Yan Meng

Knockdown of Stat3 expression using RNAi inhibits growth of laryngeal tumors in vivo

AbstractAim:To study the effect of pSilencer 1.0-U6-siRNA-stat3 on the growth of human laryngeal tumors in nude mice.Methods:Hep2 cells were transplanted into nude mice, then at the time of tumor formation, growth rates were observed. After the tumor formed, pSilencer 1.0-U6-siRNA-stat3 was injected. Tumor volumes were calculated, and growth curves were plotted. Representative histological sections were taken from mice bearing transplantation tumors in both treated and control groups, and start3, pTyr-stat3, Bcl-2, cyclin D1, and survivin expression were detected by Western blotting. survivin mRNA levels were detected by Northern blotting, hematoxylin and eosin staining and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay to confirm the apoptosis of tumors.Results:In nude mice, pSilencer 1.0-U6-siRNA-stat3 significantly suppressed the growth of tumors compared with controls (P<0.01). In suppressed stat3 expression, and downregulated BcL2, cyclin D1, and survivin expression within the tumor. This significantly induced apoptosis of the tumors.Conclusion:pSilencer 1.0-U6-siRNA-stat3 was able to inhibit the growth of transplanted human laryngeal tumors in nude mice and induce apoptosis.

Surface electrocardiogram and action potential in mice lacking urea transporter UT-B

UT-B is a urea transporter protein expressed in the kidney and in many non-renal tissues including erythrocytes, brain, heart, bladder and the testis. The objective of this study was to determine the phenotype of UT-B deletion in the heart. UT-B expression in the heart was studied in wild-type mice vs UT-B null mice by utilizing RT-PCR and Western blot. A surface electrocardiogram (ECG) recording (lead II) was measured in wild-type mice and UT-B null mice at the ages of 6, 16 and 52 weeks. For the action potential recording, the ventricular myocytes of 16 w mice were isolated and recorded by floating microelectrode method. The sodium current was recorded by the patch clamp technique. RT-PCR and Western blot showed the UT-B expression in the heart of wild-type mice. No UT-B transcript and protein was found in UT-B null mice. The ECG recording showed that the P-R interval was significantly prolonged in UT-B null mice ((43.5 ± 4.2), (45.5 ± 6.9) and (43.8 ± 7.6) ms at ages of 6, 16 and 52 weeks) vs wild-type mice ((38.6 ± 2.9), (38.7 ± 5.6) and (38.2 ± 7.3) ms, P<0.05). The atrial ventricular heart block type II and III only appeared in the aging UT-B null mice (52 w old). The amplitude of action potential and Vmax decreased significantly in UT-B null mice ((92.17 ± 10.56) and (101.89 ± 9.54) mV/s) vs those in wild-type mice (vs (110.51 ± 10.38) and (109.53 ± 10.64) mV/s, P<0.05). The action potential duration at 50% and 90% (APD50 and APD90) was significantly prolonged in UT-B null mice ((123.83 ± 11.17) and (195.43 ± 16.41) ms) vs that in wild-type mice ((108.27 ± 10.85) and (171.00 ± 15.53) ms, P<0.05). The maximal sodium current decreased significantly in UT-B null mice (−8.80 ± 0.92) nA vs that in wild-type mice ((−5.98 ± 1.07) nA, P<0.05). These results provide the first evidence that UT-B deletion causes progressive heart block in mice.

Panaxadiol Saponin and Dexamethasone Improve Renal Function in Lipopolysaccharide-Induced Mouse Model of Acute Kidney Injury

Background Acute kidney injury (AKI) is a serious complication of systemic inflammatory response syndrome (SIRS), which has a high mortality rate. Previous studies showed that panaxadiol saponin (PDS) and Dexamethasone have similar anti-inflammatory properties and protect cardiopulmonary function in lipopolysaccharide (LPS)-induced septic shock rats. In the present study, we investigated whether PDS or Dexamethasone has a similar role in improving kidney function in LPS-induced AKI mice. Methods and Results Mice subjected to LPS (10 mg/kg) treatment exhibited AKI demonstrated by markedly increased blood urea nitrogen and creatinine levels compared with controls (P<0.01). However, PDS and Dexamethasone induce similar reverse effects on renal function, such as reduced serum creatinine and blood urea nitrogen levels compared with the LPS group (P<0.05). PDS decreased the production and release of tumor necrosis factor (TNF)-α and interleukin (IL)-6 by inhibiting the NF-κB signaling pathway, down-regulating inducible nitric oxide synthase protein expression levels and inhibiting oxidative stress. In most anti-AKI mechanisms, PDS and dexamethasone were similar, but PDS are better at inhibition of TNF production, promote SOD activity and inhibition of IKB phosphorylation. In addition, nuclear glucocorticoid receptor expression was markedly enhanced in PDS and Dexamethasone treatment groups. Further research is required to determine whether PDS can combine with the glucocorticoid receptor to enter the nucleus. Conclusion This study demonstrated that PDS and dexamethasone have similar reverse amelioration for renal functions, and have potential application prospects in the treatment of sepsis-induced AKI.

Differential protein expression in heart in UT-B null mice with cardiac conduction defects.

Cardiac conduction defects were found in transgenic mice deficient in urea transporter UT‐B. To investigate the molecular mechanisms of the conduction defects caused by UT‐B deletion, we studied the protein expression profiles of heart tissue (comprising most conduction system) in wild‐type versus UT‐B null mice at different ages. By two‐dimensional electrophoresis‐based comparative analysis, we found that more than dozen proteins were modulated (>two‐fold) in the myocardium of UT‐B null mice. Out of these modulated proteins, troponin T (TNNT2) presented significant changes in UT‐B null mice at early stage prior to the development of P‐R interval elongation, while the change of atrial natriuretic peptide (ANP) occurred only at late stage in UT‐B null mice that had the AV block. These data indicate that UT‐B deletion caused the dynamic expression regulation of TNNT2 and ANP, and these proteins may provide new clues to investigate the molecular events involved in cardiac conduction.

Plasmid-Based Stat3 siRNA Delivered by Functional Graphene Oxide Suppresses Mouse Malignant Melanoma Cell Growth.

RNA interference (RNAi) has been used for cancer gene therapy in recent years. However, the application of RNAi is hindered in the absence of safe and efficient gene delivery. In this article, a novel vehicle of graphene oxide functionalized with polyethylenimine and polyethylene glycol (GO-PEI-PEG) was successfully synthetized and then used to deliver plasmid-based Stat3 siRNA. The carrier can readily bind plasmid with high transfection efficiency. Moreover, molecular biology studies reveal that Stat3-related gene and protein expressions were significantly inhibited, suggesting that the formation of GO-PEI-PEG complexes could be utilized as a promising gene delivery in cancer therapy.

UT-B-deficient mice develop renal dysfunction and structural damage

BackgroundUrea transporter UT-B is the major urea transporter in erythrocytes and the descending vasa recta in the kidney. In this study, we investigated the effects of long-term UT-B deficiency on functional and structural defect in the kidney of 16-and 52-week-old UT-B-null mice.MethodsUT-B-knockout mice were generated by targeted gene disruption and lacked UT-B protein expression in all organs. The urinary concentrating ability of mice was studied in terms of daily urine output, urine osmolality, and urine and plasma chemistries. Changes in renal morphology were evaluated by hematoxylin and eosin staining.ResultsThe UT-B-null mice showed defective urine concentrating ability. The daily urine output in UT-B-null mice (2.5 ± 0.1 ml) was 60% higher and urine osmolality (985 ± 151 mosm) was significantly lower than that in wild-type mice (1463 ± 227 mosm). The 52-week-old UT-B-null mice exhibited polyuria after water deprivation, although urine osmolality was increased. At 52 weeks of age, over 31% of UT-B-null mice exhibited renal medullary atrophy because of severe polyuria and hydronephrosis.ConclusionsLong-term UT-B deficiency causes severe renal dysfunction and structural damage. These results demonstrate the important role of UT-B in countercurrent exchange and urine concentration.

Extrarenal Phenotypes of the UT-B Knockout Mouse

The urea transporter UT-B is expressed in multiple tissues including erythrocytes, kidney, brain, heart, liver, colon, bone marrow, spleen, lung, skeletal muscle, bladder, prostate, and testis in mammals. Phenotype analysis of UT-B-null mice has confirmed that UT-B deletion results in a urea-selective urine-concentrating defect (see Chap. 9 ). The functional significance of UT-B in extrarenal tissues studied in the UT-B-null mouse is discussed in this chapter. UT-B-null mice present depression-like behavior with urea accumulation and nitric oxide reduction in the hippocampus. UT-B deletion causes a cardiac conduction defect, and TNNT2 and ANP expression changes in the aged UT-B-null heart. UT-B also plays a very important role in protecting bladder urothelium from DNA damage and apoptosis by regulating the urea concentration in urothelial cells. UT-B functional deficiency results in urea accumulation in the testis and early maturation of the male reproductive system. These results show that UT-B is an indispensable transporter involved in maintaining physiological functions in different tissues.

Quantitative proteomic study of myocardial mitochondria in urea transporter B knockout mice

In previous research, we showed that 16‐week‐old urea transporter B (UT‐B) null mice have an atrial–ventricular conduction block, and hypothesized myocardial mitochondrial dysfunction. To investigate the mechanism of this block, we examined the proteomic differences in the myocardial mitochondria of UT‐B null and wild‐type mice with nanoscale LC–MS/MS. Of 26 proteins clearly downregulated in the UT‐B null mice, 15 are involved in complexes I, III, IV, and V of the respiratory chain, which would strongly reduce the activity of the electron transport chain. Excess electrons from complexes I and III pass directly to O2 to generate ROS and deplete ROS‐scavenging enzymes. Myocardial intracellular ROS were significantly higher in UT‐B null mice than in wild‐type mice (p < 0.01), constituting an important cause of oxidative stress injury in the myocardia of UT‐B null mice. The mitochondrial membrane potential (ΔΨm) was also lower in UT‐B null mice than in wild‐type mice (p < 0.05), causing oxidative phosphorylation dysfunction of complex V and insufficient ATP in the myocardial cells of UT‐B null mice. HADHA (a trifunctional protein) and HSP60 were also downregulated in the UT‐B null myocardial mitochondria. These results confirm that mitochondrial dysfunction underlies the pathogenesis of the atrial–ventricular conduction block in UT‐B null mice.

Urea transport B gene induces melanoma B16 cell death via activation of p53 and mitochondrial apoptosis

Urea Transporter B (UT‐B) is a membrane channel protein that mediates the rapid transmembrane transport of urea and participates in urine concentration. Urea Transporter B is expressed in skin, but we found that there is little expression in human melanoma tissue. In this study, we examined the effects of UT‐B overexpression in melanoma. The results indicated that there is no UT‐B mRNA expression in B16 cells, and UT‐B overexpression repressed B16 cell proliferation and induced apoptosis in vitro. We show that UT‐B overexpression causes increased reactive oxygen species production, which may be caused by mitochondria dysfunction. The mitochondrial membrane potential (ΨΔm) was lower in UT‐B‐overexpressing B16 cells. The proteins involved in complexes I, III, IV and V of the respiratory chain were clearly downregulated in UT‐B‐overexpressing B16 cells, which would strongly reduce the activity of the electron transport chain. We found that mitochondrial release of cytochrome C into the cytoplasm also increased, indicating that apoptosis had been activated. In addition, UT‐B overexpression reduced AKT phosphorylation and MDM2 expression and increased p53 expression; p53 activation may be involved in the anticancer effects of UT‐B overexpression. Urea Transporter B overexpression also inhibited tumor growth in vivo. In conclusion, we demonstrated that UT‐B may be related to the occurrence of melanoma and play a role in tumor development.