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AIM: To study the protective effect of fenofibrate on diabetic retinal neurodegeneration and observe its effect on miR-26a-5p and its target gene PTEN in the retinal of diabetic mice.METHODS: Diabetic mice models were established and they were gavaged by fenofibrate. H& E staining and transmission electron microscopy were used to observe the impairments of retinal neurons. Real-time PCR was used to examine the expression of miR-26a-5p, and Western blotting was employed to measure the expression of phosphatase and tensin homologue(PTEN)in the retina of diabetic mice. The expression level of nuclear factor-κB(NF-κB), interleukin-1β(IL-1β)and the morphology of neural tissues were observed.RESULTS: When compared with the diabetic mice, fenofibrate significantly attenuated the damage to retinal ganglion cells and the atrophy of retinal nerve fiber layer. While the level of miR-26a-5p was increased and the levels of PTEN and inflammatory mediators were significantly decreased in the retina of fenofibrate treated diabetic mice, with significant statistical significance(P<0.05).CONCLUSIONS: Fenofibrate protects against diabetic retinal neurodegeneration by upregulating miR-26a-5p and inhibiting PTEN, attenuating the inflammatory response and alleviating retinal cell injury.
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Background Exposure to arsenic can damage trophoblast cells and thus induce abortion, but the mechanism is not known. Objective To investigate the role of miR-145 and PTEN/AKT/mTOR pathway in arsenic-induced abortion and trophoblast cell damage in rats. Methods In the animal experiment, twenty SD pregnant rats were randomly divided into a normal control group (saline gavage) and an arsenic-induced abortion group (10.65 mg·kg−1 sodium arsenite solution was administered by gavage, and the gavage volume was 10 mL·kg−1), with 10 rats in each group. After the miscarriage occurred in the arsenic-induced abortion group (5-6 d after exposure), placental tissues were collected from the two groups. The mRNA expression levels of microRNA-145 (miR-145), phosphatase and tensin homologue (PTEN), kinase B (AKT), mammalian target of rapamycin (mTOR) were detected by real-time quantitative PCR (RT-PCR), and the protein expression levels of PTEN, AKT, mTOR, p-AKT, and p-mTOR were detected by Western blotting. For the in vitro study with immortalized human trophoblast cell line (HTR-8/SVneo cells), a control group, an arsenic exposure group, an miR-145 overexpression group, and an arsenic exposure+miR-145 overexpression group were prepared and cultured for 72 h with 37 °C and 5% CO2, at cell density of 5×105 cells per well, and the arsenic exposure concentration was 20 μmol·L−1. The MTT method was applied to detect cell viability, crystal violet staining to detect the number of monoclonal formation, flow cytometry to detect the level of apoptosis, Image J Angiogenesis Analyzer 1.8.0 plug-in to evaluate total blood vessel length and total blood vessel number; the detection indexes and methods of genes and proteins were the same as "animal experiment". Results (1) In the animal experiment, compared with the normal control group, the expression level of miR-145 mRNA in the placenta tissues of the arsenic-induced abortion group was increased (P<0.05), and the expression levels of PTEN, AKT, mTOR mRNA and proteins, and p-AKT and p-mTOR proteins were decreased (P<0.05). (2) For the in vitro study, compared with the control group, the cell viability rate, number of monoclonal formation, total vessel length, and total vessel number were decreased, and the apoptosis rate was increased in the arsenic exposure group, the miR-145 overexpression group, and the arsenic exposure+miR-145 overexpression group (P<0.05). Compared with the arsenic exposure group and the miR-145 overexpression group, the cell viability rate, number of monoclonal formation, total vessel length, and vessel number were decreased, and the apoptosis rate was increased in the arsenic exposure+miR-145 overexpression group (P<0.05). Compared with the control group, the levels of miR-145 mRNA in the arsenic exposure group, the miR-145 overexpression group, and the arsenic exposure+miR-145 overexpression group increased (P<0.05), the expression levels of PTEN, AKT, mTOR mRNA and protein and the expression levels of p-AKT and p-mTOR protein were decreased (P<0.05); compared with the arsenic exposure group and the miR-145 overexpression group, the level of miR-145 mRNA in the arsenic exposure+miR-145 overexpression group was increased (P<0.05), and the levels of PTEN, AKT, mTOR mRNA and protein as well as p-AKT and p-mTOR protein were decreased (P<0.05). Conclusion miR-145 might be related to abortion due to arsenic exposure. miR-145 could inhibit the proliferation and angiogenesis of trophoblast HTR-8/SVNEO cells, and promotes their apoptosis; the mechanism may be related to the inhibition of PTEN/AKT/mTOR pathway.
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Background: Gastric cancer is a common gastrointestinal malignant tumor. MiR-21 can regulate the expression of phosphatase and tensin homologue (PTEN) protein and induce apoptosis of various tumor cells. Aims: To explore effect of miR-21 on proliferation, apoptosis and invasion of gastric cancer cells by targeting PTEN. Methods: SGC-7901 cells in logarithmic growth phase were divided into miR-21 mimic group, miR-21 inhibitor group, negative control group and blank control group. RT-PCR and Western blotting were used to detect mRNA and protein expressions of miR-21 and PTEN, respectively. The luciferase reporter gene assay was used to validate the targeted regulatory relationship between miR-21 and PTEN. Gastric cancer SGC-7901 cells were divided into blank control group, NC group, miR-21 group, PTEN group and miR-21+PTEN group. The proliferation activity was determined by CCK-8 assay, apoptosis was detected by TUNEL assay, and invasion and migration ability of cells was detected by Transwell and scratch test, respectively. The expressions of Ki-67, PCNA, ratio of cleaved caspase-3, Bcl-2, Bax, PTEN, p-PI3K/PI3K and p-Akt/Akt proteins were detected by Western blotting. A tumorigenesis model of nude mice was established to detect the effect of overexpression of miR-21 on the volume and mass of transplanted tumor. Results: MiR-21 could negatively regulate expression of PTEN. Compared with NC group, cell proliferation, invasion and migration rates were significantly increased in miR-21 group (P<0.05), apoptosis rate was significantly decreased (P<0.05), expressions of Ki-67, PCNA, ratio of Bcl-2/Bax, p-PI3K/PI3K and p-Akt/Akt proteins were significantly increased (P<0.05), expressions of PTEN and cleaved caspase-3 was significantly decreased (P<0.05). Cell proliferation, invasion and migration rates were significantly decreased in PTEN group (P<0.05), apoptosis rate was significantly increased (P<0.05), expressions of Ki-67, PCNA, ratio of Bcl-2/Bax, p-PI3K/PI3K and p-Akt/Akt proteins were significantly decreased (P<0.05), expressions of PTEN and cleaved caspase-3 were significantly increased (P<0.05). Compared with PTEN group, cell proliferation, invasion and migration rates were significantly increased in miR-21+PTEN group (P<0.05), apoptosis rate was significantly decreased (P<0.05), expressions of Ki-67, PCNA, ratio of Bcl-2/Bax, p-PI3K/PI3K and p-Akt/Akt proteins were significantly increased (P<0.05), and expressions of PTEN and cleaved caspase-3 were significantly decreased (P<0.05). Compared with the control group, overexpression of miR-21 significantly increased the volume and mass of transplanted tumor in nude mice (P<0.05). Conclusions: MiR-21 can negatively regulate PTEN expression, activate PI3K/Akt signaling pathway, promote proliferation, invasion and migration of gastric cancer cells, and inhibit cell apoptosis.
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Objective: To investigate the effect of herba artemisiae eapillaris extracts (HACE) on the expression of phosphatase and tensin homologue deleted chromatosome 10 (PTEN) in the kidney tissue of the diabetic rats, and to clarify the pharmacological mechanism of protective effect of HACE on the kidney of the diabetic rats. Methods: Thirty Wistar rats were randomly divided into control group (/i=6). model group ( n= 12) and HACK group (n=12). The rats in HACK group were intragastrically administrated with HACK (5 g • kg !) one time every day. and the rats in control group and model group were intragastrically administrated with the same volume of normal saline. The diabetic rat models were duplicated by streptozotocin (STZ). After 4 months of administration, the urinary albumin excretion rates and urinary total protein excretion rates of the rats in various groups were detected. The pathomorphology of the kindey tissue of the rats in various groups were observed by HE staining and the distribution of renal extracellular matrix (ECM) was detected by PAS and Mas son staining. The expressions of PTEN protein in kidney tissue of the rats in various groups were detected by immunohistostaining and the expression levels of PTEN protein in kidney tissue of the rats in various groups were detected by Western blotting method. Results: Compared with model group, the glomerular mesangial aggregation, positive staining of PAS as well as Masson aggregation and basement membrane thickening of the rats in HACE group were significantly reduced, and the 24 h urinary albumin excretion rate was significantly decreased ( P0. 05). The immunohistochemistry staining results showed that the expression level of PTEN protein in kidney tissue of the rats in control group was higher, the expression level of PTEN protein in the kidney tissue of the rats in model group was decreased, and the expression level of PTEN protein in kidney tissue of the rats in HACE group was increased to normal. The Western blotting results showed that the expression level of PTEN protein in the renal cortex tissue tissue of the rats in model group was significantly decreased compared with control group C P<0. 05) ; compared with model group, the expression level of PTEN protein in renal cortex tissue of the rats in HACE group was significantly increased C P<-0. 05). Conclusion: HACE can increase the expression of PTEN protein in kidney tissue of the rats, which may be one of the mechanisms of its protective effect on the kidney in the diabetic rats.
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Objective To study the effect of simvastatin on the expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and β-catenin in myocardial cells of rabbits with chronic heart failure (CHF).Methods Twenty-four male New Zealand rabbits were randomly divided into control group,CHF model group and simvastatin treatment group,with 8 rabbits in each group.The rabbits in CHF model group and simvastatin treatment group were injected with adriamycin (2.0 mg · kg-1) ria ear rein once a week for six weeks,and from the seventh week were injected with adriamycin (1.5 mg · kg-1)once a week for another six weeks to establish the CHF model;the rabbits in control group were injected with the same volume saline.The rabbits in simvastatin treatment group were given simvastatin (1.5 mg · kg-1 · d-1) by intragastric administration at the time point of first injection of adriamycin for 12 weeks;the rabbits in CHF model group and control group were given the same volume saline for 12 weeks.The left ventricular structure and function were determined by color doppler uhrasonography after the modeling.Then the rabbits were sacrificed and the left ventricular walls were taken to observe the changes of myocardial cell structures by hematoxylin-eosin staining.The positive expression rate of PTEN and β-catenin protein was calculated by immunohistochemistry staining.The expression of PTEN and β-catenin mRNA was detected real-time quantitative polymerase chain reaction.Results Compared with the control group,the left ventricular end-systolic dimension (LVESD),left ventricular end-diastolic dimension(LVEDD) were increased and the left ventricular ejection fraction(LVEF) was decreased in the CHF model group and simvastatin treatment group(P ＜ 0.05).Compared with the CHF model group,the LVESD,LVEDD were decreased and the LVEF was increased in the simvastatin treatment group(P ＜ 0.05).The positive expression rate of PTEN protein in myocardial cells of rabbits in control group,CHF model group and simvastatin treatment group was (16.36 ± 0.54) ％,(41.63 + 0.72) ％ and (24.17 ± 0.51) ％ respectively;the positive expression rate of β-catenin protein in myocardial cells of rabbits in control group,CHF model group and simvastatin treatment group was (21.73 ± 0.46)％,(52.26 ±+ 0.72) ％ and (38.42 + 0.56) ％ respectively.The positive expression rates of PTEN and β-catenin protein in myocardial cells of rabbits in CHF model group and simvastatin treatment group were significanlty higher than those in the control group(P ＜ 0.05);the positive expression rates of PTEN and β-catenin protein of myocardial cell in simvastatin treatment group were significantly lower than those in the CHF model group (P ＜ 0.05).The epression of PTEN mRNA and β-catenin mRNA in myocardial cells of rabbits in control group,CHF model group and simvastatin treatment group was 1.91 ± 0.30,4.61 ± 0.71,3.49 ± 0.64 and 1.51 ± 0.21,2.48 ± 0.34,1.51 ±+ 0.25.The expression of PTEN and β-catenin mRNA in myocardial cells of rabbits in CHF model group and simvastatin treatment group were significanlty higher than those in the control group (P ＜ 0.05);the expression of PTEN and β-catenin mRNA in myocardial cells of rabbits in simvastatin treatment group were significantly lower than those in the CHF model group (P ＜ 0.05).Conclusion Simvastatin can inhibit myocardial apoptosis,improve cardiac function of CHF rabbits.It may be related to inhibiting the expression of PTEN and β-catenin.
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AIM:To explore the regulatory effect of microRNA-3666 (miR-3666) on the expression of its tar-get gene phosphatase and tensin homologue deleted on chromosome ten (PTEN) in leukemic cells.METHODS: miR-3666 expression levels in normal peripheral blood mononuclear cells and leukemic cells were determined by quantitative real-time PCR.miR-3666 targeting PTEN 3'-untranslated region (3'UTR) was predicted by TargetScan software .3'UTR of PTEN was inserted in the dual luciferase reporter vector psiCHECK 2.The reporter activity was evaluated by the Dual-Lu-ciferase Reporter Assay System after the luciferase promoter vector and miRNA were co -transfected into HEK293T cell line. K562 cells were transfected with synthetic miR-3666 inhibitor ( anti-miR-3666) or a synthetic control miRNA ( anti-miR-C) .The expression of PTEN protein in the above transfected K 562 cells was determined by Western blotting .RESULTS:miR-3666 was up-regulated in the human leukemic cell lines and primary leukemic cells compared to normal peripheral blood mononuclear cells .The results of dual luciferase assays validated PTEN as a specific target gene of miR-3666.Inhi-bition of miR-3666 resulted in an up-regulation of PTEN protein expression in the K 562 cells.CONCLUSION:miR-3666 is over-expressed in leukemic cells .The abnormal over-expression of miR-3666 may play a key role in leukemia due to the down-regulation of PTEN .
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Objective To investigate the anti-proliferation mechanisms of Phenethyl isothiocyanate (PEITC)in human lung cancer cells line NCI-H446. Methods Human lung cancer cell line NCI-H446 was cultured in vitro,and treated with 10,30,50μmol/L PEITC for 24 h respectively. Cell viability and apoptosis were analyzed by methylthiazolyldiphenyl-tetrazolium bromide (MTT)assay and flow cytometry,respectively. Phosphorylation of Akt,activation of caspase-3 and caspase-9 in NCI-H446 cells,and production of cytochrome C in cytoplasm were detected by Western blot. Expression of phosphatase and tensin homologue (PTEN)was detected by real-time PCR. Results PEITC significantly reduced NCI-H446 cells proliferation in dose-dependent manner (P<0.05 ),and apoptosis was also inducted after PEITC administration. PEITC also markedly induced expression of caspase-3 and caspase-9,as compared with control group. And the level of cytochrome C in the cytoplasm was also increased after treatment with PEITC.Furthermore,PEITC treatment significantly increased the mRNA level of PTEN in NCI-H446 cells. Conclusion PEITC may be used as a potential anti-lung cancer agent by regulationg PI3K/AKT pathway and increasing PETN expression.
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Background and purpose: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) gene is a kind of tumor suppressors, which has been reported to be underexpressed in endometrial carcinoma (EC) tissues by several reports. However, the biological effects and possible mechanisms of PTEN on EC have been known less. In this study, we tried to investigate the effects and possible mechanisms of PTEN on the invasion and migration of endometrial carcinoma cells and to provide a potential target for endometrial carcinoma therapy. Methods:The recombinant plasmid pIRES2-ZsGreen1-PTEN was rebuilt by gene recombination technology;The plasmid was transferred into HEC-1B cells and the cells transfected with pIRES2-ZsGreen1 plasmid were used as control;The expression of PTEN was observed by fluorescence microscope and Western blot assay;Cell migration and invasion was determined by the wound healing assay, transwell migration and invasion assays respectively;The Western blot analysis was performed to detect the expression of ATP-dependent tyrosine kinase (AKT), phosphorylated-AKT (p-AKT) and matrix metalloproteinase-2 (MMP-2). Results:The agarose gel electrophoresis showed a stripe of 1.2 kb which was same to PTEN cDNA;The sequence analysis showed the PCR products owned the same sequence with the coding region of PTEN cDNA in GenBank, suggesting the recombinant plasmid was constructed successfully;The green light of cells observed by fluorescence microscope and the Western blot analysis showed the expression of PTEN was upregulated in the cells transfected with the recombinant plasmid, suggesting the plasmid expressed successfully in HEC-1B cells;The wound healing assay as well as transwell migration assay showed ectopic expression of PTEN suppressed cell migration;The invasive capacity of HEC-1B cells was significantly decreased upon transfection with PTEN plasmid compared to control and untreated groups;Moreover, compared with the control groups, the expression of p-AKT and MMP-2 was downregulated, while there was no significant alteration of the expression of AKT. Conclusion:PTEN could suppress cell migratory and invasive ability of endometrial carcinoma cells by suppressing the phosphorylation of AKT followed by the decrease of MMP-2.
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PURPOSE: We investigated the effects of phosphatase and tensin homologue deleted on chromosome 10 gene phosphatase and tensin homologue deleted on chromosome 10 gene (PTEN) expression on the cell proliferation and on the responsiveness of troglitazone in osteosarcoma cells. MATERIALS AND METHODS: Western blotting alnalysis was performed to detect the expression of PTEN in U-2OS cells treated with troglitazone. WST (water-soluble tetrazolium) assay was used to evaluate cell proliferation. Flow cytometry was used to determine cell apoptosis. Further, transfection of wild-type PTEN plasmid DNA was used to upregulate PTEN expression. RESULTS: Troglitazone treatment induced growth inhibition of U2-OS cells in a dose- and time-dependent manner. Troglitazone increased the expression of PTEN in a dose-dependent manner. PTEN upregulation induced by troglitazone treatment resulted in cell growth inhibition and apoptosis in U-2OS cells. PTEN over-expression by plasmid transfection enhanced these effects of troglitazone. Moreover, no changes were observed in the mutant type-PTEN group. CONCLUSION: Upregulation of PTEN is involved in the inhibition of cell growth and induction of cell apoptosis by troglitazone. Further, PTEN over-expression can cause cell growth inhibition in osteosarcoma cells and these cell growth inhibitions could be enhance by troglitazone treatment.
Subject(s)
Humans , Apoptosis , Blotting, Western , Cell Proliferation , Chromans , Chromosomes, Human, Pair 10 , DNA , Flow Cytometry , Microfilament Proteins , Osteosarcoma , Plasmids , Thiazolidinediones , Transfection , Up-RegulationABSTRACT
Phosphatase and tensin homologue deleted on chromosome 10(PTEN) is a tumor suppressor which can inhibit proliferation and migration and control apoptosis in a number of cell types,mainly through inhibiting the phosphoinositide 3-kinase(PI3K) signaling pathway.A number of in vitro and in vivo studies has been instrumental in uncovering a direct correlation between deregulated PTEN/PI3K signaling and changes in neuronal morphogenesis,which is likely to have profound bearings upon the pathogenesis of neurological symptoms.This review outlines recent work on the function of PTEN during the formation and maintenance of neuronal circuits in the brain.
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0.05).Conclusion Neither rs1903858 nor rs701848 of the PTEN gene has no association with laryngocarcinoma in Chinese Han population.
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Phosphatase and tensin homology deleted on chromosome 10 (PTEN) is a tumor suppressor with dual protein and lipid phosphatase activity.PTEN is involved multisystem diseases and cancer,though the mechanisms of PTEN regulation are far from clear.Recent advances concerning regulation of PTEN including protein-protein interaction,phosphorylation,ubiquitination,oxidation and acetylation will be discussed.
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Purpose:To clarify roles of overexpression of phosphorylated Akt(p-Akt) and loss of phosphatase and tensin homologue deleted on chromosome 10(PTEN) expression in biological behavior of non-small-cell lung cancer(NSCLC). Methods:Immunohistochemical staining was used to determine the expression of p-Akt and PTEN in 20 cases of normal lung tissues and 102 patients with NSCLC.Results:Negative p-Akt expression and positive PTEN expression were found in normal lung tissues, while the positive incidences of p-Akt expresssion and loss of incidences of PTEN expresssion in NSCLC were 41.2% (42/102) and 46.1% (47/102)respectivtly.The overexpression of p-Akt and loss of PTEN expression were correlated to degree of differention of cancer, metastasis(including lymph node), and clinical stages. A significant negative correlation was observed between expression of p-Akt and PTEN(Phi r=-0.425,P