Exosomal miR-221 derived from hydroquinone-transformed malignant human bronchial epithelial cells is involved in cell viability of recipient cells.

miR-221, an oncogenic microRNA, can promote cell proliferation and is highly expressed in various types of tumors. However, the role of exosomal miR-221 in benzene-caused carcinogenesis remains elusive. Our study was designed to investigate whether exosomes secreted by the hydroquinone (HQ; an active metabolite of benzene)-transformed malignant cells can transmit miR-221 to normal recipient cells and its possible effects on cell viability. Our investigation revealed that expression levels of miR-221 were significantly increased in HQ-transformed malignant cells relative to normal controls. Furthermore, exposure of control cells to exosomes that were derived from HQ-transformed malignant cells increased miR-221 levels and promoted their proliferation. Analyses of the biological potency of exosomes derived from HQ-transformed malignant cells in which miR-221 levels were decreased using an inhibitor, showed that both miR-221 levels and proliferation of recipient cells were decreased, but still were higher than those of normal 16HBE cells. Our study indicates that exosomal miR-221 derived from HQ-transformed malignant human bronchial epithelial cells is involved in the proliferation of recipient cells.

MicroRNA-1225-5p inhibits the development and progression of thyroid cancer via targeting sirtuin 3.

The objective of this study was to grab the expression levels and biological function of microRNA-1225-5p in human thyroid cancer. The miR-1225-5p expression in thyroid cancer tissue and cell lines was detected, followed by detecting the effects of overexpression of miR-1225-5p on the proliferation, apoptosis, migration and invasion of thyroid cancer cells. Moreover, the target relationship between miR-1225-5p and sirtuin 3 (SIRT3) was investigated. Besides, the effect of miR-1225-5p on the activation of Wnt/ß-catenin pathway was elucidated. The miR-1225-5p expression was downregulated in thyroid cancer tissues and cell lines. Overexpression of miR-1225-5p significantly inhibited TPC-1 cell proliferation, increased cell apoptosis, and suppressed migration and invasion. In addition, SIRT3 was verified as a functional target of miR-1225-5p. SIRT3 expression was overtly increased in thyroid cancer tissues and cell lines. Overexpression miR-1225-5p and SIRT3 at the same time could significantly reverse the effects of miR-1225-5p overexpression alone on the malignant phenotypes of thyroid cancer cells. Furthermore, overexpression of miR-1225-5p significantly inhibited the activation of the Wnt/ß-catenin pathway, which was remarkably reversed after overexpression of SIRT3. Our data reveal that downregulation of miR-1225-5p may promote tumor cell proliferation and metastasis in thyroid cancer via a direct targeting of SIRT3. Activation of the Wnt/ß-catenin pathway may be a key mechanism to mediate the role of miR-1225-5p/SIRT3 axis in thyroid cancer. miR-1225-5p may serve as a potential anti-cancer target for thyroid cancer treatment.

Quantitative assessment of the association between L769L and S836S polymorphisms at RET gene and medullary thyroid carcinoma risk.

RET single nucleotide polymorphisms (SNPs) have been implicated in the pathogenesis and progression of medullary thyroid carcinoma (MTC). Epidemiologic studies have evaluated the association between RET L769L and S836S polymorphisms and predisposition to MTC. However, the results were inconclusive. A literature search was performed using the PubMed database for relevant studies published through October 31, 2013. A total of 13 eligible studies were selected for this meta-analysis, including 1,117 cases and 1,916 controls for L769L and 1,230 cases and 2,246 controls for S836S. The carrier frequency of the variant alleles was 26.3 % in patients with MTC and 24.6 % in controls for L769L polymorphism, and 6.6 % in patients with MTC and 5.0 % in controls for S836S polymorphism. In our pooled analysis of all these studies, the results of our meta-analysis suggested that the RET L769L variant was not significantly associated with an elevated MTC risk (odds ratio (OR) 1.06, 95 % confidence interval (CI) 0.94-1.19). And there was no evidence for the association between the S836S variant and MTC risk (OR 1.20, 95 % CI 0.97-1.49). Moreover, no significant differences were found when considering patients or controls heterozygous or homozygous for RET L769L and S836S polymorphisms. In conclusion, this meta-analysis suggests that RET L769L and S836S polymorphisms may not be associated with MTC development.

Transmission electron microscopic morphological study and flow cytometric viability assessment of Acinetobacter baumannii susceptible to Musca domestica cecropin.

Multidrug-resistant (MDR) Acinetobacter baumannii infections are difficult to treat owing to the extremely limited armamentarium. Expectations about antimicrobial peptides' use as new powerful antibacterial agents have been raised on the basis of their unique mechanism of action. Musca domestica cecropin (Mdc), a novel antimicrobial peptide from the larvae of Housefly (Musca domestica), has potently active against Gram-positive and Gram-negative bacteria standard strain. Here we evaluated the antibacterial activity of Mdc against clinical isolates of MDR-A. baumannii and elucidate the related antibacterial mechanisms. The minimal inhibitory concentration (MIC) of Mdc was 4 µg/mL. Bactericidal kinetics of Mdc revealed rapid killing of A. baumannii (30 min). Flow cytometry using viability stain demonstrated that Mdc causes A. baumannii membrane permeabilization in a concentration- and time-dependent process, which correlates with the bactericidal action. Moreover, transmission electron microscopic (TEM) examination showed that Mdc is capable of disrupting the membrane of bacterial cells, resulting in efflux of essential cytoplasmic components. Overall, Mdc could be a promising antibacterial agent for MDR-A. baumannii infections.

The therapeutic effect of vascular endothelial growth factor gene- or heme oxygenase-1 gene-modified endothelial progenitor cells on neovascularization of rat hindlimb ischemia model.

OBJECTIVE: To explore the therapeutic potential of endothelial progenitor cells (EPCs) transfected with vascular endothelial growth factor A (VEGFA) and heme oxygenase-1 (HO-1) on rat hindlimb ischemia model. METHODS: Eukaryotic expression vectors encoding VEGFA or HO-1 were constructed and introduced into EPCs isolated from rat bone marrow. In total, 150 Sprague Dawley rat hindlimb ischemia models were established and randomized into five groups which were injected via tail vein with phosphate-buffered saline (PBS), nontransfected EPCs, VEGFA-modified EPCs, HO-1-modified EPCs, and both VEGFA- and HO-1-modified EPCs, respectively. The microvessel density, the expressions of VEGFA and HO-1 in the ischemic limbs, the recovery of blood flow as evaluated by laser-Doppler perfusion imaging, and the rate of limb salvage were compared among different groups. RESULTS: Transplantation of both VEGFA- and HO-1-modified EPCs in recipient rats significantly increased the microvessel density (expressed as capillaries/m(2) at day 21 after operation, group vascular endothelial growth factor (VEGF)+HO-1, 357 ± 14.1; group VEGF, 253.7 ± 9.9; group HO-1, 255.5 ± 12.5; group EPC, 210.7 ± 10.3; group PBS, 144.3 ± 9.3; P < .001), the expressions of VEGFA and HO-1 in ischemic tissue, the recovery of blood flow (at day 21, VEGF+HO-1 group, 85.4 ± 17.8%; VEGF group, 51.2 ± 13.2%; HO-1 group, 50.4 ± 12.9%; EPC group, 39.9 ± 8.5%; PBS group, 28.3 ± 7.8%; P < .001), and the rate of limb salvage (VEGF+HO-1 group, 94.4%; VEGF group or HO-1 group, 63.6%; EPC group, 50.0%; PBS group, 11.1%), compared with transplantation of either VEGFA- or HO-1-modified EPCs alone, or of nontransfected EPCs, or PBS injection. The order of therapeutic effectiveness on ischemic limbs was VEGFA- + HO-1-modifed EPC > either VEGFA- or HO-1-modified EPC alone > nontransfected EPC > PBS. CONCLUSIONS: VEGFA-modified EPC and HO-1-modified EPC synergized with each other in promoting angiogenesis in ischemic limbs of rat hindlimb ischemia model. In addition to VEGF, the introduction of HO-1 in EPC-based transplantation may serve as a novel and useful therapeutic strategy for ischemic disease of lower extremity.

Ultrasonic Characteristics Improve Prediction of Central Lymph Node Metastasis in cN0 Unifocal Papillary Thyroid Cancer.

Background: Prediction of central lymph node metastasis (CLNM) is vital for clinical decision-making processes in clinically N0 (cN0) unifocal papillary thyroid carcinoma (PTC), but the sensitivity of preoperative detection of CLNM is limited. The aim of the present study was to determine whether there are ultrasonic (US) characteristics associated with CLNM. Methods: In total, 1657 PTC patients (514 men and 1143 women) were enrolled in the present study between January 2018 and May 2021. The patients met the following inclusion criteria based on preoperative detection: suspected nodule confirmed as PTC by biopsy; the nodule was unifocal and less than 4 cm in diameter; no prior neck radiation exposure; no extrathyroidal extension; and no CLNM or distant metastases on imaging. All the enrolled patients underwent total thyroidectomy with prophylactic central lymph node dissection (CLND). A postoperative pathological diagnosis was made. Results: CLNM was found in 58.4% of male patients and 36.9% of female patients. In univariate analysis, size, adjacent anterior capsule, distance to the lower pole and color Doppler flow imaging (CDFI) were considered risk factors for the male and female groups (p < 0.05). In multivariate analyses, size, adjacent anterior capsule, distance to the lower pole and CDFI were independent risk factors for male patients. For females, the independent risk factors included size, adjacent anterior capsule, distance to the lower pole and CDFI. Conclusion: In the present cohort, US imaging characteristics, including size, adjacent anterior capsule, distance to the lower pole and CDFI, were identified to be potentially beneficial in preoperative clinical decision-making processes for cN0 unifocal PTC patients.

Surgical Outcome and Malignant Risk Factors in Patients With Thyroid Nodule Classified as Bethesda Category III.

Background: Thyroid nodules are a very common finding in the general population. Fine-needle aspiration (FNA) has been recommended as the initial test for the evaluation of thyroid nodules. The trend on reporting as atypia of undetermined significance (AUS) has been significantly increased, but the malignant risk is still controversial among different studies. The aim of this study is to investigate the malignancy risk of thyroid nodules reported as Bethesda category III (AUS/FLUS) on initial FNA. Method: We reviewed 272 papillary thyroid cancer (PTC) patients with suspicious thyroid nodules who underwent fine-needle aspiration and received surgical treatment during 2019 to 2020. Results: One hundred ten (40.4%) patients were diagnosed with PTC. Multivariate analysis showed that microcalcification (p = 0.037, OR = 2.260, 95% CI: 1.051-4.860), shape (p = 0.003, OR = 4.367, 95% CI: 1.629-11.705), diameters (p = 0.002, OR = 0.278, 95% CI: 0.123-0.631), anti-thyroglobulin antibodies (TGAb) (p = 0.002, OR = 0.150, 95% CI: 0.046-0.494), anti-thyroid peroxidase antibody (A-TPO) (p = 0.009, OR = 4.784, 95% CI: 1.486-15.401), and nodule goiter (p < 0.001, OR = 0.100, 95% CI: 0.046-0.217) were independent malignant risk factors in patients with thyroid nodule classified as Bethesda category III. Conclusion: In this study, malignant risk factors in patients with thyroid nodule classified as Bethesda category III were significantly associated with preoperative serum TGAb, A-TPO, microcalcification, irregular shape, and nodule diameters. Nodules with malignant factors should be carefully elevated; surgery may be the better option for those patients.

Predictive Factor of Large-Volume Central Lymph Node Metastasis in Clinical N0 Papillary Thyroid Carcinoma Patients Underwent Total Thyroidectomy.

Large-volume central lymph node metastasis (large-volume CLNM) is associated with high recurrence rate in papillary thyroid carcinoma (PTC) patients. However, sensitivity in investigating large-volume CLNM on preoperative ultrasonography (US) is not high. The aim of this study is to investigate the clinical factors associated with large-volume CLNM in clinical N0 PTC patients. We reviewed 976 PTC patients undergoing total thyroidectomy with central lymph node dissection during 2017 to 2019. The rate of large-volume LNM was 4.1% (40 of 967 patients). Multivariate analysis showed that male gender and young age (age<45 years old) were independent risk factors for large-volume CLNM with odds ratios [(OR), 95% confidence interval (CI)] of 2.034 (1.015-4.073) and 2.997 (1.306-6.876), respectively. In papillary thyroid microcarcinoma (PTMC), capsule invasion was associated with large-volume CLNM with OR (95% CI) of 2.845 (1.110-7.288). In conventional papillary thyroid cancer (CPTC), tumor diameter (>2cm) was associated with large-volume CLNM, with OR (95% CI) 3.757 (1.061-13.310), by multivariate analysis. In ROC curve analysis on the diameter of the CPTC tumor, the Area Under Curve (AUC) =0.682(p=0.013), the best cut-off point was selected as 2.0cm. In conclusion, male gender and young age were predictors for large-volume CLNM of cN0 PTC. cN0 PTMC patient with capsule invasion and cN0 CPTC patient with tumor diameter >2cm were correlated with large-volume CLNM. Total thyroidectomy with central lymph node dissection may be a favorable primary treatment option for those patients.

Adsorption of Pb(II) from aqueous solution by polyacrylic acid grafted magnetic chitosan nanocomposite.

A creative combination of chitosan with polyacrylic acid (PAA) improves the acidity resistance of chitosan and increases its potential in the field of adsorption. In order to facilitate recovery, magnetic nanoparticles were incorporated in CS-PAA to obtain a magnetic-CS-PAA (MCS-PAA) nanocomposite. The physical and chemical characteristics of the composite adsorbent MCS-PAA were determined by SEM, TEM, FTIR, EDX, XRD, and XPS. This environmental-friendly, magnetic, composite adsorbent showed significantly better adsorption performance than those of the individual adsorbents alone. The maximal adsorption capacity was 204.89 mg/g according to the Langmuir isotherm model, when the concentration of Pb(II) was 100 mg/L at the equilibrium time of 70 min. The main adsorption mechanism was the complexation between the carboxyl, amino, and hydroxyl groups in MCS-PAA and Pb(II). Further, introduction of PAA also improved the acid resistance of CS. The new adsorbent MCS-PAA is thus expected to facilitate a wider range of applications for chitosan in the adsorption of Pb(II).

Exosomes derived from normal human bronchial epithelial cells down-regulate proliferation and migration of hydroquinone-transformed malignant recipient cells via up-regulating PTEN expression.

The gene encoding the tumor suppressor, phosphatase and tensin homolog (PTEN), located on chromosome 10, is frequently expressed at low levels in various tumors, resulting in the stimulation of cell proliferation and migration. However, the role of exosomal PTEN in cell-cell communication during the progress of benzene-induced carcinogenesis remains unclear. The goal of this study was to explore whether exosomes derived from normal human bronchial epithelial cells (16HBE) could transmit PTEN to hydroquinone-transformed malignant recipient cells (16HBE-t) and its possible effects on cell proliferation and migration. Consistent with PTEN expression being down-regulated in transformed cells, we found that its expression was significantly decreased in 16HBE-t relative to 16HBE cells and that purified exosomes secreted by 16HBE, up-regulated PTEN levels in recipient 16HBE-t cells. Thus, down-regulating their proliferation and migration. Further, when exosomes derived from 16HBE cells that had been treated with the PTEN inhibitor SF1670, were incubated with recipient 16HBE-t cells, they exhibited decreased PTEN levels, with a corresponding increase in their proliferation and migration. In conclusion, our study demonstrates that exosomes derived from 16HBE cells can down-regulate proliferation and migration of recipient 16HBE-t cells via transferring PTEN.

Surgical Repair of Aortic Rupture Caused by Retained Neurointerventional Guidewire.

Retention of guidewire during a neurointervention procedure is extremely rare and potentially lethal. We report a successful operation on an aortic rupture caused by a retained guidewire from a neurointervention procedure 2 years ago. During the operation, we took out the guidewire from both the aortic arch and femoral artery. Postoperative treatment was uneventful. At the 10-month follow-up, the patient remained asymptomatic and computed tomography showed no abnormality.

Intravenous leiomyomatosis with different surgical approaches: Three case reports.

BACKGROUND: Intravenous leiomyomatosis (IVL) is a rare and complicated disease, which requires surgery by a multidisciplinary team. However, the optimal surgical approach has not been determined. CASE SUMMARY: Here we report three cases of IVL treated with different surgical approaches. All patients presented with circulation symptoms. Two patients had lower extremity edema and the other had cardiopalmus. The diagnosis of IVL was confirmed based on the imagining examinations and pathological findings. All patients underwent surgical treatment and were discharged without any complications. CONCLUSION: Preoperative examination is crucial for surgical planning and surgical approach is dependent on the patient's condition and tumor involvement.

Diagnosis and treatment of 34 cases of blunt duodenal injuries.

OBJECTIVE: To summarize the experience on diagnosis and treatment of blunt duodenal injuries, and thus to improve the therapeutic skills. METHODS: Clinical data of 34 cases of blunt duodenal injuries admitted to our hospital from 1990 to 2006 were retrospectively analyzed. RESULTS: Among them, 28 cases were cured, 20 cases presented with complications, and 6 cases died. The causes of death were: duodenal or pancreatic fistula in 2 cases, intra-and retroperitoneal infection complicating septicopyemia in 2, disseminated intravascular coagulation in 1 and multiple organ failure in 1. CONCLUSIONS: Misdiagnosis and missed diagnosis predispose to happen for blunt duodenal injuries. Early recognition and surgical intervention are critical to a successful rescue.

Clinical diagnosis and treatment of abdominal visceral injury complicated by craniocerebral injury.

OBJECTIVE: To improve the cure rate of patients with abdominal visceral injury complicated by craniocerebral injury. METHODS: Clinical data of 176 cases of abdominal visceral injury complicated by craniocerebral injury were retrospectively analyzed. RESULTS: In this series, 44 cases died and the mortality was 25.0. The main cause of death is abdominal visceral injury combined with shock and severe craniocerebral injury. CONCLUSIONS: It is essential to improve the cure rate by accurate diagnosis at early stage. Abdominal paracentesis and CT should be performed promptly and dynamically. Priority should be given to the treatment of life-threatening injuries.

Diagnosis and treatment of pancreatic trauma.

OBJECTIVE: To improve diagnosis and therapeutic efficacy for pancreatic trauma. METHODS: We retrospectively analyzed 71 cases of pancreatic injuries received in our department from 1987 to 2004. Different surgical procedures were performed according to different patterns of pancreatic injuries. Among them, 31 cases were defined as Grade I or II injury according to the pancreatic organ injury score developed by American Association for the Surgery of Trauma and were performed pancreas debridement and drainage; 26 cases belonged to Grade III injury and were performed distal pancreatectomy plus external drainage; 10 cases of Grade IV injury in whom 8 were performed distal Roux-en-Y pancreaticojejunostomy and 2 were performed Whipple operation; 4 cases of Grade V injury in whom 1 was performed restoration of duodenal damage, suture of proximal pancreatic laceration and distal Roux-en-Y pancreaticojejunostomy, and 3 were performed Whipple operation. RESULTS: Sixty-six cases were cured, of whom 14 had postoperative complications, and 5 cases died. The causes of death were of pancreatic fistula in 2 cases, upper gastrointestinal bleeding in 1 case, ARDS in 1 case, and serious abdominal infection in 1 case. CONCLUSIONS: Preoperative diagnosis for pancreatic trauma is rather challenging. Prompt explorative laparotomy is still a reliable diagnostic method for pancreatic trauma. In order to improve curative rate, different surgical procedures should be undertaken according to different sites and grades of pancreatic traumas.

Management of femoral artery pseudoaneurysm due to addictive drug injection.

OBJECTIVE: To study surgical management for patients with femoral pseudoaneurysm resulting from addictive drug injection. METHODS: Clinical data of 34 patients with femoral pseudoaneurysm resulting from addictive drug injection were retrospectively reviewed. RESULTS: Thirteen patients underwent bypass graft (end to side) of external iliac artery and superficial femoral artery using expanded polytetrafluoroethylene (ePTFE). Three patients who had an autogenous saphenous vein graft in situs, one of whom was then performed an ePTFE graft when rupture and bleeding occurred at the anastomotic site. Color Doppler image showed patent grafted blood vessels in all the patients after operation. Eighteen patients had their femoral arteries ligated. Limbs of all the 34 patients were saved. CONCLUSIONS: Ligating femoral artery is an effective way to treat femoral artery pseudoaneurysm if autogenous saphenous vein graft or artificial vessel graft is not applicable.

Protein kinase C beta mediates CD40 ligand-induced adhesion of monocytes to endothelial cells.

Accumulating evidence supports the early involvement of monocyte/macrophage recruitment to activated endothelial cells by leukocyte adhesion molecules during atherogenesis. CD40 and its ligand CD40L are highly expressed in vascular endothelial cells, but its impact on monocyte adhesion and the related molecular mechanisms are not fully understood. The present study was designed to evaluate the direct effect of CD40L on monocytic cell adhesion and gain mechanistic insight into the signaling coupling CD40L function to the proinflammatory response. Exposure of cultured human aortic endothelial cells (HAECs) to clinically relevant concentrations of CD40L (20 to 80 ng/mL) dose-dependently increased human monocytic THP-1 cells to adhere to them under static condition. CD40L treatment induced the expression of vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein expression in HAECs. Furthermore, exposure of HAECs to CD40L robustly increased the activation of protein kinase C beta (PKCß) in ECs. A selective inhibitor of PKCß prevented the rise in VCAM-1 and THP-1 cell adhesion to ECs. Moreover, stimulation of ECs to CD40L induced nuclear factor-κB (NF-κB) activation. PKCß inhibition abolished CD40L-induced NF-κB activation, and NF-κB inhibition reduced expression of VCAM-1, each resulting in reduced THP-1 cell adhesion. Our findings provide the evidence that CD40L increases VCAM-1 expression in ECs by activating PKCß and NF-κB, suggesting a novel mechanism for EC activation. Finally, administration of CD40L resulted in PKCß activation, increased VCAM-1 expression and activated monocytes adhesiveness to HAECs, processes attenuated by PKCß inhibitor. Therefore, CD40L may contribute directly to atherogenesis by activating ECs and recruiting monocytes to them.

Stable transfection of estrogen receptor-alpha suppresses expression of cyclooxygenase-2 and vascular endothelial growth factor-C in MDA-MB-231 breast cancer cells.

BACKGROUND: Estrogen receptor (ER)-negative breast cancer cells are more aggressive than ER-positive cells. Elevated levels of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor-C (VEGF-C) expression have been detected in cultured human breast cancer cells and are associated with negative hormone receptor status. In this study, we created ERalpha stable transfectants in MDA-MB-231 cells to explore the effect of ERalpha on cell growth and COX-2 and VEGF-C expression. METHODS: The green fluorescent protein (GFP)-ERalpha plasmids were stably transfected into ER-negative MDA-MB-231 cells. The proliferation and migration of untransfected MDA-MB-231 cells, ERalpha-transfected MDA-MB-231 cells and ER-positive MCF-7 cells were determined. The expression of COX-2, and the levels of VEGF-C mRNA and the VEGF-C secretion concentration were assayed in these cell lines. RESULTS: The proliferation and migration capacities of ERalpha-tranfected MDA-MB-231 cells were significantly decreased (P < 0.05). The expression of COX-2 was significantly lower in ERalpha-tranfected MDA-MB-231 cells than in untranfected MDA-MB-231 cells. The mRNA and protein levels of VEGF-C were lower in ERalpha-tranfected MDA-MB-231 cells than in untransfected MDA-MB-231 cells (P < 0.05). CONCLUSIONS: ERalpha stable transfection inhibits proliferation and migration capacities of MDA-MB-231 cells and decreases expression of COX-2 and VEGF-C. The decreases of proliferation and migration capacities may be related to suppression of COX-2 and VEGF-C expression.

Gambogic acid induces G0/G1 cell cycle arrest and cell migration inhibition via suppressing PDGF receptor ß tyrosine phosphorylation and Rac1 activity in rat aortic smooth muscle cells.

AIM: Gambogic acid (GA) is the major active compound of Gamboge, a brownish or orange resin exuded from Garcinia hanburryi tree in Southeast Asia. Previous studies have demonstrated that GA exhibits potent anticancer effects by inducing cell cycle arrest or apoptosis in many types of cancer cell lines and blocking angiogenesis via inhibition of vascular endothelial cell proliferation and migration. Proliferation and migration of vascular smooth muscle cells (VSMCs) are critical steps in the progress of atherosclerosis and restenosis after angioplasty. In the present study, we investigated whether GA has an inhibitory effect on the proliferation and migration of VSMCs and its possible mechanism. METHODS: The inhibitory effect of GA on the proliferation induced by PDGF-BB and EGF was measured by using Cell number counting assay and [(3)H]-thymidine incorporation. The effects of GA on the cell cycle progression and viability stimulated by PDGF-BB and EGF were also analyzed by flow cytometry analysis. The inhibitory effect of GA on the migration stimulated by PDGF-BB was measured by transwell chamber assay. The effect of GA on the Cell cycle regulatory molecules (cyclinD1, cyclinE, CDK2, CDK4), PDGFR and its downstream signaling molecules including ERK1/2, PLCγ1, AKT and JNK was measured by western blotting. The effect of GA on the Rac1 activity was measured by using GST-pulldown assay. The effects of GA on the tyrosine phosphorylation stimulated by PDGF-BB and EGF and the capacity of GA binding with PDGF-BB and EGF were also measured. RESULTS: We found that GA significantly inhibited proliferation, migration and DNA synthesis in primary cultured rat aortic VSMCs at concentrations of 0.25, 0.5, 1.0 and 2.0 µmol/L after stimulation of 50 µg/L platelet-derived growth factor-BB (PDGF-BB). GA induced G0/G1 phase arrest in the cell cycle progression of VSMCs. No obvious necrosis or apoptosis was found with GA treatment. The expressions of CDK2, CDK4, cyclin D1 and cyclin E, cell cycle regulatory molecules, were significantly suppressed by GA treatment in a concentration-dependent manner. The phosphorylation of PDGF receptor ß (PDGFR-ß) and the activities of downstream intracellular signaling molecules including phospho-ERK, phospho-PLCγ1, phospho-AKT, phospho-JNK and GTP-Rac1 were also inhibited by GA pretreatment. GA inhibited PDGFR-ß phosphorylation through inhibiting the activity of tyrosine directly, rather than indirectly via binding PDGF-BB. CONCLUSIONS: The results of this study provide preliminary evidence that the inhibitory effects of GA on VSMCs proliferation and migration may be mediated through multiple signal pathways controlled by PDGF-Rß activation and its downstream intracelluar signaling.

[Construction of an expression vector carrying short hairpin RNA targeting hTERT gene and its effects on breast cancer cell telomerase activity and proliferation in vivo].

OBJECTIVE: To construct a RNA interference expression vector targeting human telomerase reverse transcriptase gene (hTERT) gene and investigate its effects on telomerase activity and proliferation in breast cancer cells in vitro. METHODS: The shRNA sequences targeting hTERT gene were designed and recombined into pSuper-retro-puro vector. The breast cancer cell lines MCF-7 and MDA-MB231 were transfected with the recombined vector, and the telomerase activity of the cells was tested by telomerase repeat sequence amplification-enzyme linked immunosorbent assay (TRAP-ELISA). The proliferation of the transfected cells was assessed using MTT and soft-agar clone formation assays. RESULTS: The recombinant plasmids pSuper-retro-puro-TERT RNAi#1 and #2 were successfully constructed as confirmed by enzymatic digestion and DNA sequencing. The telomerase activity in the transfected breast cancer cells were down-regulated significantly as compared with that in negative control cells (Plt;0.005). The transfection resulted in significant inhibition of the proliferation of both MCF-7 and MDA-MB231 cells as detected by MTT assay (Plt;0.05) and soft agar clone formation assay (Plt;0.001). CONCLUSION: Transfection with the recombinant plasmid containing the shRNA targeting hTERT gene can down-regulate telomerase activity and inhibit proliferation of breast cancer cells in vitro, suggesting the potential of gene therapy targeting telomerase in the treatment of breast cancer.