JMJD2A contributes to breast cancer progression through transcriptional repression of the tumor suppressor ARHI.

INTRODUCTION: Breast cancer is a worldwide health problem and the leading cause of cancer death among females. We previously identified Jumonji domain containing 2A (JMJD2A) as a critical mediator of breast cancer proliferation, migration and invasion. We now report that JMJD2A could promote breast cancer progression through transcriptional repression of the tumor suppressor aplasia Ras homolog member I (ARHI). METHODS: Immunohistochemistry was performed to examine protein expressions in 155 cases of breast cancer and 30 non-neoplastic tissues. Spearman correlation analysis was used to analyze the correlation between JMJD2A expression and clinical parameters as well as several tumor regulators in 155 cases of breast cancer. Gene and protein expressions were monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Results from knockdown of JMJD2A, overexpression of JMJD2A, Co-immunoprecipitation (Co-IP) assay, dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) elucidated molecular mechanisms of JMJD2A action in breast cancer progression. Furthermore, the effects of ARHI overexpression on JMJD2A-mediated tumor progression were investigated in vitro and in vivo. For in vitro experiments, cell proliferation, wound-healing, migration and invasion were monitored by cell counting, scratch and Boyden Chamber assays. For in vivo experiments, control cells and cells stably expressing JMJD2A alone or together with ARHI were inoculated into mammary fat pads of mice. Tumor volume, tumor weight and metastatic nodules were measured by caliper, electronic balance and nodule counting, respectively. RESULTS: JMJD2A was highly expressed in human breast cancers and positively correlated with tumor progression. Knockdown of JMJD2A increased ARHI expression whereas overexpression of JMJD2A decreased ARHI expression at both protein and mRNA levels. Furthermore, E2Fs and histone deacetylases were involved in the transcriptional repression of ARHI expression by JMJD2A. And the aggressive behavior of JMJD2A in breast cancers could be reversed by re-expression of ARHI in vitro and in vivo. CONCLUSION: We demonstrated a cancer-promoting effect of JMJD2A and defined a novel molecular pathway contributing to JMJD2A-mediated breast cancer progression.

C/EBP-α ameliorates CCl(4)-induced liver fibrosis in mice through promoting apoptosis of hepatic stellate cells with little apoptotic effect on hepatocytes in vitro and in vivo.

CCAAT enhancer binding protein-α (C/EBP-α) is a transcript factor that regulates adipocyte differentiation and induces apoptosis in hepatic stellate cells (HSCs) in vivo and in vitro. However, the effect of C/EBP-α on hepatocytes in vivo remains unknown. This study investigated whether C/EBP-α exerts different apoptotic effects on hepatocytes and HSCs in vitro and in vivo. An adenovirus vector-expressing C/EBP-α gene was constructed, and a rat hepatic stellate cell lines (HSC-T6) and hepatocytes were transfected. A CCl(4)-induced liver fibrosis model in mice was also utilized. C/EBP-α induced apoptosis in hepatocytes and HSCs, but a significant difference between these cell types was observed in vitro. The mitochondrial pathway was involved in the apoptotic process and was predominant in HSC-T6 apoptosis. In the CCl(4)-induced mice liver fibrosis model, the administration of Ad-C/EBP-α decreased extracellular matrix deposition, including collagen and hydroxyproline content, and γ-GT levels, a marker of liver damage, were reduced significantly. Immunohistochemistry and TUNEL assay results showed an increase of apoptosis in HSCs, but hepatocytes were less affected. C/EBP-α induced differential apoptotic effects in hepatocytes and HSCs in vitro and in vivo. This differential effect could be a potential target for the treatment of hepatic fibrosis with little hepatic toxicity.

Cellular prion protein promotes glucose uptake through the Fyn-HIF-2α-Glut1 pathway to support colorectal cancer cell survival.

Cellular prion protein (PrPc) is a glycosylphosphatidylinositol-anchored membrane protein that has various physical functions, including protection against apoptotic and oxidative stress, cellular uptake of copper ions, transmembrane signaling, and adhesion to the extracellular matrix. In this study, we show that PrPc is highly expressed in colorectal adenocarcinomas. Transcriptome profiling of PrPc-depleted DLD-1 cells revealed downregulation of glucose transporter 1 (Glut1). PrPc is shown to be involved in regulating Glut1 expression through the Fyn-HIF-2α pathway. As Glut1 is the natural transporter of glucose and is required for the high glycolytic rate seen in colorectal tumors, silencing of PrPc reduced the proliferation and survival rate of colorectal cancer cells in vitro. In vivo, knockdown of PrPc by hydrodynamic injection with a cocktail of PrPc-shRNA-encoding plasmids also inhibited tumorigenicity in a xenograft model in nude mice. In summary, our data characterize a novel molecular mechanism that links PrPc expression to the regulation of glycolysis. Targeting PrPc will therefore be a promising strategy to overcome the growth and survival advantage in colorectal tumors.

RACK1 promotes breast carcinoma migration/metastasis via activation of the RhoA/Rho kinase pathway.

We aimed to gain a mechanistic understanding of the role of RACK1 in breast carcinoma migration/metastasis. Migration assays were conducted in breast carcinoma cell lines. siRNA targeting RACK1 as well as the Rho kinase inhibitor were also applied. Immunoprecipitation and immunofluorescence were used to study the RACK1/RhoA interaction. GTP-Rho pull-down assays were performed to assess the activation of RhoA. We also conducted immunohistochemistry in 160 breast carcinoma samples. Experiments in vitro showed that RACK1 promotes migration via interaction with RhoA and activation of the RhoA/Rho kinase pathway. Immunohistochemistry in 160 samples revealed that RACK1 is strongly correlated with accepted tumor spread indicators and RhoA (all P < 0.05). Kaplan-Meier survival analysis indicated a correlation between higher RACK1 expression and shorter survival times (P < 0.001). RACK1 is a prognostic factor that promotes breast carcinoma migration/metastasis by interacting with RhoA and activating the RhoA/Rho kinase pathway.

RACK1: A superior independent predictor for poor clinical outcome in breast cancer.

We aimed to investigate the expression of RACK1 in breast cancer, evaluate its role in predicting prognosis and compare with commonly used biomarkers: Ki67, ER, PR and HER-2 for patients with breast cancer. The RACK1 expression and its clinical significance were examined in 160 breast carcinoma patients using immunohistochemistry. Correlations of RACK1 expression with other commonly used biomarkers and survival analyses were assessed. Immunohistochemistry results showed that the number of RACK1 cases scoring 0, 1, and 2 were 66, 54, and 40, respectively. RACK1 staining was strongly related to clinical stage, histological grade, Ki67, ER, PR and HER-2 (all p < 0.05). Consistently, all of the cases exhibiting RACK1 staining score 0 were survivors, whereas the majority (55.0%) of those exhibiting RACK1 staining score 2 were deaths. Kaplan-Meier survival analysis of 160 cases revealed a correlation between higher RACK1 expression levels and shorter overall survival times (p < 0.001). Univariate and multivariate analyses revealed that RACK1, tumor size, lymph node metastasis, and HER-2 were independent prognostic factors (all p < 0.05). Interestingly, receiver operator characteristic (ROC) curves showed that the ROC areas for RACK1, Ki67, ER, PR and HER-2 were 0.833, 0.766, 0.446, 0.387, and 0.689, respectively, and the superiority of RACK1 in sensitivity and specificity as biomarker was demonstrated. To our knowledge, it is the first time to investigate the expression of RACK1, and identified that RACK1 is a superior independent biomarker for diagnosis and prognosis comparing with currently widely used diagnostic index in breast carcinoma.

Overexpression and involvement of special AT-rich sequence binding protein 1 in multidrug resistance in human breast carcinoma cells.

Special AT-rich sequence binding protein (SATB) 1 has been proposed to act as a determinant for the acquisition of metastatic activity by controlling expression of a specific set of genes that promote metastatic activity. Here we found that SATB1 expression is upregulated in multidrug-resistant breast cancer cells that exhibit higher invasive potential than the parental cells. Apart from accelerating metastasis and inducing epithelial-mesenchymal transition, SATB1 was demonstrated to confer resistance to both P-glycoprotein-related and P-glycoprotein-non-related drugs on MCF7 cells, which was accompanied by decreasing accumulation of adriamycin in SATB1-overexpressing transfectants. SATB1 depletion could partially reverse the multidrug resistance (MDR) phenotype of MCF7/ADR in vitro and in vivo. The SATB1-induced P-glycoprotein-mediated MDR could be reversed by treatment with anti-P-glycoprotein mAb. Moreover, SATB1 plays an important role in anti-apoptotic activity in MCF7/ADR cells in response to adriamycin treatment, which suggests another mechanism contributing to SATB1-related MDR of breast cancers. These data provide new insights into the mode by which breast tumors acquire the MDR phenotype and also imply a role for SATB1 in this process.

RACK1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo.

A yeast two-hybrid system was utilized to identify novel PI3K p110alpha-interacting proteins, of which receptor of activated protein kinase C1 (RACK1) was chosen for successive detailed analyses. Our aim was to investigate the function(s) of RACK1 and its involvement in mechanisms of breast carcinoma proliferation and invasion/metastasis. Experiments in breast carcinoma cell lines stably transfected with RACK1, as well as nude mouse models, showed that RACK1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo. Conversely, knockdown of RACK1 by siRNA in vitro inhibited proliferation, migration, and invasion. In cell lines stably transfected with RACK1, p-AKT, cyclin D1, cyclin D3, and CD147 expression, as well as MMP2 activity, were elevated. RACK1-induced migration could be inhibited by the addition of Rho-kinase inhibitor. In 160 breast carcinoma cases, survival analyses established that RACK1 is an independent prognostic factor for poor outcome (P < 0.001). In conclusion, RACK1 is an independent prognosis-related factor and promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo.

Loss of BCL2L10 protein expression as prognostic predictor for poor clinical outcome in gastric carcinoma.

AIMS: BCL2L10 protein is an apoptosis-related member of the Bcl-2 protein family. The clinical significance of its expression in gastric carcinoma is poorly understood. The aim was to investigate BCL2L10 expression and its clinical and prognostic significance in gastric carcinoma patients. METHODS AND RESULTS: Immunohistochemistry, real-time polymerase chain reaction (PCR) and immunoblotting all revealed extensive loss of BCL2L10 expression in gastric cancer cells. The scaled BCL2L10 expression data was categorized into three groups (groups 0-2) to facilitate statistical analysis. A significant correlation was observed between the lower BCL2L10 expression group and shorter disease-free survival (P=1.956×10(-18)). Multivariate regression analysis showed that loss of BCL2L10 protein expression [P=4.883×10(-8), hazard ratio (HR)=0.252] is an independent prognostic predictor of gastric carcinoma. The receiver operator characteristic (ROC) curve showed that the area for BCL2L10 protein was 0.817 (P=8.331×10(-14)), indicating that loss of BCL2L10 protein expression is an excellent prognostic predictor of gastric carcinoma. CONCLUSIONS: Loss of BCL2L10 protein expression predicts poor clinical outcome in gastric carcinoma.

Twist1-mediated adriamycin-induced epithelial-mesenchymal transition relates to multidrug resistance and invasive potential in breast cancer cells.

PURPOSE: Besides its therapeutic effects, chemotherapeutic agents also enhance the malignancy of treated cancers in clinical situations. Recently, epithelial-mesenchymal transition (EMT) has attracted attention in studies of tumor progression. We aimed to test whether transient Adriamycin treatment induces EMT and apoptosis simultaneously in cancer cells, clarify why the same type of cells responds differentially (i.e., apoptosis, EMT) to Adriamycin treatment, and elucidate the role of Twist1, the master regulator of EMT, in this process. EXPERIMENTAL DESIGN: In unsynchronized MCF7 cells or cells synchronized at different phases, apoptosis, EMT, and concurrent events [multidrug resistance (MDR) and tumor invasion] after Adriamycin or/and Twist1 small interfering RNA treatment were examined in vitro and in vivo. The Adriamycin-induced Twist1 expression and the interaction of Twist1 with p53-Mdm2 were examined by immunoblotting and immunoprecipitation, respectively. RESULTS: We showed in vitro that Adriamycin induced EMT and apoptosis simultaneously in a cell cycle-dependent manner. Only the cells undergoing EMT displayed enhanced invasion and MDR. Twist1 depletion completely blocked the mesenchymal transformation, partially reversed MDR, and greatly abolished invasion induced by Adriamycin. Also, we confirmed in vivo that Twist1 RNA interference improved the efficacy of Adriamycin for breast cancers. Further, Twist1 reduction in Adriamycin-treated cells promoted p53-dependent p21 induction and disrupted the association of p53 with Mdm2. CONCLUSIONS: Our studies show the diverse responses to Adriamycin treatment in cells at different phases, suggest an unrecognized role of EMT in regulating MDR and invasion, and show the efficacy of Twist1 RNA interference in Adriamycin-based chemotherapies for breast cancer.

[Relationship between breast cancer molecular subtypes with clinicopathological characteristics and prognosis].

OBJECTIVES: This study try to subclassify breast cancer into different prognostic subgroups according to immunohistochemical algorithm and discuss the relationship between subtypes and biological and clinical behavior and prognosis. METHODS: One hundred and twenty-eight cases of infiltrative ductal carcinoma were studied using immunohistochemical staining with an antibody panel of ER, PR, HER2 and CK5/6 and subclassified referring to previous reports, and the 9 cases of HER2 positive subtype were tested using FISH. RESULTS: The expression of ER, PR, HER2, and CK5/6 was detected in 67%, 45%, 27% and 27% cases, respectively. All cases were subclassified into five subgroups, with luminal A (55%), luminal B (20%), HER2 positive (7%), basal-like (10%) and unclassified cases (8%). Nine HER2 positive cases all showed amplification of HER2 gene. It was demonstrated that the luminal A group was associated with the best prognosis but the basal-like group worst by univariate analysis. Multivariate analysis demonstrated that both the clinical stage and immunohistochemical subtypes of tumor were related to overall survival. Menses status were different among these subtypes. CONCLUSION: According to the expression of ER, PR, HER2 and CK5/6, infiltrative ductal carcinoma could be subclassified into five subgroups with different biological features and outcome, having a role in evaluating the prognosis and guiding the clinical treatment.

Dual role of Ski in pancreatic cancer cells: tumor-promoting versus metastasis-suppressive function.

Ski used to be defined as an oncogene that contributes to the resistance of tumor cells to transforming growth factor-beta (TGF-beta)-induced growth arrest. As TGF-beta has a dual effect on tumor growth with both tumor-suppressing and -promoting activity depending on the stage of carcinogenesis and the cell type, the precise role of Ski in carcinogenesis remains unclear. In this study, we show that downregulation of Ski through lentivirus-mediated RNA interference decreases tumor growth both in vitro and in vivo, yet promotes cell invasiveness in vitro, and lung metastasis in vivo in the pancreatic cancer cell line SW1990, which contain wild-type Smad4 expression, and the BxPC3 cell line, which is Smad4 deficient. We also show that the downregulation of Ski increases TGF-beta-induced transcriptional activity, which is associated with increased TGF-beta-dependent Smad2/3 phosphorylation, and results in an altered expression profile of TGF-beta-inducible genes involved in metastasis, angiogenesis and cell proliferation and epithelial-mesenchymal transition. Immunohistochemical analysis of specimens from 71 patients with pancreatic adenocarcinoma showed a significant association between overexpression of Ski and decreased patient survival time (P = 0.0024). Our results suggest that Ski may act as a tumor proliferation-promoting factor or as a metastatic suppressor in human pancreatic cancer.

Low-level expression of Smad7 correlates with lymph node metastasis and poor prognosis in patients with pancreatic cancer.

BACKGROUND: Whether Smad7 acts as a tumor proliferation promoting factor or as a metastatic suppressor in human pancreatic cancer remains unclear. This study aims to determine the prognostic value of Smad7 in patients with pancreatic adenocarcinoma. METHODS: Surgical specimens obtained from 71 patients with pancreatic adenocarcinoma were immunohistochemically assessed for Smad7, Ki-67, MMP2, CD34, and Smad4 expression. The relationship between Smad7 expression and the clinicopathological characteristics of patients with pancreatic adenocarcinoma were also evaluated. RESULTS: Fifty-one of 71 specimens (71.8%) were Smad7 positive and 20 specimens were Smad7 negative. Negative expression of Smad7 correlated with lymph node metastasis, liver metastasis after surgery, and a poor survival rate (P = 0.0004, 0.0044, and 0.0003, respectively). We also found an inverse correlation between the expression of Smad7 and MMP2 (P = 0.0189). Multivariate analysis revealed that Smad7 expression was an independent prognostic factor [hazard ratio (HR) 0.3902; 95% confidence interval (CI) 0.1839-0.8277; P = 0.0142]. Furthermore, in both Smad4-negative and Smad4-positive groups, survival of patients with Smad7-positive tumors was significantly better than those with Smad7-negative tumors (both P < 0.0001). CONCLUSIONS: We conclude that low-level expression of Smad7 in pancreatic cancer is significantly associated with lymph node metastasis, high MMP2 expression, and poor prognosis.

Over-expression of ubiquitin carboxy terminal hydrolase-L1 induces apoptosis in breast cancer cells.

Ubiquitin carboxy terminal hydrolase-L1 (UCH-L1) belongs to the UCH proteases family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system. Previous research showed that UCH-L1 was expressed in mouse retinal cells and testicular germ cells, and its function was associated with apoptosis. But it is still unclear whether UCH-L1 is concerned with apoptosis in tumor cells. In order to clarify the role of UCH-L1 in tumor cells, multi-drug resistance (MDR) human breast carcinoma cell line MCF7/Adr, that expresses relatively high UCH-L1, and its parental cell line MCF7, that expresses relatively low UCH-L1, were chosen for this study. We transfected pcDNA3.1-UCH-L1 plasmid and UCH-L1 siRNA into MCF7 and MCF7/Adr cells, respectively. Using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, western blot, Hoechst 33258 staining assay and flow cytometry, we found that over-expression of UCH-L1 in MCF7 cells induced apoptosis. On the other hand, silencing of UCH-L1 in MCF7/Adr cells led to the opposite effect. Moreover, to explore the mechanism underling these observations, we further investigated the expression of phospho-Akt and its downstream signal phospho-IkB-alpha and other signal molecules including Fas, Fas-L, Trail, DR4, DR5, Bax, cytochrome C, active caspase-3, phospho-p53, phospho-Mdm-2, Bcl-2, Bcl-xL, p21 and p27. The results indicated that the process of apoptosis triggered by UCH-L1 is, at least in part, probably through Phosphoinositide 3-kinase (PI3K)/Akt signal pathway. Our findings suggest that modulating the ubiquitination and deubiquitination pathway could be a novel method for tumor therapy.

Cervical actinomycosis with spinal cord compression. Case report and literature review.

Cervical actinomycosis with spinal cord compression is extremely rare. The clinical presentation of spinal actinomycosis may be nonspecific and back pain is the most consistent early symptom. Here, we present such a case with fever, pain in the neck and upper back, progressive weakness and numbness in all 4 limbs with difficulty ambulating, constipation and uroschesis. Correct diagnosis is difficult because the clinical and radiological findings of actinomycosis closely resemble metastatic tumors and other infectious processes. Timely surgical debridement and decompression contributed to the prompt improvement of the patient's conditions, and histopathological demonstration of the inflammatory granulation tissue and Gram-positive sulfur-containing filamentous bacteria led to the correct diagnosis of actinomycosis. The diagnosis must be made promptly because delayed treatment can result in irreversible neurologic damage or death. Timely and long-term antibacterial therapy is essential for the complete recovery of the patient with actinomycosis.

Interaction between CD147 and P-glycoprotein and their regulation by ubiquitination in breast cancer cells.

BACKGROUND: Multidrug-resistant cancer cells overexpressing P-glycoprotein (P-gp) display variations in invasive and metastatic ability through the upregulation of the extracellular matrix metalloproteinase (MMP) inducer (CD147). However, the direct linkage between these two proteins is still unclear. METHODS: We used immunoprecipitation, immunofluorescence analysis, migration and invasion assays, drug sensitivity assay and Western blot to measure the physical and functional interaction between P-gp and CD147. Then we transfected vectors carrying ubiquitin C-terminal hydrolase L1 (UCH-L1) or UCH-L1 siRNA into MCF7 and MCF7/Adr cells, respectively, and investigated the role of UCH-L1 in the regulation of the expression and degradation of P-gp, CD147 and MMP-1, MMP-2, and MMP-9 by quantitative real-time polymerase chain reaction, Western blot and immunoprecipitation. RESULTS: In this paper, we showed that P-gp and CD147 interacted with each other, and that the ubiquitin-proteasome pathway played an important role in the turnover of them. In addition, we found that inhibition of N-glycosylation increased the ubiquitination and degradation of P-gp and CD147, and affected their function. UCH-L1 not only regulated the expression of P-gp, CD147 and MMP-1, MMP-2, and MMP-9, but also the ubiquitination and degradation of P-gp and CD147 in breast cancer cells. CONCLUSION: Our results demonstrate a mechanism underlying the linkage between multidrug resistance and tumor metastasis, and suggest for the first time that modulating the ubiquitination of P-gp and CD147 might be a novel method for tumor therapy.

Up-regulation of CD147 and matrix metalloproteinase-2, -9 induced by P-glycoprotein substrates in multidrug resistant breast cancer cells.

Treatment of animals bearing multidrug resistant (MDR) tumor cells with P-glycoprotein (P-gp) substrates could worsen host survival. It is assumed that this is due to increased tumor metastasis. To clarify the mechanism(s) underlying this observation, the MDR human breast cancer cell line, MCF-7/AdrR, and its sensitive parental line, MCF-7, was treated with various concentrations of P-gp substrate drugs (vincristine, paclitoxel, adriamycin) and a P-gp non-substrate drug (bleomycin) in serum-free media. Increased production of CD147, and matrix metalloproteinases (MMP)-2, -9 was observed only in MDR cancer cells exposed to P-gp substrates, as determined using real-time polymerase chain reaction, western blotting and zymography. Correspondingly, P-gp substrates significantly enhanced the in vitro invasion abilities of MCF-7/Adr cells. It was also found that the drug-induced promotion of CD147, and MMP-2, -9 was consistent with increased expression of epidermal growth factor receptor (EGFR) and that inhibition of either EGFR or P-gp activity could significantly interrupt the downstream effects, and so inhibit in vitro invasion abilities motivated by P-gp substrates. These results imply that treatment of MDR tumors with P-gp substrates could adversely affect therapeutic outcomes through modulating the production of CD147, MMP-2, -9, and EGFR, and suggest that this effect may be initiated by the transporter function of P-gp.

Roles of EGFR-Stat3 signal pathway in carcinogenesis of experimental hepatoma in rats.

The purpose of this study is to investigate if the EGFR-Stat3 signal pathway contributes to the carcinogenesis of hepatoma in rats. Hepatoma was induced in rats by 3'Me-DAB as a model. EGFR, TGFalpha, Stat3, p-Stat3 in different stages of carcinogenesis were detected by immunohistochemistry and Western blot. In situ hybridization was applied to investigate the expression of Stat3 mRNA. The expressions of signal molecules were assessed by KS400 Image Analysis system. The data were statistically evaluated. EGFR, TGFalpha, Stat3 were highly expressed in the stages of liver necrosis and repairment. All hepatocellular carcinoma cases revealed elevated expression of EGFR, TGFalpha. Elevation of Stat3 mRNA and protein levels were identified, increase of activation of Stat3 was also observed. In HCC, there was positive correlation between p-Stat3 level and the expression of TGFalpha and PCNA. Increased expression of Bcl-2 (P < 0.05) coincided with elevated level of p-Stat3. Therefore, the EGFR-Stat3 signal pathway was related to the development of hepatoma in rats. TGFalpha-EGFR autocrine ring formation may lead to the activation of Stat3 and in turn, promote proliferation and regulate the transcription of genes regulating cell apoptosis and cell cycle.

The effect of TGF-ß1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells.

The aim of this study was to investigate whether transforming growth factor-ß1 (TGF-ß1) could induce alveolar epithelial-mesenchymal transition (EMT) in vitro, and whether Smad7 gene transfer could block this transition. We also aimed to elucidate the possible mechanisms of these processes. The Smad7 gene was transfected to the rat type II alveolar epithelial cell line (RLE-6TN). Expression of the EMT-associated markers was assayed by Western Blot and Real-time PCR. Morphological alterations were examined via phase-contrast microscope and fluorescence microscope, while ultrastructural changes were examined via electron microscope. TGF-ß1 treatment induced a fibrotic phenotype of RLE-6TN with increased expression of fibronectin (FN), α-smooth muscle actin (α-SMA) and vimentin, and decreased expression of E-cadherin (E-cad) and cytokeratin19 (CK19). After transfecting the RLE-6TN with the Smad7 gene, the expression of the mesenchymal markers was downregulated while that of the epithelial markers was upregulated. TGF-ß1 treatment for 48 h resulted in the separation of RLE-6TN from one another and a change into elongated, myofibroblast-like cells. After the RLE-6TN had been transfected with the Smad7 gene, TGF-ß1 treatment had no effect on the morphology of the RLE-6TN. TGF-ß1 treatment for 48 h resulted in an abundant expression of α-SMA in the RLE-6TN. If the RLE-6TN were transfected with the Smad7 gene, TGF-ß1 treatment for 48 h could only induce a low level of α-SMA expression. Furthermore, TGF-ß1 treatment for 12 h resulted in the degeneration and swelling of the osmiophilic multilamellar bodies, which were the markers of type II alveolar epithelial cells. TGF-ß1 can induce alveolar epithelial-mesenchymal transition in vitro, which is dependent on the Smads signaling pathway to a certain extent. Overexpression of the Smad7 gene can partially block this process.

[Effect of Smad7 on transforming growth factor-beta1-induced alveolar epithelial to mesenchymal transition].

OBJECTIVE: To investigate whether transforming growth factor beta1 (TGF-beta1) can induce in vitro alveolar epithelial-mesenchymal transition (EMT), and whether Smad7 gene transfer can block this transition and the possible signaling mechanism. METHODS: Rat alveolar type II epithelial cells of the line RLE-6TN were cultured. TGF beta1 (3 ng/mL) was added into the culture fluid. Lipofectamine 2000 was used to transfect Smad7 gene to the RLE-6TN cells. The expression of the markers of the epithelial cells, including E-cadherin and cytokeratin-19 (CK19), and markers of mesenchymal cells, including fibronectin (FN), vimentin, and alpha-smooth muscle actin (alpha-SMA) were assayed using Western blotting and real-time PCR. The morphological alterations were examined by phase-contrast microscope while the ultrastructure changes were examined by electron microscope. RESULTS: Smad7 was successfully transfected to the RLE-6TN cells. Before transfection TGF-beta1 treatment could lead to the expression upregulation of the mesenchymal markers and downregulation of the epithelial markers at the levels of both mRNA and protein, and after transfection, the mesenchymal makers were downregulated while the epithelial markers were upregulated. Before the transfection, TGF-beta1 treatment could lead to the expression upregulation of phosphorylated Smad2/3 which did not obvious change following transfection. TGF beta1 treatment could induce the EMT process of the RLE-6TN cells. Smad7 gene transfected into the RLE-6TN cells could block the process of EMT. Exposed of the RLE-6TN cells to TGF beta1 resulted in degeneration, tumefaction, and gradual disappearance of the osmiophilic multilamellar bodies, markers of type II alveolar epithelial cells. CONCLUSION: Under TGF beta1 treatment, RLE-6TN undergoes a conversion process into myofibroblasts in vitro with the conversion mechanism related to Smad signaling pathway, and transfection of Smad7 gene can partly reverse this process.