressure cold plasma (APCP) generated by helium ionization is effective in the inactivation bacteria without any visible changes in cells and tissues (1) and the aim of the presented study is to ascertain if the same doses of APCP are able to promote wound healing. At this purpose we tested the effects of APCP exposure on dermal fibroblasts viability obtained from 3 different donors by means of MTT and Tunnel test, in presence or absence of a potent antioxidant such as 5mM N-acetylcystein (NAC). To define the cellular response to APCP, we then analyzed by real-time PCR the expression changes of selected genes involved in oxidative stress-related response (OGG1, GPX1) and wound repair (IL1beta, IL6, TNFα, FGF2, TGFβ, SMAD3, α-SMA, collagen I) in the plasma treated cells cultures. Our results demonstrated that in all the three different fibroblasts cultures, 2 min APCP application did not cause any variation of viability 2 h after treatment and increased significantly after 24 h (p<0.05). In fibroblasts of two donors we also found at this time a significant increase of viability of cell exposed for 2 min to APCP when compared to the untreated control. Moreover, NAC pretreatment positively affected the viability of one cell preparations. Evaluation of apoptosis (Tunel test) on all fibroblast preparations treated with 2 min APCP and monitored after 24 h revealed that the number of apoptotic nuclei was comparable to those detected in untreated cells. RT-PCR analysis of cells two hours after APCP treatment revealed only a transient increasing in GPX1, IL6, FGF2 and TNFα mRNA levels. In conclusion, cell exposure of 2 min APCP, a dose that inactivates skin pathogens, maintain or increase their viability, but revealed a transitory alteration of the expression of some genes involved in oxidative stress and wound repair.

The expression and significance of Smad4 and Smad7 in newborn rats with hyperoxia-induced chronic lung diseases

Objective To investigate the expression and significance of Smad4 and Smad7 in newborn rats with hyperoxia-induced chronic lung disease(CLD).Methods Sixty-four newborn Wistar rats 12 h after birth were divided into high-oxygen group (n =32) and air group (n =32,control group) by random number table method.The high-oxygen group was placed in the oxygen glass tank with continuous infusion of oxygen.And 1,3,7,14 d after experiment,tracheal separated,the chest opened to expose heart and lung,slices were Masson staining,undergo dynamic observation of the pulmonary pathological changes under light microscope.Lung fibrosis score was carried out to determine the degree of pulmonary fibrosis,and immunohistochemical technique was used to detect Smad4 and Smad7 protein expression in lung tissue.The expression levels of Smad4 and Smad7 protein in lung tissue were detected with Western blot.Results Compared with the air group,there was statistically significant difference in pulmonary fibrosis score on day 7 (2.67 ± 0.21 vs 0.58 ± 0.17) and day 14 (4.48 ± 0.24 vs 0.63 ± 0.13) in high-oxygen group (P ＜ 0.05) ; Smad4 and Smad7 was main in visible lung epithelial cells and interstitial fibroblasts.Smad4 expression in the high-oxygen group gradually enhanced,compared with the air group (P ＜ 0.05) on day 7 (122.35 ± 10.3 vs 140.08 ±7.77) and day 14(129.7 ± 7.33 vs 144.99 ± 6.49).Smad7 expression in the high-oxygen group first increased and then decreased,expression in the high-oxygen group increased on day 7 (122.35 ± 10.29 vs 130.56 ±9.8),and compared decreased with the air group(P ＜0.05) on day 14(132.16 ±4.38 vs 126.22 ±6.49).Conclusion The newborn rat exposed hyperoxia,the up-regulation of Smad4 protein expression and the down-regulation of Smad7 protein expression are imposible closely related to the happen and development of CLD pulmonary fibrosis. Key words: Smad4 protein; Smad7 protein; Pulmonary fibrosis; Hyperoxia; Rat, newborn

Construction of A549 cell lines silence expressing miR-26a

Objective To construct a lentiviral expression vector stably down-regulating has-miR-26a-5p in human lung adenocarcinoma cell line(A549). Methods Specific complementary sequence of mature hsa-miR-26a-5p was synthesized in vitro. Restriction enzyme cutting sites of Age I and EcoRI was added to this sequence, which was then cloned into the plasmid of GV280 by double digestion to obtain the lentiviral vector named GV280-miR-26a-down. The recombinant plasmid was confirmed by DNA sequencing, subsequently cotransfected into 293T cell with viral pac-kaging plasmid pGC-LV vector, pHelper 1.0 vector, and pHelper 2.0 vector. Virus titer was measured according to the expression level of green fluorescent protein. The lentivirus down-regulating miR-26a was transfected into A549 cells which was screened by puromycin subsequently to obtain stable cell line. One of miR-26a's target gene SMAD1 was detected by Western blot to ensure the function of this A549 cell line. Results The lentivirus vector of GV280-miR-26a-down was successfully constructed confirmed by DNA sequencing, and then was packaged in 293T cells to produce lentiviral particles and the virus titer reached to 2×1012 TU/L. The efficiency of recombinant lentivirus transfecting A549 cells was up to 95%. Nearly 100% A549 cells expressed GFP after being screened by 2.5 mg/L puromycin for about 2 weeks. Western blot results showed that the expression of miR-26a's target gene SMAD1 significantly upregulated in miR-26a-down group compared with viruses in the control group and the blank control group. Conclusion The lentivirus vector of GV280-miR-26a-down and the A549 cell line stably down-regulating has-miR-26a-5p can be successfully constructed, which provides the basis for further study of miR-26a. Key words: miR-26a; A549 cell line; Lentivirus vector

ACVRL1 gene variant in a patient with vein of Galen aneurysmal malformation.

Although mutations in the RASA1 gene in vein of Galen aneurysmal malformation (VGAM) and an endoglin gene mutation in a VGAM patient with a family history of hereditary hemorrhagic telangiectasia (HHT) have been identified, most VGAM cases have no mutation in these genes. We sought to detect mutations in other genes related to HHT. We screened for mutations in RASA1 and three genes (endoglin, activin receptor-like kinase 1 (ACVRL1), encoding ALK1, and SMAD4) related to HHT in four VGAM patients. One variant (c.652 C>T p.R218W) in ACVRL1 was identified. Immunoblotting revealed that the ALK1-R218W protein could not promote SMAD1/5/8 phosphorylation by BMP9 stimulation. On the other hand, wild-type ALK1 could enhance the phosphorylation as expected. Furthermore, the transcriptional activation of ALK1-R218W was less efficient than that of wild-type ALK1. We identified 1 variant in ACVRL1 in a VGAM patient. These findings suggest that the ACVRL1 variant-R218W may be associated with the pathogenesis of VGAM.