RNA interfering retinoblastoma-like protein 2 gene induces apoptosis of human cardiac stem cells mediated by DNA methyltransferase

AIM: Retinoblastoma-like protein 2 (Rbl-2) plays an important role in the cell proliferation and DNA methyltransferase (DNMT) may involve in the regulation of differentiation in embryonic stem cells. This study is to investigate the effect of knocking down Rbl-2 by specific siRNA on apoptosis in human cardiac stem cells.METHODS: The siRNA of Rbl-2 (siRbl-2) was transfected into human cardiac stem cells. The mRNA expression of Rbl-2 and DNMT-3B was detected by real-time RT-PCR 48 h after transfection. The DNMT-3B protein expression and the activation of caspase-3 were determined by Western-blotting. The cell apoptosis was analyzed by flow cytometry with annexin V-FITC/PI staining. RESULTS: Knocking down of Rbl-2 gene increased the expression of DNMT-3B in human cardiac stem cells, and induced cell apoptosis. Compared with negative control group, caspase-3 was activated and cleaved caspase-3 was increased in the stem cells transfected with siRbl-2. The cleaved caspase-3 accounted for more proportions of total caspase-3 in transfected cells than that in non-transfected cells (P<0.01). The apoptotic rate was also increased significantly in transfected group (P<0.01).CONCLUSION: Rbl-2 plays an important role in the regulation of survival and apoptosis in human cardiac stem cells. This regulatory mechanism may involve in epigenetic modification, which is mediated by DNMT.     

SHP-2 regulates PC12 cell survival and apoptosis in response to NGF via activation of ERK and JNK

AIM: To investigate the central role of the protein tyrosine phosphatase SHP-2 in the survival of PC12 cells upon NGF treatment and apoptosis after NGF withdrawal. METHODS: PC12 cells were treated with SHP-2 inhibitor (NSC87877). Cell survival rate was detected by MTT method and apoptosis was determined by flow cytometry. Moreover, pIRES-GFP (vector alone), pIRES-GFP-SHP-2 (wild type) and pIRES-GFP-SHP-2C459S (dominant negative mutant form) were transfected into PC12 cells. ERK, p-ERK, JNK and p-JNK were immunoblotted at 1 h in the presence of NGF and 5 h after NGF withdrawal. RESULTS: SHP-2 potentially enhanced survival and attenuated apoptosis of PC12 cells. The activation of ERK was significantly enhanced with NGF treatment either in the group without SHP-2 inhibitor or the SHP-2 wild type group. On the other hand, phosphorylation level of JNK was significantly increased after NGF withdrawal when PC12 cells were treated with SHP-2 inhibitor or transfected with SHP-2 mutant. CONCLUSION: SHP-2 may play a central role in mediating the survival and apoptosis of PC12 cells upon NGF exposure and withdrawal. The underlying mechanisms may be through the activation of ERK and JNK.     

Dithiothreitol induces apoptosis of BRL-3A cells by activation of calpain-2/caspase-12 signaling pathway

AIM: To investigate the expression changes of glucose-regulated protein 78 (GRP78), calpain-2, caspase-12 and caspase-3 in dithiothreitol (DTT)-induced endoplasmic reticulum stress in rat normal liver cell line BRL-3A, and to explore their effects on apoptosis of BRL-3A cells. METHODS: BRL-3A cells were treated with 2.5 mmol/L DTT to induce endoplasmic reticulum stress. The mRNA expression of GRP78, calpain-2, caspase-12 and caspase-3 was detected by real-time PCR. The protein expression of GRP78, calpain-2, caspase-12 and caspase-3 was detected by cell immunofluorescence technique. The protein levels of cleaved caspase-12 and cleaved caspase-3 were determined by Western blot. The apoptosis of BRL-3A cells was analyzed by flow cytometry. RESULTS: After treatment with DTT for 12 h and 24 h, the mRNA expression of GRP78, calpain-2 and caspase-12 in the BRL-3A cells was obviously increased compared with normal control group (P<0.01), and no significant change of caspase-3 mRNA after treatment with DTT for 12 h and 24 h was observed. The results of immunofluorescence technique and Western blot showed that the protein levels of GRP78, calpain-2, caspase-12, cleaved caspase-12, caspase-3 and cleaved caspase-3 were obviously elevated after treatment with DTT for 12 h and 24 h(P<0.05). In addition, the increased apoptotic rate was also found in the BRL-3A cells treated with DTT for 12 h and 24 h (P<0.05). CONCLUSION: The activation of calpain-2/caspase-12 signaling pathway may be involved in apoptosis of BRL-3A cells induced by DTT.     

Effect of Rip2 overexpression on apoptosis in human pancreatic cancer cell line Panc-1

AIM: To observe the effect of receptor-interacting protein 2 (Rip2) overexpression on human pancreatic cancer cell line Panc-1. METHODS: pEGFP-C2 and pEGFP-Rip2 plasmids were respectively transfected into the Panc-1 cells using JetPRIME reagent. The cells were divided into control group, pEGFP-C2 group and pEGFP-Rip2 group. The apoptosis in the cells was detected 48 h after transfection by flow cytometry. Rip2 level and the expression of apoptosis-related proteins, Bax, cytoplasmic cytochrome c (Cyt-c) and Bcl-2, were analyzed by Western blot. The activity of caspase-3 was measured by colorimetric method. RESULTS: Rip2 protein expression significantly increased in the cells transfected with control and pEGFP-C2 plasmids. The apoptotic rate in pEGFP-Rip2 group was higher than that in control group and pEGFP-C2 group, whereas no significant difference of apoptotic rate was observed between control group and pEGFP-C2 group. The protein expression of Bax and cytoplasmic Cyt-c was remarkably increased and the protein expression of Bcl-2 was obviously decreased in pEGFP-Rip2 group as compared with control group and pEGFP-C2 group. The activity of caspase-3 in pEGFP-Rip2 group was obviously increased as compared with control group and pEGFP-C2 group. CONCLUSION: Overexpression of Rip2 is able to induce apoptosis in the Panc-1 cells, and the mechanism may be related to the up-regulation of Bax and cytoplasmic Cyt-c protein expression, down-regulation of Bcl-2 protein expression and enhancement of caspase-3 activity, thus activating intrinsic apoptotic pathway.     

Influence of Bcl-2 inhibitor on Astragalus injection-reduced expression of caspase-3 in rat hippocampal neurons with oxygen-glucose deprivation and reoxygenation

AIM: To observe the influence of Bcl-2 inhibitor on the expression of caspase-3 reduced by Astra-galus injection in rat hippocampal neurons with oxygen-glucose deprivation and reoxygenation (OGD/R). METHODS: The primary rat hippocampal neurons cultured in vitro for 8 d were chosen and randomly divided into 6 groups: normal control group, model group (OGD/R group), Astragalus injection group, Astragalus injection solvent (sterile deionized water)group, Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group. The cells in all groups were tested 24 h after they were treated with reoxygenation after deprived of oxygen and glucose for 30 min except normal control group. The cell type and rate of positive cells were observed by immunohistochemistry. The protein levels of Bcl-2 and cleaved caspase-3 in the hippocampal neurons were measured by Western blotting. The mRNA expression of caspase-3 was detected by RT-PCR. RESULTS: Compared with normal control group, the caspase-3 positive rate of the cells, the protein levels of Bcl-2 and cleaved caspase-3, and the mRNA expression of caspase-3 in model group enhanced significantly (P < 0.05). Compared with model group, the expression of Bcl-2 in Astragalus injection group obviously enhanced, while the caspase-3 positive rate of the cells, the protein level of cleaved caspase-3 and the mRNA expression of caspase-3 in the Astragalus injection group decreased significantly (P < 0.05). No significant difference in injection solvent group, Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group was observed (P > 0.05). The expression of Bcl-2 was decreased sharply in Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group. CONCLUSION: Bcl-2 inhibitor antagonizes the inhibitory effect of Astragalus injection on caspase-3 expression in rat hippocamal neurons with OGD/R, which may be one of the possible target for the inhibitory action of Astragalus injection on the apoptosis of rat hippocampal neurons induced by OGD/R.     

Effects of NOD8 on H2O2-induced apoptosis in human hepatocytes

AIM: To observe the effects of NOD8 on H2O2-induced apoptosis in human L02 hepatocytes. METHODS: pEGFP-C2 and pEGFP-NOD8 plasmids were transfected into L02 cells by JetPRIME, respectively. The apoptosis of these transfected cells was induced by H2O2. The cells were divided into pEGFP-C2 group, pEGFP-C2+H2O2 group and pEGFP-NOD8+H2O2 group. MTT assay was used to detect the cell viability. NOD8 protein expression was determined by Western blotting. The cell apoptosis was observed by Hoechst 33342 staining and apoptotic rate was evaluated by flow cytometry. The caspase-3 activity was analyzed by a colorimetric method. RESULTS: L02 cells were stimulated by H2O2 at concentrations of 0.2～2 mmol/L for 6 h, and H2O2 at concentration of 1 mmol/L was chosen to induce apoptosis determined by MTT assay. The protein expression of NOD8 significantly increased in the cells transfected with pEGFP-NOD8 plasmid. More cellular nucleus with strong blue fluorescence by Hoechst 33342 staining in pEGFP-C2+ H2O2 group were observed, indicating that apoptosis was increased, while the apoptosis in pEGFP-NOD8+H2O2 group significantly reduced. The apoptotic rate in pEGFP-C2+H2O2 group was obviously increased, whereas that in pEGFP-NOD8+H2O2 group was significantly decreased. The caspase-3 activity in pEGFP-C2+H2O2 group was remarkably increased. By contrast, the activity of caspase-3 was significantly reduced in pEGFP-NOD8+H2O2 group.CONCLUSION: NOD8 inhibits H2O2-induced apoptosis in L02 cells and the mechanism may be related to inhibition of caspase-3 activity.     

Modulation of bacterial redox protein azurin induces human osteosarcoma cell apoptosis by Fas antigen and caspase-8

AIM: To determine the role of Fas antigen and caspase-8 in modulating apoptosis of osteosarcoma cells induced by bacterial redox protein azurin. METHODS: The human osteosarcoma cell line U2OS was treated with bacterial redox protein azurin (150 mg/L) for 6, 12, 24 and 48 h, respectively. Cell immunohistochemistry and quantitative image pattern analysis were applied for detecting the expression of Fas antigen. Caspase-8 activity was detected using caspase-8 fluorescent assay kit. The apoptotic rate was measured by FCM. RESULTS: Compared with the control group, the expression of Fas antigen and activity of caspase-8 significantly increased in U2OS cells treated with 150 mg/L azurin (P<0.01). There was positive correlation between the expression of Fas antigen and activity of caspase-8 (r=0.873, P<0.01). The increased Fas antigen expression had the function to transfer apoptotic signals and the anti-Fas antibody promoted azurin induced apoptosis through increasing Fas antigen expression. IETD-FMK blocked the activation of procaspase-8 and reduced apoptosis of U2OS cells in the presence of azurin or anti-Fas antibody. The apoptotic rates in azurin group and anti-Fas antibody group were significantly different from the inhibitor group (P<0.01). CONCLUSION: The Fas-dependent signalling is the important pathway of azurin inducing apoptosis in U2OS cells. The up-regulation of csepase-8 may play an important role.     

Effect of transcription factor BACH2 over-expression on viability and apoptosis of human acute lymphoblastic leukemia T lymphocytes

AIM To investigate the effect of over-expression of BTB and CNC homology 2 （BACH2） on the viability and apoptosis of human acute lymphoblastic leukemia T lymphocytes CCRF-CEM. METHODS CCRF-CEM cells were divided into 3 groups： control group, empty vector group, and BACH2 over-expression group. The BACH2 over-expression vector was transfected into CCRF-CEM cells of BACH2 over-expression group by liposome transfection method. The difference in mRNA expression of BACH2 between CCRF-CEM cells and peripheral blood mononuclear cell （PBMC） was detected by qPCR. CCK8 assay was performed to evaluate the viability of CCRF-CEM cells. Flow cytometry was used to analyzed the apoptosis of CCRF-CEM cells. The protein expression of BACH2 and cyclin D3 in the CCRF-CEM cells was observed by immunofluorescence staining. The protein expression of cyclin D3, Bcl-2 and caspase-3 was determined by Western blot. RESULTS The mRNA expression of BACH2 in CCRF-CEM cells was significantly lower than that in PBMC （P<0.05）. Compared with control group, BACH2 over-expression significantly suppressed the viability,increased the apoptotic rate and caspase-3 expression, and decreased the expression of cyclin D3 and Bcl-2 in CCRF-CEM cells （P<0.05）. CONCLUSION BACH2 expression is decreased in T lymphocytes of human acute lymphoblastic leukemia. Over-expression of BACH2 inhibited the viability of human acute lymphoblastic leukemia T lymphocyte and induced apoptosis.     

Effect of CHOP expression knock-down on apoptosis of renal tubular epithelial cells

AIM:To study the effect of C/EBP homologous protein (CHOP) on the apoptosis of renal tubular epithelial HK2 cells. METHODS:The serum mRNA levels of CHOP in the patients with acute kidney injury and healthy controls were detected by qPCR. In vitro, renal tubular epithelial HK2 cells were divided into control group, negative group (transfected with negative control siRNA), si-CHOP group (transfected with CHOP siRNA), and induced by transforming growth factor-β1 (TGF-β1). The viability of the cells was measured by MTT assay, and the apoptotic rate was analyzed by flow cytometry. The protein levels of nuclear antigen Ki-67, proliferating cell nuclear antigen (PCNA), caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with the healthy controls, the serum mRNA levels of CHOP in the patients with acute kidney injury were increased significantly (P<0.05). Transfection with CHOP siRNA significantly decreased the expression of CHOP in the renal tubular epithelial HK2 cells (P<0.05). Knock-down of CHOP expression by siRNA significantly increased the viability of renal tubular epithelial HK2 cells (P<0.05), decreased the apoptotic rate (P<0.05), increased the expression of Ki-67 and PCNA (P<0.05), and down-regulated the protein level of cleaved caspase-3 (P<0.05). CONCLUSION:The serum mRNA levels of CHOP were increased in the patients with acute kidney injury. Knock-down of CHOP expression inhibits the apoptosis of renal tubular epithelial cells by regulating the expression of proliferation-and apoptosis-related proteins.     

Caspase-8 small hairpin RNA attenuates apoptosis of human bone marrow mesenchymal stem cells under conditions of serum deprivation and hypoxia

AIM：To investigate the effect of caspase-8 small hairpin RNA (shRNA) on attenuating apoptosis of human mesenchymal stem cells (hMSCs). METHODS：Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed. Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR. The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid, which was linearized and transfected into HEK293 cells for packaging and amplification of the recombinant adenovirus rAd-Cap8 shRNA. The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting. Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hMSCs under the conditions of serum deprivation and hypoxia. The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR. RESULTS：The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR. The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant adenovirus (rAd)-Cap8 shRNA successfully. rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 expression in hMSCs. Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the apoptotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia, with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2. CONCLUSION：Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia.     

Determination of T cell cycle and the expression of bcl- 2 in asthmatic mice and its significance

AIM: To investigate the changes of T cell cycle, the expression of bcl- 2 in allergic asthmatic mice and the effects of dexamethasone on them. METHODS: An animal model with asthma was established by means of ovalbumin sensitizing-challenging. CD3 expression in spleen and lymphocytes in bronchoalveolar lavage fluid (BALF), T cell cycle and Bcl-2 expression in spleen were detected by flow cytometry. RESULTS: In BALF lymphocytes and spleen lymphocytes, CD3 expression rate in the asthmatic group was significantly higher than that of control group. In BALF lymphocytes, CD3 expression rate in the asthma plus dexamethasone group was significantly lower than that of the asthmatic group. However, in spleen lymphocytes, CD3 expression rate in the asthma plus dexamethasone group was significantly higher than that of the asthmatic group. In spleen lymphocytes, the cell count in S phase, G₂+M phase and apoptosis rate of T cell from the asthmatic group were significantly higher than that from the control group. Cell count in S phase, G₂+M phase and apoptosis rate of T cell from the asthmaplus dexamethasone group was significantly lower than that from the asthmatic group. The Bcl-2 expression rate of T cell from the asthmatic group was significantly higher than that from the control group. CONCLUSIONS: In the allergic asthmatic mice model, T cell count, proliferation and activation of T cells, apoptosis rate of T cells in spleen lymphocytes increase, meanwhile bcl- 2 expression also increases significantly. There was no significant effect of dexamethasone on the bcl- 2 expression. The therapeutic effects of dexamethasone on asthma may be not due to the inhibition of the bcl- 2 expression in T cells.     

Expression of SCD-1 in cervical carcinoma and effect of its down-regulation on proliferation and apoptosis of cervical carcinoma cells

AIM:To examine the expression of stearoyl-CoA desaturase-1 (SCD-1) in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki, and to investigate the effect of down-regulation of SCD-1 on the proliferation and apoptosis of cervical carcinoma cells. METHODS:The expression of SCD-1 was detected by Western blotting in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki. SCD-1 siRNA and control siRNA were utilized to transfect CaSki cells by Lipofectamine 2000, and SCD-1 protein level was determined by Western blotting after transfection. Furthermore, CCK-8 and flow cytometry were utilized to investigate the changes of cell proliferation and apoptosis after transfection with SCD-1 siRNA in CaSki cells. Subsequently, the activities of caspase-3 and caspase-9 were analyzed by Caspase-Glo3/7 and 9 detection kit after transfection with SCD-1 siRNA in CaSki cells. Finally, the protein expression of Bcl-2 and Bax was detected by Western blotting. RESULTS:The protein expression of SCD-1 in cervical squamous cell carcinoma tissues was significantly higher than that in normal cervical tissues, and the protein expression of SCD-1 in the 3 cervical carcinoma cell lines was obviously higher than that in normal cervical tissues, in which CaSki cells displayed the highest SCD-1 protein level. In addition, the protein expression of SCD-1 in SCD-1 siRNA group was significantly lower than that in untreated group and control siRNA group. Compared with untreated group and control siRNA group, the proliferation of CaSki cells was markedly inhibited in SCD-1 siRNA group. Early apoptotic rate in SCD-1 siRNA group was evidently higher than that in untreated group and control siRNA group. The activities of caspase-3 and caspase-9, and the level of Bax protein were significantly elevated, and the protein level of Bcl-2 was obviously reduced after transfection with SCD-1 siRNA in CaSki cells. CONCLUSION: SCD-1 may play an important role in the occurrence and development of cervical carcinoma, and its down-regulation, which mediates cell proliferation inhibition and apoptosis, may be tightly associated with the activities of caspase-3 and caspase-9, and the protein expression of Bcl-2 and Bax.     

Effects of the grub extract on apoptosis of MCF-7 human breast cancer cell line

AIM: To investigate the apoptotic pathway of MCF-7 breast cancer induced by the grub extract in vitro.METHODS: MTT assay was used to determine the effect of the grub extract on proliferation of MCF-7 human breast cancer cell line and cell toxicity. Morphological changes of the apoptosis in cancer cells were observed by HE staining through invert microscope, light microscope, AO/EB double fluorescent staining under fluorescent microscope. FCM was used to assay the change of apoptotic rate. The expression of Bcl-2, Fas, caspase-9, caspase-3 in apoptotic pathway was detected with immunocytochemical method before and after exposure to the grub extract, and the effect of that on apoptotic pathway was explored.RESULTS: (1) The MTT test showed that the growth of MCF-7 human breast cancer cell line was significantly inhibited by the grub extract in dose and time dependent manners. The inhibitory rate in exposure group was significantly different from that in control group (P<0.01). (2) Morphological changes of apoptosis including nuclear condensation, fragment and apoptosis body formation were observed by invert microscope. (3) The MCF-7 human breast cancer cells in experimental group by HE staining showed nuclear condensation and blue-black, cytoplasm slight red, nuclear chromatin condensation and fragment shape, apoptosis body formations. (4) Apoptosis in the experimental group was observed by AO/EB double fluorescent staining under fluorescent microscope. (5) FCM assay indicated that apoptotic rate increased significantly in time dependent manner in experimental group. (6) The expression of Bcl-2 was down-regulated, while that of Fas, caspase-3, caspase-9 was up-regulated, compared with control group (P<0.01).CONCLUSION: (1) The proliferation of MCF-7 human breast cancer cell line can be inhibited significantly by the grub extract in vitro. (2)The mechanism of effect of the grub extract on MCF-7 human breast cancer cell line might be mediated by down-regulation of Bcl-2 and up-regulation of Fas, caspase-3, caspase-9. This type of apoptosis starting and performing is through death receptor pathway and mitochondrial pathway.     

Berberine promotes anti-proliferative effects of doxorubicin in T24 bladder cancer cells by enhancing apoptosis

AIM: To observe the effect of berberine (Ber) on doxorubicin (DOX)-induced apoptosis in bladder cancer T24 cells. METHODS: The cells were exposed to DOX in the presence or absence of different concentrations of Ber. The viability of the cells was determined by CCK-8 assay. The apoptosis was measured by Hoechst 33258 staining and the protein levels of cleaved caspase-3, cleaved caspase-9, Bcl-2 and Bax were detected by Western blotting.RESULTS: Ber enhanced the inhibitory effect of DOX on the viability of T24 cells and promoted DOX-induced apoptosis in T24 cells. DOX increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, all of which were enhanced by treatment with Ber. In contrast, Ber exposure further decreased the expression of Bcl-2 in DOX-treated T24 cells.CONCLUSION: Ber enhances the anti-proliferative effects of DOX through promoting apoptosis in bladder cancer cells.     

Effects of tetrahydroxystilbene-2-O-β-D-glucoside on apoptosis and expression of bcl-2/bax/caspase-3 in HUVECs treated with homocysteine

AIM：To explore the effects of tetrahydroxystilbene-2-O-β-D-glucoside (TSG) from Polygonum multiflorum on the apoptosis and the mRNA expression of bcl-2, bax and caspase-3 in human umbilical vein endothelial cells (HUVECs) treated with homocysteine (Hcy). METHODS：Cultured HUVECs were treated with Hcy (3 mmol/L) to establish a Hcy-damaged model. HUVECs in TSG treated groups were pre-incubated with TSG at concentrations of 1 μmol/L and 10 μmol/L for 2 h before treated with Hcy. Cell nuclear damage was detected by Hoechst 33342 staining. Cell apoptosis was determined by flow cytometry. The mRNA expression of bcl-2, bax and caspase-3 was measured by real-time fluorescence quantitative RT-PCR. RESULTS： After treatment with Hcy at concentration of 3 mmol/L, the nuclear damage and apoptotic rate of HUVECs were higher than that in normal group. The expression of bcl-2 was lower, and the expression of Bax and caspase-3 was higher than that in normal group. On the other hand, pre-incubation with TSG at concentrations of 1 μmol/L and 10 μmol/L decreased the nuclear damage and cell apoptosis, increased the expression of bcl-2, and decreased the expression of bax and caspase-3 as compared with the cells only treated with Hcy. CONCLUSION：TSG reduces the apoptosis of HUVECs induced by Hcy, and the mechanism might be associated with regulating the expression of bcl-2, bax and caspase-3.     

Focal adhesion kinase antisense oligodeoxynucleotide promotes apoptosis in human pulmonary artery smooth muscle cells

AIM: To investigate whether focal adhesion kinase (FAK) takes part in the intracellular signaling pathway involved in apoptosis of human pulmonary artery smooth muscle cells (HPASMCs). METHODS: Cultured HPASMCs stimulated by fibronection (40 mg/L) were passively transfected with antisense-FAK oligodeoxynucleotides (ODNs). Expression of FAK and caspase-3 proteins was detected by immunoprecipitation and Western blotting. In addition, apoptosis were measured by TUNEL. RESULTS: The protein expression of FAK in HPASMCs decreased and caspase-3 expression upregulated in HPASMCs in antisense-FAK ODNs group. At the same time, antisense-FAK ODNs transfection increased the rate of cell apoptosis. CONCLUSION: These results suggest that FAK may be related with the apoptosis of HPASMCs. Antisense-FAK ODNs inhibit HPASMC proliferation and may facilitate their apoptosis via caspase-3 signaling pathway.     

Effect of HIPK2 gene on viability,apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation

AIM: To investigate the effect of homeodomain-interacting protein kinase 2 (HIPK2) on the viabi-lity, apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation (H/R). METHODS: HIPK2 small interfering RNA (siRNA) was transfected into NRK-52E cells by Lipofectamine^TM 2000, and normal control group (control group) and negative control group (HIPK2-NC group) were set up. After H/R, the cell viability was measured by CCK-8 assay, the apoptotic rate and Ca²⁺ fluorescence intensity were analyzed by flow cytometry, and the protein levels of Ki67, cleaved caspase-3, caspase-12, Bcl-2, Bax, p-JAK2 and p-STAT3 were determined by Western blot. RESULTS: Compared with control group, the protein expression of HIPK2 in the NRK-52E cells was significantly decreased after transfection with HIPK2 siRNA (P<0.05). Compared with control group, the cell viability and the protein expression of Ki67 and Bcl-2 in H/R group were also significantly decreased, and the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly increased (P<0.05). Compared with H/R group, the cell viability and the protein expression of Ki67 and Bcl-2 in HIPK2-siRNA+H/R group were significantly increased, while the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly decreased (P<0.05). CONCLUSION: Inhibition of HIPK2 gene expression promotes H/R-induced growth of NRK-52E renal tubular epithelial cells, and reduces the apoptosis. The mechanism is related to down-regulating the JAK2/STAT3 signaling pathway.     

Roles of microRNA-29a in expression of Bcl-2 and Mcl-1 in rat cardiomyocytes

AIM: To investigate the regulatory mechanisms of microRNA-29a (miR-29a) on the expression of Bcl-2 and Mcl-1 in rat cardiomyocytes (CM cells). METHODS: The CM cells were isolated from the hearts of newborn rats and transfected with miR-29a mimic (100 nmol/L) by Lipofectamine RNAiMAX. The expression of Bcl-2 and Mcl-1 at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and Western blotting. The luciferase assay was performed in HEK293T cells and CM cells, which were co-transfected with plasmid DNA and miRNA using Lipofectamine 2000. RESULTS: Transfection of miR-29a mimics significantly reduced the expression levels of Bcl-2 and Mcl-1 in CM cells as compared with the control cells (P<0.05). In addition, HEK293T cells co-transfected with miR-29a mimic and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid significantly reduced the luciferase activity as compared with control group (P<0.05). While CM cells transfected with miR-29a inhibitor and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid in succession, the luciferase activity was increased inversely (P<0.05). CONCLUSION: miR-29a may regulate apoptosis by targeting the bcl-2 and mcl-1 genes.