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Background and purpose:A large number of studies have showed that retinoblastoma gene 1 (RB1) can inhibit the occurrence and development of many tumors, including neuroblastoma, small cell lung cancer, osteosar-coma, pancreatic cancer, breast cancer and so on. RB1 is also closely related to the regulation of cell cycle, differentia-tion, senescence, apoptosis, growth inhibition, etc. The goal of this article is to elucidate whether miR-222 promotes cell proliferation and invasion by targeting RB1, further to explore the molecular mechanism that miR-222 functions as an oncogene in retinoblastoma cells.Methods:miR-222 (miR-222 mimics) and RB1-wt, miR-NC and RB1-wt, miR-222 and RB1-mut, miR-NC (a controlled miR-222 mimics) and RB1-mut were co-transfected into Y79 cells, and luciferase activity was detected by single photon. Retinoblastoma cells were transfected with miR-222 mimics and miR-NC, and the expressions of RB1 protein were detected by Western blot. Retinoblastoma cell proliferation assays were performed by MTS assay when miR-222, miR-NC, RB1 (pcDNA3.1-RB1), vector (pcDNA3.1), miR-222+RB1 and miR-NC+vec-tor were transfected into Y79 cells. The growth and invasion ability of Y79 cells with ectopic expression of miR-222 were evaluated by MTS and Transwell invasion assays.Results:This study demonstrated that miR-222 could promote the luciferase activity of RB1-wt. The expression levels of luciferase reporter gene activity in Y79 cells after transfection with miR-222+RB1-wt were higher than those in the negative control cells (miR-NC+RB1-wt) (P0.05). After transfection with miR-222 mimics for 48 h, Transwell invasion assay showed that the number of cells through the basement membrane was (193±10). Compared with the control group (144±11), it could signiifcantly accelerate the invasion of Y79 cells (P0.05).Conclusion:miR-222 promotes cell proliferation and invasion by targeting RB1 expression in retinoblastoma cells.
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Objective To investigate whether miR-200b suppresses proliferation and induces apoptosis of non-small cell lung cancer cells by targeting DNMT3A. Methods A qRT-PCR was employed for detecting the expression of miR-200b in different non-small cell lung cancer cells and human bronchial epithelial cells. A549 cells were transfected with miR-200b mimics, scramble, DNMT3A-siRNA and control-siRNA, respectively. The scramble and control-siRNA were served the negative control of miR-200b mimics and DNMT3A-siRNA, respectively. Western blot assay was conducted to detect the expression of DNMT3A protein in A549 cells. MTT and Annexin V/propidium iodide staining were employed to detect the proliferation ability and apoptosis rate of A549 cells. The effects of miR-200b mimics and DNMT3A-siRNA on the proliferation and apoptosis rate of A549 cells were compared between groups. Results Results of qRT-PCR showed that the expression of miR-200b was significantly down-regulated in A549, H1299, L78 and H460 cells than that of 16HBE cells. Among them, the most obviously reduction was found in A549 cells (P<0.05). Western blot assay showed that the level of DNMT3A protein was inhibited by restored miR-200b or knock-down DNMT3A in A549 cells. After transfection of miR-200b mimics or knock-down DNMT3A for 48 h, 72 h and 96 h, MTT showed that the OD values, which reflected the optical density of cell proliferation were significantly lower than those in the control group (P<0.05). Annexin V/propidium iodide staining showed that apoptosis rates of A549 cells after transfection of miR-200b mimics or knock-down DNMT3A were (23.33%±0.90%and 20.41%±0.70%), compared with the control group (5.28%± 0.55%and 5.68%±0.47%, P<0.01). Conclusion miR-200b suppresses cell proliferation and induces apoptosis by targeting DNMT3A in non-small cell lung cancer.
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Background Oxydative stress is an important pathogenesis of age-related macular degeneration.Resent evidences indicate that docosahexaenoic acid (DHA) plays an important role during the development of retinal photoreceptor cells and protect the cells against oxydative stress by inducing the expression of heme oxygenase-1 (HO-1).However,whether DHA can induce the expression of HO-1 in human retinal pigment epithelium (RPE) cells is unelucidated.Objective This study was to investigate the effect of DHA on the expression of HO-1 in RPE cells and its molecular mechanism.Methods Human RPE cell line ARPE-19 was cultured in vitro and treated with 30,50,100 and 120 μmol/L DHA for 4 to 24 hours,respectively,and the cells were cultured without DHA as the control group.The cytotoxicity of DHA was detected by lactate dehydrogenase(LDH),and the expression of HO-1 mRNA and protein were detected by real-time PCR and Western blot assay,respectively.The enzymatic activity of HO-1 was detected by colorimetry.The reactive oxygen species (ROS) proportion in the cells was detected using fluorescence probe H2 DCFDA,and immunofluorescence technology was adopted to detect the nuclear translocation of nuclear facotor-E2-related factor 2 (Nrf2).The expression of Nrt2 protein in the cells was detected by Western blot after intervention of ROS inhibitor N-acetylcysteine (NAC) and transfection of Nrf2 small interfering RNA (siRNA).Results The LDH leakage rate was significantly different after 0,3,50,100 and 120 μmol/L DHA treated the cells for 24 hours (F=8.14,P＜0.05),and the LDH leakage rate in the 120 μmol/L DHA group was significantly higher than that of 0,30,50 and 100 μmol/L DHA group (all at P＜0.05).The relative expression levels of HO-1 mRNA and HO-1 protein or HO-1 enzymatic activity in the cells were significantly different among different concentrations of DHA group in 8 hours after treatment (F=16.24,P＜0.05;F=11.34,P＜0.05;F=11.81,P＜0.05),and the expressions of these factors were considerably higher in the 30,50 and 100 μ mol/L DHA group than those in the 0 μmol/L DHA group (all at P＜0.05).The ROS relative fluorescence intensity and nuclear Nrf2 positive cells proportion were statistically significant among different concentrations of DHA groups (F =11.08,P ＜ 0.05;F=16.42,P＜0.05),and the ROS relative fluorescence intensity and nuclear Nrf2 positive cells proportion were evidently higher in the 30,50 and 100 μmol/L DHA group than those in the 0 μmol/L DHA group (all at P＜0.05).The relative expression levels of HO-1 protein and the proportion of nuclear Nrf2 positive cells were significantly lower in the NAC pretreated 100 μmol/L DHA group than those in the 100 μmol/L DHA group.In addition,the HO-1 relative expression level and the positive cells proportion of nuclear Nrf2 were significantly lower in the of Nrf2 siRNA transfection group than those in the blank siRNA transfection group (both at P＜0.05).Conclusions DHA with concentration below 100 μ mol/L can protect RPE cells from oxidative stress by inducting the expression of HO-1 in the cells via ROS/Nrf2 pathway.
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AIM:To observe the effect of docosahexaenoic acid ( DHA) on H2 O2-induced apoptosis in human retinal pigment epithelium cells and its molecular mechanism .METHODS: Human retinal pigment epithelium cell line ARPE-19 was cultured in vitro, and 12.5 mmol/L H2 O2 was used to mimic the oxidative stress condition .The cells were treated with 30~100μmol/L DHA for 4~24 h.The expression of heme oxygenase-1 (HO-1) at mRNA and protein levels was detected by real-time PCR and Western blot , respectively .The enzymic activity of HO-1 was measured by colorimetry . Production of reactive oxygen species ( ROS) was determined by fluorescent probe .Activation of NF-E2-related factor 2 (Nrf2) was examined by immunofluorescence method .Apoptosis of ARPE-19 cells was analyzed by flow cytometry .RE-SULTS:The mRNA and protein expression and the enzymic activity of HO-1 were significantly increased in the ARPE-19 cells after DHA treatment .Meanwhile , nuclear translocation of Nrf 2 was also observed .Apoptosis appeared and ROS was produced upon H2O2 incubation.In contrast, DHA at 100 μmol/L significantly abrogated H2O2-induced apoptosis and ROS production.Furthermore, silencing of HO-1 by specific siRNA, or treatment with ZnPP, an inhibitor of HO-1, partly counteracted the protective effect against H 2 O2-induced apoptosis and ROS production .CONCLUSION: DHA protects retinal pigment epithelial cells against oxidative stress via induction of heme oxygenase -1 expression after Nrf2 activation .
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Objective To observe the role of heme oxygenase (HO)-1 on the protective effect of C-phycocyanin (CPC) on doxorubicin (DOX)-induced myocardial cells injury by Nvf2/HO-1 pathway. Methods 60 SD rats were randomly divided into control group, DOX group, CPC group and tin protoporphyrin IX (SnPP, an inhibitor of HO-1) group. The control group was injected with normal saline injection,while the DOX group was administrated with doxorubicin by intraperitoneal injection in a cumulative dose of 15 mg/kg for two weeks. For the CPC rats, 20, 40 and 60 mg/kg of CPC was administrated. The level of creatine kinase (CK) and lactate dehydrogenase (LDH) were detected, and the activity of HO-1 and caspase-3 were also examined. Expression of HO-1 and activation of Nrf 2 were detected by Western blot. Results Compared with control group, serum levels of CK, LDH and Caspase-3 activity in DOX group were significantly increased(P<0.05), but HO-1 in cardiac muscle was only increased slightly. upregulation. Treatment with CPC could significantly ameliorated the CK, LDH and Caspase-3 activity, and markedly induce HO-1 expression and its activity. The reduction of CK, LDH and Caspase-3 activity by CPC could be reversed by treatment of the HO-1 inhibitor, SnPP. Furthermore, CPC sould also induce Nrf 2 activation. Conclusion The protective effect of CPC on doxorubicin-induced myocardial cells in jury via Nrf 2 induced HO-1 HO-1 expression.
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Objective To study the protective effect mechanism of Rosa laevigata Michx (RLM) on cardiotoxicity induced by adriamycin in rats. Method 30 SD rats were randomly into control group, doxorubicin group and RLM groups. The control group was injected with normal saline injection, while the model group was injected with adriamycin intraperitoneally at the dosage of 15 mg/kg every other day. For the RLM groups,1~5 g/kg RLM were given after adriamycin injection. The survival rate, plasma BNP was observed. Apoptosis of cardiomyocyte was detected by instituted-labeled DNA (TUNEL). The activity of GSH-PX, CAT and total SOD in the myocardium tissue were also observed. The expression level of CuZn-SOD , bcl-2 and bax were detected by real-time PCR. Results The survival rate was significantly improved in SD rats treated with RLM compared with that in the adriamycin group (P<0.01). The BNP level was increased when treated by adriamycin (P<0.01), and decreased after RLM administration (P<0.01). RLM could also upregulate the expression of the CuZn-SOD mRNA level, and enhance the activity of GSH-PX, CAT and T-SOD compared with that in adriamycin group. Adriamycin could induce myocardial cells apoptosis, as demonstrated by TUNEL. RLM could inhibit adriamycin-induced apoptosis, bax mRNA expression, and increase bcl-2 expression and bcl-2/bax ratio. Conclusion RLM exhibit some antioxidant activity through many stages, and the anti-apoptosis activity may be related to affect the expression of bax and bcl-2 expression.
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Objective To investigate the effect and molecular mechanism of Andrographolide (AD) on matrix metalloproteinase-9 (MMP-9) expression in human colon cancer H 3255 cells. Methods Human lung cancer H3255 cell line weve cultured in vitro, and treated with 1.0, 3.0, 5.0μmol/L AD for 24 h, untreated cells was used as blank control. Cell viability, cell migration and cell invasion were analyzed by MTT assay, scratch healing assay and transwell membrane assay, respectably. Expression of MMP-9 mRNA was analyzed by RT-PCR. Protein expression and phosphorylation of Akt were detected by Western blot. Activity of NF-κB and MMP-9 were analyzed by luciferase reporter assay. Results AD could significantly reduced H 3255 cells invasion and migration without affecting the viability of cells, as demonstrated by scratch healing and transwell membrane assay. Furthermore, Western blot and RT-PCR results showed that AD could markedly inhibited MMP-9 activity and its expression in both protein and mRNA levels. AD could attenuated Akt’s phosphorylation and the activity of NF-κB. Moreover, LY 294002, an inhibitor of PI3 K, could significantly inhibited NF-κB transcriptional activity and MMP-9 expression. In addition, different concentrations of AD could inhibit the promote activity of MMP-9. Conclusion AD was a potential anti-invasive agent by inhibiting MMP-9 involved in PI3 K/NF-κB pathways.
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BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) can be induced toward the chondrogenic lineages with chondrogenic medium contained transforming growth factor-β_1(TGF-β_1). However, it remains concerned about the disadvantage with use of TGF-β_1 in vitro, which can induce chemotaxis, activate inflammatory cells, cause local defect and want an ideal matrix to deliver proteins. Therefore, with advanced gene-delivery techniques, TGF-β_1 can be long-lasting expressed by transduced stem cells, which is very useful for Chondrogenic Tissue Engineering.OBJECTIVE: To master the method of construction and transfection of eukaryotic expression vector for rat transforming growth factor-β_1 and to study the possibility of gene transfection to ADSCs with this vector.METHODS: Recombining DNA techniques were applied to construct recombinant plasmid pcDNA3.1-TGF-β_1. And this plasmid was verified by restriction endonuclease mapping and DNA sequencing; Then the plasmid with TGF-β_1 gene or not was transfected into ADSCs by use of LipofectamineTM2000. After infection, transduced ADSCs were diluted and cultured with neomycin (G418). Gene transfer efficiency compared on the basis of green fluorescent protein expression was assessed.RESULTS AND CONCLUSION: Digestion of the plasmid with double restriction endo- nuclease XboⅠand Hind Ⅲ showed about two specific electrophoretic strips (1.35 bp and 5.4 kb), and the sequence of the rat TGF-β_1 gene in recombinant was concorded with that reported in Genbank. There were 80 percent of the cells which were transduced, and the expressions of mRNA and protein of TGF-β_1 in ADSCs were discovered positively. These indicated that the eukaryotic expression vector for rat TGF-β_1 (i.e. pcDNA3.1-TGF-β_1) , which is able to transfect the ADSCs, can be constructed through genetic recombination.
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@#Resveratrol is a kind of polyphenolic compounds, and widely exists in plants.It can be provided with comprehensive physiological and pharmacological effects. In this paper, the authors reviewed the effect of resveratrol on myocardial ischemia-reperfusion injury, the effective treatment of atherosclerotic diseases and coronary heart disease, and discuss the protective effect and the mechanism of resveratrol on cardiovascular disease through the effect of the electrophysiology and the accommodation of the blood vessel.
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Objective To compare the therapeutic effect of Microendoscopic Discectomy (MED) with love's method for the treatment of lumbar disk herniation. Methods Pre- and postoperative JOA score, the length of incision, blood lost, operative time existed weight of disk, body temperature variation and time on getting out of bed after operation were compared between the MED group and the love's method group randomized 35 cases respectively since December 1999. Results The preoperative JOA scores in the MED group and the love's method group were 12.1?1.8 and 12.1?1.4, respectively (t=0.437, P=0.663); The postoperative JOA scores in both groups were 26.9?1.6 and 26.1?5.3, respectively (t=1.81, P=0.0077); The length of incision were (2.0?0.1)cm and (5.0?0.6)cm, respectively (t=26.72, P
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Objective:To evaluate the therapeutical effects of sodium hyaluronate and triamcinolone acetonide on secondary temporomandibular synovitis.Methods:67 patients with secondary temporomandibular synovitis were divided into two groups:A group received the intraarticular injection of sodium hyaluronate and B group received triamcinolone acetonide injection.The effects of two groups were observed.Results:Patients were followed up at 1 week,2 months and 3 months after being treated respectively.Although 1 week after treatment,A group showed better curative effect(P0.05).After 3 months,B group showed better effects than A group(P