Targeted suppression of miRNA-21 inhibit K562 cells growth through PTEN-PI3K/AKT signaling pathway

Objective To investigate the K562 cells biological function and related molecular changes in PTEN-PI3K/AKT signaling pathway of leukemia K562 cells by inhibiting the miRNA-21 expression to explore its pathogenesis of leukemia.Methods The chemical synthetic miRNA-

Effects of bone marrow-derived mesenchymal stemcells engraftment on vascular endothelial cell growthfactor in lung tissue and plasma at early stage of smoke inhalation injury

BACKGROUND： This study was undertaken to determine the effect of mesenchymal stem cells （MSCs） engraftment on vascular endothelial cell growth factor （VEGF） in lung tissue, plasma and extravascular lung water at early stage of smoke inhalation injury.METHODS： A rabbit smoke inhalation injury model was established using a home-made smoke inhalation injury generator, and rabbits were divided into two groups randomly： a control group （S group, n=32） and a MSCs treatment group （M group, n=32）. 10 ml PBS was injected via the ear marginal vein immediately at injury into the S group. Third generation MSCs with a concentration of 1×107/10 ml PBS were injected via the ear marginal vein immediately at injury into the M group. VEGF in peripheral blood and lung tissue were measured at 0 （baseline）, 2, 4 and 6 hours after injection respectively and analyzed. The right lungs of rabbits were taken to measure lung water mass fraction.RESULTS： In the lung tissue, VEGF decreased gradually in the S group （P〈0.05） and signi？ cantly decreased in the M group （P〈0.05）, but it increased more signi？ cantly than the values at the corresponding time points （P〈0.05）. In peripheral blood, VEGF increased gradually in the S group （P〈0.05） and markedly increased in the M group （P〈0.05）, but it decreased more signi？ cantly than the values at corresponding time points （P〈0.05）.CONCLUSION： MSCs engraftment to smoke inhalation injury could increase VEGF in lung tissue, decrease VEGF in plasma and reduce extravascular lung water, indicating its protective effect on smoke inhalation injury.

Effects of glucocorticoids on the growth and chemosensitivity of carcinoma cells are heterogeneous and require high concentration of functional glucocorticoid receptors

AIM： To determine how glucocorticoids （GCs） may affect the growth and chemosensitivity of common carcinoma cells. METHODS： The effect of dexamethasone （DEX） on growth and chemosensitivity was assessed in 14 carcinoma cell lines. The function of GC receptors （GR） was assessed by MMTV reporter assay. Overexpression of GR was done by in vitro transfection and expression of a GR-expressing vector. Immunohistochemical stain of tissues and ceils were done by PA1-511A, an anti-GR monodonal antibody. RESULTS： DEX inhibited cell growth of four （MCF-7, MCF- 7/MXR1, MCF-7/TPT300, and HeLa）, increased cisplatin cytoxicity of one （SiHa）, and decreased dsplatin cytotoxicity of two （H460 and Hep3B） cell lines. The GR content of the seven cell lines affected by DEX was significantly higher than those of the seven cell lines unaffected by DEX （5.2±2.5×10^4 sites/cell vs1.3±1.4×10^4 sites/cell, P= 0.005）. Only two DEX-unresponsive cell lines {NPC-TW01 and NPC- TW04） oontained high GR amounts in the range （1.9-8.1×10^4 sites/cell） of the seven DEX-responsive cell lines. The GR function of NPC-TW01 and NPC-TW04, however, was foundto be impaired. The importance of high cellular amount of GR in mediating DEX susceptibility of the cells was further exemplified by GR dose-dependent drug resistance to cisplatin of AGS, a cell line with low GR content and was unaffected by DEX before transfection of GR-expressing vector. Immunohistochemical studies of human cancer tissues showed that 5 of the 45 （11.1%） breast cancer and 43 of the 85 （50.6%） non-small cell lung cancer had high GR contents at the ranges of the GC-responsive carcinoma cell lines. CONCLUSION： The growth and chemosensitivity of human carcinomas with high GR contents may be affected by GC. However, in light of the heterogeneous and even contradictive effects of GC on these cells, routine examination of GR contents of human carcinoma tissues may not be clinically useful until other markers that help predict the ultimate effect of GC on individual patients are identified.

mad-overexpression down regulates the malignant growth and p53 mediated apoptosis in human hepatocellular carcinoma BEL-7404 cells

Mad protein has been shown as an antagonist of cMyc protein in some cell lines. The effect of Mad protein to the malignant phenotype of human hepatoma BEL7404 cell line was investigated experimentally. An eukarryotic vector pCDNA Ⅲ containing full ORF fragmentof mad cDNA was transfected into targeted cells. Under G418 selection, stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells. DNA synthesis, cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cel1s were partially inhibited in comparison to control cells.Flow Cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase, resulting in the retardation of cell proliferation. RT-PCR detected a marked inhibition of the expression of cdc25A, an important regulator gene of G0/G1to S phase in cell cycle. It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-7404-M1 cells in the absence of serume.Thus, Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL7404 cells.madoverexpression and regulation of cell growth and apoptosis

Different distributions of interstitial cells of Cajal and platelet-derived growth factor receptor-α positive cells in colonic smooth muscle cell/interstitial cell of Cajal/plateletderived growth factor receptor-α positive cell syncytium in mice

AIM To investigate the distribution and function of interstitialcells of Cajal(ICCs) and platelet-derived growth factor receptor-α positive(PDGFRα+) cells in the proximal and distal colon.METHODS The comparison of colonic transit in the proximal and distal ends was performed by colonic migrating motor complexes(CMMCs). The tension of the colonic smooth muscle was examined by smooth muscle spontaneous contractile experiments with both ends of the smooth muscle strip tied with a silk thread. Intracellular recordings were used to assess electrical field stimulation(EFS)-induced inhibitory junction potentials(IJP) on the colonic smooth muscle. Western blot analysis was used to examine the expression levels of ICCs and PDGFRα in the colonic smooth muscle.RESULTS Treatment with NG-nitro-L-arginine methyl ester hydrochloride(L-NAME) significantly increased the CMMC frequency and spontaneous contractions, especially in the proximal colon, while treatment with MRS2500 increased only distal CMMC activity and smooth muscle contractions. Both CMMCs and spontaneous contractions were markedly inhibited by NPPB, especially in the proximal colon. Accordingly, CyPPA sharply inhibited the distal contraction of both CMMCs and spontaneous contractions. Additionally, the amplitude of stimulationinduced nitric oxide(NO)/ICC-dependent slow IJPs(sIJPs) by intracellular recordings from the smooth muscles in the proximal colon was larger than that in the distal colon, while the amplitude of electric field stimulationinduced purinergic/PDGFRα-dependent fast IJPs(fIJPs) in the distal colon was larger than that in the proximal colon. Consistently, protein expression levels of c-Kit and anoctamin-1(ANO1) in the proximal colon were much higher, while protein expression levels of PDGFRα and small conductance calcium-activated potassium channel 3(SK3) in the distal colon were much higher.CONCLUSION The ICCs are mainly distributed in the proximal colon and there are more PDGFRα+ cells are in the distal colon, which generates a pressure gradient between the two ends of the colon to propel the feces to the anus.

Retinal ganglion cell neuroprotection by growth factors and exosomes:lessons from mesenchymal stem cells

Retinal ganglions cells （RGCs） are responsible for propagating electrochemical information from the eye to the brain along their axons which make up the optic nerve. The loss of RGCs is characteristic in several conditions such as glaucoma and trau- matic optic neuropathy and leads to visual loss and blindness. While no therapy exists to directly treat RGCs, the use of bone marrow-derived mesenchymal stem cells （BMSCs） has shown promise in eliciting significant RGC neuroprotection.

Nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews

Neural stem cells promote neuronal regeneration and repair of brain tissue after injury,but have limited resources and proliferative ability in vivo.We hypothesized that nerve growth factor would promote in vitro proliferation of neural stem cells derived from the tree shrews,a primate-like mammal that has been proposed as an alternative to primates in biomedical translational research.We cultured neural stem cells from the hippocampus of tree shrews at embryonic day 38,and added nerve growth factor（100 μg/L） to the culture medium.Neural stem cells from the hippocampus of tree shrews cultured without nerve growth factor were used as controls.After 3 days,fluorescence microscopy after DAPI and nestin staining revealed that the number of neurospheres and DAPI/nestin-positive cells was markedly greater in the nerve growth factor-treated cells than in control cells.These findings demonstrate that nerve growth factor promotes the proliferation of neural stem cells derived from tree shrews.

LRP6 in mesenchymal stem cells is required for bone formation during bone growth and bone remodeling

Lipoprotein receptor-related protein 6 （LRP6） plays a critical role in skeletal development and homeostasis in adults. However, the role of LRP6 in mesenchymal stem cells （MSCs）, skeletal stem cells that give rise to osteoblastic lineage, is unknown. In this study, we generated mice lacking LRP6 expression specifically in nestin＋ MSCs by crossing nestin-Cre mice with LRP6 flox mice and investigated the functional changes of bone marrow MSCs and skeletal alterations. Mice with LRP6 deletion in nestin＋ cells demonstrated reductions in body weight and body length at I and 3 months of age. Bone architecture measured by microCT （uCT） showed a significant reduction in bone mass in both trabecular and cortical bone of homozygous and heterozygous LRP6 mutant mice. A dramatic reduction in the numbers of osteoblasts but much less significant reduction in the numbers of osteoclasts was observed in the mutant mice. Osterix＋ osteoprogenitors and osteocalcin＋ osteoblasts significantly reduced at the secondary spongiosa area, but only moderately decreased at the primary spongiosa area in mutant mice. Bone marrow MSCs from the mutant mice showed decreased colony forming, cell viability and cell proliferation. Thus, LRP6 in bone marrow MSCs is essential for their survival and proliferation, and therefore, is a key positive regulator for bone formation during skeletal growth and remodeling.

Vascular endothelial growth factor 165b expression in stromal cells and colorectal cancer

AIM:To characterize the implications of vascular endothelial growth factor(VEGF)-A in stromal cells and colorectal cancer and the expression of VEGF-A splice variants.METHODS:VEGF-A expression in tumor and stromal cells from 165 consecutive patients with colorectal cancer was examined by immunohistochemistry.The association between VEGF-A expression status and clinicopathological factors was investigated.Twenty freshfrozen samples were obtained for laser capture microdissection to analyze the splice variants of VEGF-A.RESULTS:VEGF-A was expressed in 53.9% and 42.4% of tumor and stromal cells,respectively.VEGF-A expression in tumor cells(t-VEGF-A) was associated with advanced clinical stage(stage 0,1/9;stage 1,2/16;stage 2,32/55;stage 3,38/66;stage 4,16/19,P < 0.0001).VEGF-A expression in stromal cells(s-VEGF-A) increased in the earlier clinical stage(stage 0,7/9;stage 1,6/16;stage 2,33/55;stage 3,22/66;stage 4,5/19;P = 0.004).Multivariate analyses for risk factors of recurrence showed that only s-VEGF-A expression was an independent risk factor for recurrence(relative risk 0.309,95% confidence interval 0.141-0.676,P = 0.0033).The five-year disease-free survival(DFS) rates of t-VEGF-A-positive and-negative cases were 51.4% and 62.9%,respectively.There was no significant difference in t-VEGF-A expression status.The five-year DFS rates of s-VEGF-A-positive and-negative cases were 73.8% and 39.9%,respectively.s-VEGFA-positive cases had significantly better survival than s-VEGF-A-negative cases(P = 0.0005).Splice variant analysis revealed that t-VEGF-A was mainly composed of VEGF165 and that s-VEGF-A included both VEGF165 and VEGF165b.In cases with no venous invasion(v0),the level of VEGF165b mRNA was significantly higher(v0 204.5 ± 122.7,v1 32.5 ± 36.7,v2 2.1 ± 1.7,P = 0.03).The microvessel density tended to be lower in cases with higher VEGF165b mRNA levels.CONCLUSION:s-VEGF-A appears be a good prognostic factor for colorectal cancer and includes VEGF165 and VEGF165b.

A partition-type tubular scaffold loaded with PDGF-releasing microspheres for spinal cord repair facilitates the directional migration and growth of cells

The best tissue-engineered spinal cord grafts not only match the structural characteristics of the spinal cord but also allow the seed cells to grow and function in situ.Platelet-derived growth factor（PDGF） has been shown to promote the migration of bone marrow stromal cells;however,cytokines need to be released at a steady rate to maintain a stable concentration in vivo.Therefore,new methods are needed to maintain an optimal concentration of cytokines over an extended period of time to effectively promote seed cell localization,proliferation and differentiation.In the present study,a partition-type tubular scaffold matching the anatomical features of the thoracic 8–10 spinal cord of the rat was fabricated using chitosan and then subsequently loaded with chitosan-encapsulated PDGF-BB microspheres（PDGF-MSs）.The PDGF-MS-containing scaffold was then examined in vitro for sustained-release capacity,biocompatibility,and its effect on neural progenitor cells differentiated in vitro from multilineage-differentiating stress-enduring cells（MUSE-NPCs）.We found that pre-freezing for 2 hours at-20°C significantly increased the yield of partition-type tubular scaffolds,and 30 μL of 25% glutaraldehyde ensured optimal crosslinking of PDGF-MSs.The resulting PDGF-MSs cumulatively released 52% of the PDGF-BB at 4 weeks in vitro without burst release.The PDGF-MS-containing tubular scaffold showed suitable biocompatibility towards MUSE-NPCs and could promote the directional migration and growth of these cells.These findings indicate that the combination of a partition-type tubular scaffold,PDGF-MSs and MUSENPCs may be a promising model for the fabrication of tissue-engineered spinal cord grafts.

Three Dimensional Simulation Method in Early Process of Division and Growth for Tumour Cells

The process of division, growth and death for tumour cell mass in the early is simulated. An integrated GUI is provided for users to set the value of each parameters, which are cell growth rates, cell mass division rates, cell mass death rates, simulate type, maximum running time, polarity and cell colour. It can display the growth process of each cell on result GUI. Also, it can display the values of each parameters for observing and analysing in current life cycle on result GUI, which are cell mass division times, cell mass death rate, cell mass division rate and cell mass growth rate. In the process of simulation, The cell growth rate is described by the approach to combine the exponential model with the linear model. In addition, a linked list data structure to store the tumour cells is used by the cellular automata for a reference to determine the position of each cell. It sets up two linked list to store the cells, one of them save the new small division cells and the other one save the big cell. That can make the painting process of cells on result GUI clearer and more organized. At last, the polarity oftumour growth is described for determining the growth direction of cells.

EDIL3 depletion suppress epithelial-mesenchymal transition of lens epithelial cells via transforming growth factor β pathway

AIM： To study the effect of discoidin I-like domaincontaining protein 3（EDIL3） depletion on the proliferation and epithelial-mesenchymal transition（EMT） in human lens epithelial cells（LECs）. METHODS： RNA interference was used to inhibit the expression of EDIL3 in human LECs in vitro. The morphology of cells was observed using an inverted microscope. Cell proliferation was assessed using Ed U kit. Cell migration was investigated using Transwell chamber and EMT of LECs was assessed using confocal microscope and Western blotting. The transforming growth factor β（TGFβ） pathway was investigated using Western blotting. RESULTS： The data showed that silencing EDIL3 expression changed LECs morphology and suppressed LECs proliferation（P〈0.05） and migration（P〈0.01）. Furthermore, the result of Western blotting showed that EDIL3 depletion reduced the expression of α-smooth muscle actin（α-SMA）（P〈0.001） and vimentin（P〈0.01）, while increased the expression of E-cadherin（P〈0.001）. EDIL3 depletion could suppress the phosphorylation of Smad2（P〈0.01） and Smad3（P〈0.01） and the activation of exracellular signal regulated kinase（ERK）（P〈0.05）. CONCLUSION： The findings indicate that EDIL3 might participate in the proliferation and EMT in LECs via TGFβ pathway and may be a potential therapeutic target for the treatment of posterior capsule opacification.

Inhibition of epidermal growth factor receptor signaling protects human malignant glioma cells from hypoxia - induced cell death

Epidermal growth factor receptor(EGFR)signaling has become an importanttarget for drug development becauseEGFR signaling enhances tumor cell proliferation,migration,and invasion and inhibits apoptosis.However,theresults of clinical trials using EGFR inhibitors in patients with solid tumors have been disappointing.Here,wereport a protective effect of the EGFR inhibitors AG1478 and PD153035 against cell death induced by acute hy-poxia,which contrasts with their proapoptotic effects under normoxia.Under hypoxic conditions,both agents re-

Vaccinia-related kinase 2 variants differentially affect breast cancer growth by regulating kinase activity

Genetic information is transcribed from genomic DNA to mRNA,which is then translated into threedimensional proteins.mRNAs can undergo various post-transcriptional modifications,including RNA editing that alters mRNA sequences,ultimately affecting protein function.In this study,RNA editing was identified at the 499th base(c.499)of human vaccinia-related kinase 2(VRK2).This RNA editing changes the amino acid in the catalytic domain of VRK2 from isoleucine(with adenine base)to valine(with guanine base).Isoleucine-containing VRK2 has higher kinase activity than the valine-containing VRK2,which leads to an increase in tumor cell proliferation.Earlier we reported that VRK2 directly interacts with dystrobrevin-binding protein(dysbindin)and results in reducing its stability.Herein,we demonstrate that isoleucine-containing VRK2 decreases the level of dysbindin than valinecontaining VRK2.Dysbindin interacts with cyclin D and thereby regulates its expression and function.The reduction in the level of dysbindin by isoleucine-containing VRK2 further enhances the cyclin D expression,resulting in increased tumor growth and reduction in survival rates.It has also been observed that in patient samples,VRK2 level was elevated in breast cancer tissue compared to normal breast tissue.Additionally,the isoleucine form of VRK2 exhibited a greater increase in breast cancer tissue.Therefore,it is concluded that VRK2,especially dependent on the 167th variant amino acid,can be one of the indexes of tumor progression and proliferation.

Chondrogenic differentiation of human bone mesenchymal stem cells treated with growth differentiation factor 5 under hypoxia

Objective To explore the feasibility and effectiveness of the self-assembly cartilage tissue engineered with chondrogenically differentiated human bone mesenchymal stem cells (hBMCs) induced by growth differentiation factor-5 (GDF-5)

The effect of endogenous transforming growth factor β_1 on the growth of bladder cancer cells in vitro

Objective To investigate the influence of endogenous transforming growth factor β1(TGFβ1) on the cell cycle regulation and proliferation of bladder cancer. Methods A constructed replication defective retroviral vector pRevTβ-AS, which carried antisense RNA of TGFβ1.was transfected to a bladder cancer cell line EJ. The proliferation and clone-formation of transferred cells were observed in vitro,and the alteration of cell cycle was also detected by flow cytometric analysis. Results TGFβ1 antisense RNA was transferred into EJ cell and expressed efficiently. After the inhibition of target gene expression in EJ cells, the reduced growth and clone-formation rates were demonstrated, and the proliferative indexes were decreased by 12 % . The ratios of GO and G1 stage cells to June 2003 Vol12 No2 the antisense RNA-transfected EJ cells were increased, simultaneously,the ratio of S stage cells to the antisense RNA-transfected EJ cells ratios were decreased, compared with the control group. Conclusion The

Effect of the implant composite of poly lactide-co-glycolide and bone mesenchymal stem cells modified by basic fibroblast growth factor on injured spinal cord in rats

Objective To investigate the effect of the implant composite of poly lactide-co-glycolide(PLGA)and bone mesenchymal stem cells (BMSCs) modified by basic fibroblast growth factor (bFGF) on injured spinal cord in rats.Methods Two hundred and

The effect of interleukin 6 on the growth of LNCaP and PC-3 prostatic carcinoma cells

Objective To investigate the effect of IL-6 on prostatic carcinoma cell lines, and differential effects on androgen-dependent and androgen-independent prostatic carcinoma cells. Methods The IL-6 producing capacities of LNCaP and PC-3 cells were determined, and effects of exogenous IL-6 and anti-IL - 6 antibodies on LNCaP and PC - 3 cells were examined. Results LNCaP produced a very small amount of IL-6, but PC-3 produced more, the concentraion of IL-6 being 190 pg/48 h per ml(1 × 106). The exogenous IL-6 inhibited LNCaP growth significantly,but had no obvious effect on PC -3 cells. Anti-IL-6 antibodies lowered PC-3 cells growth rate but had neutral effect on LNCaP. Conclusion PC-3 cells produces IL-6 massively in autocrine manner. IL-6 could be antagonized by anti-IL-6 antibodies,resulting in slowing PC-3 cells growth, and LNCaP cells growth could be inhibited by exogenous IL-6.7 refs,2 tabs.

Suppression of cell growth and invasion by miR-205 in breast cancer

MicroRNAs （miRNAs） are endogenous, small, non-coding RNAs, which are capable of silencing gene expression at the post-transcriptional level. In this study, we report that miR-205 is significantly underexpressed in breast tumor compared to the matched normal breast tissue. Similarly, breast cancer cell lines, including MCF-7 and MDA-MB- 231, express a lower level miR-205 than the non-malignant MCF-10A cells. Of interest, ectopic expression of miR-205 significantly inhibits cell proliferation and anchorage independent growth, as well as cell invasion. Furthermore, miR- 205 was shown to suppress lung metastasis in an animal model. Finally, western blot combined with the luciferase reporter assays demonstrate that ErbB3 and vascular endothelial growth factor A （VEGF-A） are direct targets for miR-205, and this miR-205-mediated suppression is likely through the direct interaction with the putative miR-205 binding site in the 3＇-untranslated region （3＇-UTR） of ErbB3 and VEGF-A. Together, these results suggest that miR- 205 is a tumor suppressor in breast cancer.