MicroRNA Profiling of Transgenic Mice with Myocardial Overexpression of Nucleolin / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Nucleolin (NCL) is the most abundant RNA-binding protein in the cell nucleolus and plays an important role in chromatin stability, ribosome assembly, ribosomal RNA maturation, ribosomal DNA transcription, nucleocytoplasmic transport, and regulation of RNA stability and translation efficiency. In addition to its anti-apoptotic properties, the underlying mechanisms associated with NCL-related roles in different cellular processes remain unclear. In this study, the effect of NCL on microRNA (miRNA) expression was evaluated by generating transgenic mice with myocardial overexpression of NCL and by analyzing microarrays of mature and precursor miRNAs from mice.</p><p><b>METHODS</b>Using microinjection of alpha-MyHc clone 26-NCL plasmids, we generated transgenic mice with myocardial overexpression of NCL firstly, and then mature and precursor miRNAs expression profiles were analyzed in NCL transgenic mice (n = 3) and wild-type (WT) mice (n = 3) by miRNA microarrays. Statistical Package for the Social Sciences version 16.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform Student's t-test, and statistical significance was determined at P < 0.05.</p><p><b>RESULTS</b>Several miRNAs were found to be differentially expressed, of which 11 were upregulated and 4 were downregulated in transgenic mice with myocardial overexpression of NCL compared to those in WT mice. Several differentially expressed miRNAs were subsequently confirmed and quantified by real-time quantitative reverse transcription-polymerase chain reaction. Bioinformatics analysis was used for the prediction of miRNA targets. Furthermore, in vitro experiments showed that NCL regulated miR-21 expression following hydrogen peroxide preconditioning.</p><p><b>CONCLUSIONS</b>Myocardial-protection mechanisms exerted by NCL might be mediated by the miRNAs identified in this study.</p>

Role of microRNA-126-5p in myocardial injury induced by doxorubicin / 中国病理生理杂志

AIM:To observe the expression of microRNA-126-5p during myocardial injury and its role in myo-cardial cell injury induced by adriamycin(also called doxorubicin, DOX).METHODS: The BALB/c mouse model of DOX-induced acute and chronic myocardial injury was established via intraperitoneal injection of DOX.HE staining was applied to observe the morphological changes of myocardial tissues.Lactate dehydrogenase(LDH)in serum was detected and PowerLab system was used to detect the influence of DOX on the changes of ±dp/dtmax.The expression of microRNA-126-5p in injured myocardial tissues and the H 9c2 cells exposed to DOX was detected by real-time PCR.Gain-and loss-of-function experiments were conducted to detect the role of microRNA-126-5p in H9c2 cells treated with DOX on LDH release and caspase-3 activation.RESULTS:In acute and chronic DOX myocardial damage models in mice,HE staining showed disarranged myocardial fibers, dissolved myofibril and inflammatory cell infiltration.Higher serum LDH level and lower ±dp/dtmaxin DOX-treated mice than those in normal mice were found.Compared with the normal mice, the expression level of microRNA-126-5p was significant increased in the myocardium with DOX-induced injury.Similarly,the expression level of microRNA-126-5p was significant increased in the H9c2 cells treated with DOX.In addition, over-expression of microRNA-126-5p decreased cell viability and promoted apoptosis,while microRNA-126-5p ablation promoted the viability and inhibited the apoptosis of H9c2 cells.CONCLUSION:The microRNA-126-5p expression is up-regulated in myocar-dial injury induced by DOX,and microRNA-126-5p inhibits cell viability and promotes apoptosis induced by DOX.

Role of peroxisome proliferator-activated receptor beta in the inhibitory effect of epidermal growth factor on apoptosis of HaCaT / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the role of EGF in regulating HaCaT apoptosis through peroxisome proliferator-activated receptor beta (PPARbeta).</p><p><b>METHODS</b>Cultured HaCaT cells were divided into different groups with different additives in culture medium as control (normal culture), TNF-alpha (with addition of 10 ng/mL TNF-alpha), EGF (with addition of 20 ng/mL EGF), EGF + TNF-alpha (cells were treated with 10 ng/mL TNF-alpha for 60 mins after the exposure to 20 ng/mL EGF for 4 hs) groups. Conjugation activity and transcription activity of PPARbeta of HaCaT cells in each group were detected by electrophoretic mobility shift assay (EMSA) and luciferase gene analysis (LGA). Protein expression of PPARbeta of HaCaT cells after transfected by missense oligonucleotide (scrODN) and antisense oligonucleotide (asODN) was determined by Western blot. Caspase-3 activity and apoptosis rate were detected by flow cytometry.</p><p><b>RESULTS</b>Conjugation and transcription activity of PPARbeta DNA were enhanced as shown in EMSA and LGA. Compared with that of cells in groups transfected by scrODN, protein expression of PPARbeta in cells of groups transfected by asODN was obviously inhibited as shown in Western blot. Caspase-3 activity of cells in TNF-alpha and EGF + TNF-alpha groups transfected by asODN was stronger than that of cells in TNF-alpha and EGF + TNF-alpha groups transfected by scrODN (P < 0.01). Apoptosis rate of cells in control, EGF, TNF-alpha, and EGF + TNF-alpha groups which were transfected by scrODN was (7.31 +/- 0.45)%, (7.43 +/- 0.21)%, (39.78 +/- 0.65)%, (28.34 +/- 0.54)% respectively, and that in those groups transfected by asODN was (8.22 +/- 0.51)%, (7.83 +/- 0.67)%, (46.78 +/- 0.48)%, (44.69 +/- 0.83)%. Apoptosis rate of cells in TNF-alpha and EGF + TNF-alpha groups transfected by asODN was respectively higher than that in TNF-alpha and EGF + TNF-alpha groups transfected by scrODN (P < 0.01).</p><p><b>CONCLUSIONS</b>EGF inhibits HaCaT KC apoptosis caused by TNF-alpha in a PPARbeta-dependent manner.</p>

Role of cell-surface nucleolin in lipopolysaccharide-stimulated expression and secretion of TNF-alpha and IL-1beta / 中南大学学报(医学版)

OBJECTIVE@#To explore the role of cell-surface nucleolin in lipopolysaccharide (LPS)-stimulated expression and secretion of TNF-alpha and IL-1beta in human THP-1 monocytes.@*METHODS@#Immuno-fluorescence assay and Western blot were used to identify the expression of nucleolin on the surface of THP-1 monocytes. Inactivation of nucleolin was induced by anti-nucleolin monoclonal antibody blockage, and the expression and secretion of TNF-alpha and IL-1beta were observed by using reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immuno-sorbent assay (ELISA)respectively in LPS-mediated human THP-1 monocyte inflammatory model.@*RESULTS@#Immuno-fluorescence showed that nucleolin was localized on the cell surface of THP-1 monocytes as indicated by dotted red fluorescence. Western blot assay indicated that nucleolin existed in the cell membrane fractions. RT-PCR assay showed that the expressions of TNF-alpha and IL-1beta mRNA significantly increased at 2 h and 3 h after the treatment with 1000 microg/L LPS. After 1 h pretreatment with anti-nucleolin antibody, the levels of TNF-alpha and IL-1beta mRNA decreased compared with an anti-nucleolin antibody untreated group and an irrelevant IgG+LPS group (P<0.05). ELISA assay showed that the pretreatment with anti-nucleolin antibody inhibited significantly the secretion of LPS-induced levels of TNF-alpha and IL-1beta after 4, 12 and 24 h treatment with 1000 microg/L LPS.@*CONCLUSION@#Nucleolin expresses on the cell surface of THP-1 monocytes and involves in the LPS-mediated expression and secretion of TNF-alpha and IL-1beta.

Changes of nucleolin expression and cellular localization during HUVEC apoptosis induced by hydrogen peroxide / 中南大学学报(医学版)

OBJECTIVE@#To investigate the expression and cellular localization of nucleolin C23 during human umbilical vein endothelial cell (HUVEC) apoptosis induced by hydrogen peroxide (H(2)O(2)).@*METHODS@#Apoptosis of HUVEC was induced by exposure to 0.5 mmol/L H(2)O(2) for different periods and detected by flow cytometry and activity of caspase-3. The mRNA and protein expression of nucleolin were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The intracellular distribution of nucleolin was observed by indirect immunofluorescence.@*RESULTS@#The percentage of apoptotic cells was increased significantly after treatment with H(2)O(2) for 12, 24 and 36 hours. The activity of caspase-3 reached the peak after treatment with H(2)O(2) for 4 h. RT-PCR showed that nucleolin C23 mRNA was decreased after 2, 4, and 8 hours treatment with H(2)O(2). Western blot showed that C23 protein level was decreased after 12 hours with an additional cleft band of 80 kD appeared after 8 hours. Density analysis showed that the 80 kD cleft band increased in a time-dependent manner. Immunofluorescence analysis demonstrated that H(2)O(2)-induced C23 redistribution from the nucleus to the cytoplasm.@*CONCLUSION@#H(2)O(2) could induce apoptosis accompanying with C23-cleavage and C23-translocation from the nucleus to the cytoplasm.

Inhibitory effect of epidermal growth factor on apoptosis in HaCaT keratinocytes induced by TNF-alpha / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the effect of epidermal growth factor (EGF) on apoptosis induced by TNF-alpha and the expression of PPARbeta in HaCaT keratinocytes.</p><p><b>METHODS</b>HaCaT keratinocytes were cultured and randomly divided into A (normal control), B (with treatment of 10 ng/ml TNF-alpha for 24 hours), C (with treatment of 20 ng/ml TNF-alpha for 24 hours), D (with treatment of 10 ng/ml TNF-alpha after 20 ng/ml EGF treatment for 4 hours), E (with treatment of 20 ng/ml TNF-alpha after 20 ng/ml EGF treatment for 4 hours) groups. The apoptosis of HaCaT keratinocytes was observed by flow cytometry. The proliferative activity of HaCaT keratinocytes was evaluated by MTT method. The activity of caspase-3 was analyzed with caspase colorimetric assay Kit. The changes in the mRNA and protein expression of PPARbeta in HaCaT keratinocytes were observed by RT-PCR and western-blotting after treatment with different concentrations (5, 10, 20, 40 ng/ml) of EGF for 4 or 24 hrs.</p><p><b>RESULTS</b>Compared with A and B groups [(32 +/- 6)%, (57 +/- 6)%], the apoptosis of HaCaT keratinocytes in D and E groups were significantly increased [(20 +/- 3)%, (28 +/- 4)%, respectively, P < 0.01], while the survival rate of HaCaT keratinocytes in D and E groups increased, and the caspase-3 activity were decreased (P < 0.01). The expression of PPARbeta mRNA and protein in HaCaT keratinocytes reached the peak with the treatment of 20 ng/ml EGF.</p><p><b>CONCLUSION</b>EGF can inhibit the apoptosis of HaCaT keratinocytes induced by TNF-alpha, and it can also increase the expression of PPARbeta.</p>

Study on the antisense oligonucleotide against peroxisome proliferator-activated receptors promoting TNF-alpha mediated apoptosis of HaCat cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of antisense phosphorothioate oligonucleotides on peroxisome proliferator-activated receptors (PPARbeta) in the TNF-alpha mediated apoptosis of HaCat cells.</p><p><b>METHODS</b>HaCat cells were resuscitated and randomly divided into normal control (without transfection), sham (merely with liposome transfection), scrODN (with transfection of 4 micromol/L PPARbeta scrODN), asODN (with transfection of 4 micromol/L PPARbeta asODN), TNF-alpha with transfection of 10 micromol/L TNF-alpha), scrODN + TNF-alpha with 10 micromol/L TNF-alpha stimulation after transfection of 4 micromol/L PPARbeta scrODN), asODN + TNF-alpha with 10 micromol/L TNF-alpha stimulation after transfection of 4 micromol/L PPARbeta asODN) groups. The mRNA and protein levels of PPARbeta were determined with RT-PCR and Western blotting, respectively. The changes in cell morphology were observed with Hoechst 33258 fluorescent staining to quantitate apoptotic rate of nuclei. The effect of PPARbeta asODN on HaCat cell viability was assayed with MTT method. Activation of caspase-3 was evaluated with caspase colorimetric analysis kit.</p><p><b>RESULTS</b>The mRNA and protein expression of PPARbeta in normal control, sham, scrODN groups were similar, but it decreased obviously in asODN group. The nuclear apoptotic rate in normal control, scrODN and asODN groups were rather low, and the caspase-3 activity in these groups was also low. After 24 hours of culture, the nuclear apoptotic rate in TNF-alpha and scrODN + TNF-alpha groups were (33.1 +/- 2.7)% and (32.9 +/- 3.0)%, respectively, while that in asODN + TNF-alpha group was obviously increased (58.8 +/- 4.6)%, with the caspase-3 activity significantly higher, but the number of live cells markedly lower than that in the former 2 groups (P < 0.05).</p><p><b>CONCLUSION</b>PPARbeta expression can promote the apoptosis of HaCat cells mediated by TNF-alpha.</p>

HSP70 inhibits smac release from the mitochondria and protects against H2O2-induced apoptosis in C2C12 myogenic cells / 中南大学学报(医学版)

OBJECTIVE@#To observe whether HSP70 could protect against H2O2-induced apoptosis in C2C12 myogenic cells by inhibiting Smac release from the mitochondria.@*METHODS@#HSP70 gene and full length Smac gene was transiently transfected in C2C12 myogenic cells by lipofectamine and the protein levels of HSP70 and Smac were analysed by Western blotting. Hoechst 33 258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. DNA ladder pattern on agarose gel electrophoresis was used to observe the DNA fragmentation. Activities of caspase-3 and caspase-9 were assayed with Western blotting. The release of Smac from the mitochondria to the cytoplasm was observed by immunofluorescence.@*RESULTS@#H2O2 ( 0.5 mmol/L ) activated caspase-3, caspase-9 8 h after the treatment and specific morphological changes of apoptosis 12 h after the treatment, and overexpression of Smac significantly promoted H2O2-induced activation of caspase-3, caspase-9 and apoptosis in C2C12 myogenic cells. HSP70 overexpression significantly inhibited H2O2-induced and Smac-promoted apoptosis, as shown by no specific DNA ladder pattern in agarose gel electrophoresis, decrease of percentage of apoptotic nuclei, and marked inactivation of caspase-3 and caspase-9. HSP70 could inhibit the release of Smac from the mitochondria to the cytoplasm 2 h after the treatment by H2O2.@*CONCLUSION@#HSP70 inhibits Smac release from the mitochondria and protects against H2O2-induced apoptosis in C2C12 myogenic cells.