Effects of DARPP-32 on the drug sensitivity of SH-SY5Y cells

AIM: To investigate the effects of DARPP-32 on the drug sensitivity of human neuroblastoma cell line SH-SY5Y. METHODS: The plasmid containing cDNA of DARPP-32 gene and the small interfering RNA eukaryotic expression vector specific to human DARPP-32 gene were constructed and transfected into SH-SY5Y cells. Stable transfectants were identified by RT-PCR and Western blotting analysis. MTT assay and flow cytometry assay were used to investigate the effects of DARPP-32 on the drug sensitivity and the drug accumulation in cell models. The expressions of Bcl-2，Bax, P-gp and MRP were analyzed by RT-PCR and Western blotting. RESULTS: The stable clones with increased or decreased DARPP-32 expression were successfully established. Up-regulation of DARPP-32 significantly enhanced the drug sensitivity and the drug accumulation of SH-SY5Y cells due to down-regulation of P-gp and Bcl-2 expressions. Down-regulation of DARPP-32 significantly reduced the drug sensitivity of SH-SY5Y cells and decreased drug accumulation in SH-SY5Y cells. CONCLUSION: DARPP-32 might mediate the sensitivity of SH-SY5Y cells to conventional chemotherapeutic agents.     

Effects of TSG101 siRNA on the growth and drug sensitivity of SH-SY5Y cells

AIM:To investigate the effects of TSG101 siRNA on the growth and drug sensitivity of human neuroblastoma cell line SH-SY5Y.METHODS:The small interfering RNA eukaryotic expression vector specific to human TSG101 gene was constructed by gene recombination,then transfected into SH-SY5Y cells.Stable transfectants were obtained by G418 screening and further identified by RT-PCR and Western blotting analysis.The growth curve was made using MTT assay.Cell cycle distribution of the transfected cells was studied by flow cytometry and the proliferative indexes were calculated.The apoptosis after CDDP treatment was detected by DNA ladder and Annexin V/propidium iodide binding analyses.The expression of Bcl-2,Bax,P-gp and MRP were analyzed by Western blotting.RESULTS:mU6pro-TSG101 siRNA was successfully constructed and transfected into SH-SY5Y cells.As detected by MTT and flow cytometry,down-regulation of TSG101 significantly suppressed the proliferation of SH-SY5Y cells with a G1 cell cycle arrest,compared with that in control (P<0.05).As detected by DNA ladder and Annexin V/propidium iodide binding analyses,down-regulation of TSG101 significantly enhanced the sensitivity of SH-SY5Y cells to CDDP-induced apoptosis,compared with that in control (P<0.05).The expression of P-gp and Bcl-2 in transfected cells were decreased as compared with that in the control,while MRP and Bax were not.CONCLUSIONS:Down-regulation of TSG101 suppresses the proliferation of SH-SY5Y cells,and enhances the sensitivity of SH-SY5Y cells to conventional chemotherapeutic agents to a degree,suggesting TSG101 may be useful for gene therapy in the future.     

Tanshinone IIA inhibits doxorubicin resistance in gastric cancer cells

AIM: To investigate the inhibitory effect and the specific mechanism of tanshinone IIA on doxorubicin (DOX)-resistant gastric cancer cells. METHODS: The sensitivity of gastric cancer cells lines to DOX was determined by MTT assay. DOX-resistant gastric cancer cell lines were established by step selection with increasing concentrations of DOX. The cell cycle arrest, apoptosis and autophagy related-markers were analyzed by flow cytometry and Western blot. The expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multi-drug resistance-associated protein 1 (MRP-1) was determined by RT-qPCR and Western blot. RESULTS: DOX-sensitive cell lines SNU-719 and SNU-601 as well as the cell lines relatively resistant to DOX including SNU-638, SNU-668, SNU-216 and SNU-620 were identified according to the IC₅₀ values of DOX for different cell lines. Two DOX-resistant cell lines SNU-719R and SNU-601R were also established. Tanshinone IIA inhibited the expression of MRP-1 in DOX-resistant cell lines. Compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in cancer cells decreased the G₂/M phase cell number, increased the protein expression of p21, decreased the protein expressions of cyclin B1 and cyclin-dependent kinase 1 (CDK1) in the SNU-719 R cells and SNU-620 cells. In addition, compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in the cancer cells increased the protein expressions of p53, Bax and LC3B-II, decreased the protein expression of Bcl-2 and p62 (P<0.05). CONCLUSION: Tanshinone IIA is an effective drug in the inhibition of DOX resistance in gastric cancer.     

Effect of sorcin expression on sensitivity of human glioma cells to cisplatin

AIM：To investigate the effect of sorcin expression on the sensitivity of human glioma cells to cisplatin. METHODS：pSilencerTM 3.1-H1-sorcin siRNA recombinant plasmid was constructed, and transfected into human glioma U251 cells. RT-PCR and Western blotting were used to analyze the expression of sorcin at mRNA and protein levels after transfection. The viability of U251 cells was measured by MTT assay. The protein expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) in U251 cells was detected by Western blotting. RESULTS：The plasmid pSilencerTM 3.1-H1-sorcin siRNA was successfully constructed, and was confirmed by restriction enzyme digestion and sequence analysis. The expression of sorcin at mRNA and protein levels was significantly decreased after sorcin siRNA was transfected into U251 cells (P<0.05). Inhibition of sorcin expression significantly decreased the viability of U251 cells treated with cisplatin (P<0.05), and the expression of P-gp and MRP1 proteins was also inhibited (P<0.05). CONCLUSION：Inhibition of sorcin expression increases the sensitivity of U251 cells to cisplatin by decreasing the expression of resistance-related proteins P-gp and MRP1, suggesting that sorcin may be associated with the resistance of glioma cells to cisplatin.     

Overexpression of α-synuclein in SH-SY5Y cells in vitro leads to oxidative stress

AIM: To observe the influence of overexpression of α-synuclein on the cultured SH-SY5Y cells. METHODS: The plasmid of α-synuclein-pcDNA3 was transfected into SH-SY5Y cells with LipofectAMINE. The expression of α-synuclein was determined by anti-α-synuclein immunocytochemistry. The intracellular reactive oxygen species was determined with 2', 7'-dichlorofluorescein diacetate (DCF-DA) by using a FACSCAN flow cytometer and fluorescent microscope. The intracellular content of reduced GSH was detected with glutathione assay kit by spectrophotometer. RESULTS: The α-synuclein was expressed in cultured SH-SY5Y cells transfected with the plasmid of α-synuclein-pcDNA3. The DCF loading analysis and the intracellular level of reduced GSH suggested that the transfected cells were under oxidative stress. CONCLUSION: Overexpression and accumulation of α-synuclein in SH-SY5Y cells increase intracellular reactive oxygen species levels, it is suggested therefore that the α-synuclein does play an important role in the pathogenesis of Parkinson's disease.     

Effects of down-regulation of AEG-1 expression on cell cycle and invasion of human cervical carcinoma cells

AIM: To investigate the effects of down-regulation of astrocyte elevated gene-1 (AEG-1) expression on cell cycle and invasion of human cervical carcinoma SiHa cells.METHODS: The protein expression of AEG-1 was detected by Western blotting in normal cervical tissues, cervical squamous cell carcinoma tissues, HeLa cells, SiHa cells and CaSki cells. Control siRNA or AEG-1 siRNA was transfected into SiHa cells, and the protein expression of AEG-1 in SiHa cells was detected by Western blotting. The changes of cell cycle distribution and cell invasion were determined by flow cytometry and Boyden chamber, respectively. The protein levels of cyclin D1, cyclin-dependent kinase 2(CDK2) and matrix metalloproteinase-9 (MMP-9) were analyzed by Western blotting.RESULTS: The protein expression of AEG-1 in cervical squamous cell carcinoma tissues was significantly higher than that in normal cervical tissues (P<0.05). Meanwhile, the protein expression of AEG-1 in the 3 cervical carcinoma cell lines was obviously higher than that in normal cervical tissues, in which SiHa cells displayed the highest AEG-1 protein level (P<0.05). In addition, AEG-1 siRNA effectively down-regulated the protein expression of AEG-1 in SiHa cells, which led to increase the percentage at G0/G1 phase and reduced the invasion of SiHa cells. Furthermore, the protein levels of cyclin D1, CDK2 and MMP-9 in AEG-1 siRNA group were markedly lower than those in non-treatment group and control siRNA group (P<0.05).CONCLUSION: Over-expression of AEG-1 may be closely associated with the occurrence and development of cervical carcinoma, and the AEG-1 down-regulation-mediated cell cycle arrest and attenuation of invasion may be tightly related to the down-regulations of cyclin D1, CDK2 and MMP-9 at protein levels.     

Effect of ClC-3 siRNA on cell cycle of HeLa cells

AIM: To investigate the roles of ClC-3 chloride channels in the regulation of cell cycle and the relationship between ClC-3 chloride channels and the cell cycle regulators, such as cyclin D1, cyclin-dependent kinase (CDK)4, CDK6, P21 and P27 in the HeLa cells.METHODS: ClC-3 genes were silenced by the siRNA technique in the HeLa cells. The transfection efficiency of ClC-3 siRNA was detected by real-time PCR. The cell cycle distribution was analyzed by the flow cytometry. The protein expression of ClC-3, P21, P27, CDK4, CDK6 and cyclin D1 was determined by Western blot.RESULTS: ClC-3 was knocked down by ClC-3 siRNA in the HeLa cells. Transfection of the cells with ClC-3 siRNA arrested the cells at G₀/G₁ phases, decreased the expression of cyclin D1, CDK4 and CDK6, and increased the expression of P21 and P27.CONCLUSION: ClC-3 plays an important role in the cell cycle of HeLa cells through the G₁-S transition point. ClC-3 may regulate the cell cycle progression by up-regulation of cyclin D1, CDK4 and CDK6 expression and/or by down-regulation of P21 and P27 expression.     

miR-24-3p targeting KLF6 gene affects viability and apoptosis of esophageal cancer cells via IL-6/STAT3 signaling pathway

AIM: To investigate the effect of microRNA-24-3p (miR-24-3p) on the viability and apoptosis of esophageal cancer cells. METHODS: The expression of miR-24-3p and KLF6 mRNA in the esophageal cancer cells TE11, Eca109 and EC9706 were detected by RT-qPCR. The protein expression of KLF6 was determined by Western blot. EC9706 cells were transfected with anti-miR-24-3p and KLF6 siRNA. The cell viability was measured by MTT assay, the apoptotic rate was analyzed by flow cytometry, and the proliferation, apoptosis and IL-6/STAT3 signaling pathways related proteins were determined by Western blot. The level of IL-6 was measured by ELISA. The dual luciferase reporter gene assay was used to verify the relationship between miR-24-3p and KLF6. RESULTS: The levels of miR-24-3p were up-regulated in the esophageal cancer cells TE11, Eca109 and EC9706 (P < 0.05), and the expression of KLF6 at mRNA and protein levels was down-regulated (P < 0.05). Knock-down of miR-24-3p expression inhibited the cell viability, induced apoptosis, and inhibited the protein levels of CDK4, cyclin D1, CDC25A, p-STAT3, Bcl-2 and IL-6, and promoted the protein expression of caspase-3 and Bax in EC9706 cells. CONCLUSION: miR-24-3p targets KLF6 gene to affect the viability and apoptosis of esophageal cancer cells by regulating IL-6/STAT3 signaling pathway.     

Stable reversal of multidrug resistance of colon cancer cells by RNAi targeting MDR1 gene

AIM: To investigate the reversal effect of RNAi targeting MDR1 gene on MDR1/P-gp-dependent multidrug resistance of colon cancer cells. METHODS: Plasmid vectors pSilencer-#4029 encoding #4029 MDR1 siRNA and pSilencer-#4123 encoding #4123 MDR1 siRNA were constructed, respectively. Plasmid vectors were transfected into COLO 320DM cell line, a colon cancer multidrug resistance cell line. Clone cells were screened by G418 and identified by real time RT-PCR and Western blotting. Cell viabilities were measured by MTT assay. Cell cycle analysis was performed with flow cytometry. Intracellular adriamycin accumulations were measured by flow cytometry. RESULTS: The expression of MDR1 mRNA and P-gp in positive clone cells (clone #4029 and clone #4123) were all inhibited. IC50 of adriamycin and vincristine for COLO 320DM parent cells were 9.616 μmol/L and 0.358 μmol/L, respectively. However, for clone #4029, they were decreased to 1.094 μmol/L and 0.023 μmol/L, respectively (P<0.01). For clone #4123, they were decreased to 0.780 μmol/L and 0.035 μmol/L, respectively (P<0.01). The PI/AI values of COLO 320DM parent cells treated with adriamycin and vincristine were 5.68 and 9.59, respectively. However, they were decreased to 2.74 and 3.59 respectively for clone #4029 (P<0.01). They also decreased to 2.75 and 3.24 respectively for clone #4123 (P<0.01). The intracellular adriamycin accumulation concentrations of COLO 320DM parent cells treated with 10 μmol/L adriamycin were 27.92. However, they were increased to 187.24 and 215.57 for clone #4029 and clone #4123, respectively (P<0.01). CONCLUSION: Stable transfection of plasmid vector encoding MDR1 siRNA stably reverses MDR1/P-gp-dependent multidrug resistance of colon cancer cells.     

Glucosylceramide synthase upregulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway in leukemia multidrug-resis-tant cell line

AIM: To investigate whether glucosylceramide synthase (GCS) regulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway, thus enhancing drug resistance of K562/A02 human leukemia multidrug resistant cell line. METHODS: siRNA targeting GCS was transfected into K562/A02 cells. Bcl-2, p-ERK and total ERK expression at mRNA and protein levels after GCS knockdown were detected by real-time PCR and Western blotting. After exposed to MEK-ERK pathway inhibitor U0126, the expression of Bcl-2 at mRNA and protein levels also was analyzed by real-time PCR and Western blotting, respectively. The viability of the cells was evaluated by CCK-8 assay. RESULTS: The expression of GCS and Bcl-2, as well as MEK/ERK signaling were significantly inhibited in K562/A02 cells by GCS siRNA transfection compared with negative control group. Inactivation of MEK/ERK signaling due to U0126 treatment decreased Bcl-2 mRNA and protein levels in a concentration-dependent manner, and sensitized K562/A02 cells to adriamycin. CONCLUSION: GCS may affect the expression of apoptosis-related gene bcl-2 by MEK/ERK signaling pathway, thus regulating multidrug resistance of human leukemia K562/A02 cells.     

Dominant negative human epidermal growth factor receptor induces G0/G1 arrest in human gastric cancer cells

AIM：To explore the effect of dominant negative epidermal growth factor receptor (DNEGFR) on the cell cycle of human gastric cancer cells. METHODS：Two human gastric cancer cell lines were used in the study. The cells were divided into 6 groups, including untreated SGC-7901 cells (US group), SGC-7901 cells stably transfected with pEGFP-N1 (ES group), SGC-7901 cells stably transfected with pEGFPN1-DNEGFR (DS group), untreated NCI-N87 cells (UN group), NCI-N87 cells stably transfected with pEGFP-N1 (EN group), and NCI-N87 cells stably transfected with pEGFPN1-DNEGFR (DN group). The cell cycle was determined by flow cytometry. The protein levels of cyclin-dependent kinase 2 (CDK2), cyclin D1, phosphorylated glycogen synthase kinase 3 beta at Ser9 ［p-GSK-3β (Ser9)］, p21 and p27 were detected by Western blotting. RESULTS：Transfection of the human gastric cancer cells with pEGFPN1-DNEGFR led to G0/G1 arrest, and down-regulated CDK2, cyclin D1, p-GSK-3β (Ser9) and up-regulated p21 and p27 as well. CONCLUSION：DNEGFR down-regulates cyclin D1 by activating GSK-3β, down-regulates CDK2, and up-regulates p21 and p27, which induce G0/G1 arrest in human gastric cancer cells in the end.     

Effects of edaravone on high glucose-induced apoptosis in SH-SY5Y cells via regulating microRNA-25

AIM: To observe the effects of edaravone on high glucose-induced apoptosis of SH-SY5Y cells and its potential mechanism. METHODS: The SH-SY5Y cells were cultured in the DMEM medium with 100 mmol/L glucose and 100 μmol/L edaravone for 24 h. The viability of the SH-SY5Y cells was detected by MTT assay. The levels of ROS in the cells were determined by DCFH-DA fluorescent probing. The apoptotic rates of the cells were analyzed by flow cytometry. The protein expression of Bax and Bcl-2 in the cells were detected by Western blot. The expression levels of micro-RNA-25 (miR-25) were determined by real-time PCR. To further clarify the target sites of edaravone on inhibiting apoptosis induced by high glucose, miR-25 inhibitor was applied to the SH-SY5Y cells and the activity of caspase-3 was measured.RESULTS: Compared with control group, the cell viability was decreased significantly in model group, and the ROS level was increased significantly. The protein expression of Bax was up-regulated significantly, while the expression levels of Bcl-2 and miR-25 were significantly down-regulated. Compared with model group, the cell viability was increased significantly in edaravone group. The ROS level was decreased significantly. Meanwhile, the expression of Bax was down-regulated, while the expression of Bcl-2 and miR-25 was up-regulated with statistical significance. The caspase-3 activity of the cells incubated with 100 mmol/L glucose and miR-25 inhibitor was increased. However, no alteration of caspase-3 activity with edaravone added simultaneously was observed. CONCLUSION: Edaravone inhibits the apoptosis of SH-SY5Y cells induced by high glucose with the potential target site of miR-25.     

Glycogen synthase kinase-3 beta overexpression enhances tau protein phosphorylation and attenuates tubulin acetylation in SH-SY5Y cells

AIM: To construct GSK3β-overexpressed SH-SY5Y cells and to observe the effects of GSK3β-overexpression on tau protein phosphorylation and tubulin acetylation in SH-SY5Y engineered cells. METHODS: The cDNA of GSK3β construct was subcloned into mammalian expression vector pBudCE4.1. The integrity of the GSK3β construct was confirmed by sequence analysis. GSK3β was transiently transfected into SH-SY5Y cells using Lipofectamine2000. Western blotting was used to measured protein levels of GSK3β and phosphorylating GSK3β, as well as, the total tau and phosphorylated tau protein and acetylated tubulin. RESULTS: 36 h after transfection, the levels of GSK3β and phosphorylating GSK3β in SH-SY5Y cells were significantly increased compared with non-transfection group and vector group. After 48 h, the levels of phosphorylated tau protein (Ser199/202, Thr231 and Thr205 residues) but not total tau protein were markedly increased in GSK3β-overexpressed SH-SY5Y cells. In addition, the level of acetylated tubulin was lower than that in non- transfection group and vector group. CONCLUSION: The over-expression of GSK3β in SH-SY5Y cells results in robust increases in tau protein phosphorylation at Ser199/202, Thr231 and Thr205 residues, and decreases in tubulin acetylation.     

Effect of PRL-2 gene transfection on proliferation and cell cycle in hepatocellular cell line

AIM: To observe the changes of proliferation and cell cycle after PRL-2 gene effectively expressed in human hepatocellular cell line.METHODS: The PRL-2 vector was transfected into CL1 cell with lipofectamine reagent,the stable expression clones were screened by G418.The expression of PRL-2 mRNA was detected by real-time PCR.The expressive protein was identified by Western blotting.The subcellular localization was demonstrated by immunochemistry.The cell cycle was measured by flow cytometry.The population doubling time (TD) was analyzed by MTT assay.The expressions of cyclin A,cyclin D1,cyclin E,p16,p21Waf1 and p27Kip1 were detected by Western blotting.The p21Waf1 mRNA was determined by real- time PCR.RESULTS: The full length ORF of PRL-2 gene was inserted into the vector pcDNA3.1 (+),transfected into CL1 cells,and expressed successfully.Real-time PCR showed stable expression of PRL-2 mRNA.Western blotting confirmed the overexpression of PRL-2 protein.The subcellular localization of PRL-2 was in the plasmid.The proportion of cells in S-phase was increased.The population doubling time was reduced (P<0.01),a significant decrease was observed both in the mRNA and the protein expression of the p21Waf1 in comparison with untransfected or vector- transfected control cells (P<0.05).The expressions of cyclin D1,cyclin E,cyclinA,p16 and p27Kip1 were not appreciably different between the control and PRL-transfected cell lines.CONCLUSION: Eukaryocytic expression vector of PRL-2 has been successfully constructed,which shows stable and effective expression in CL1 cell line.PRL -2 increases cell proliferation by stimulating progression from G1 into S phase,which is primarily associated with decreased p21Waf1.     

Prevention and treatment of AD by over-expression of neuroglobin via activating PI3K/Akt signaling pathway

AIM: To study the neuroprotective roles of neuroglobin (NGB) over-expression in the SH-SY5Y cells transfected with pAPPswe.METHODS: The plasmid pEGFP-NGB was successfully constructed and transfected into the SH-SY5Y cells, which were pretreated with pAPPswe. MTT assay was applied to detect the effect of NGB over-expression on the cell survival rates. JC-1 staining was used to detect the level of mitochondrial transmembrane potential. The cell apoptosis was analyzed by flow cytometry. The effects of NGB over-expression on the protein level of p-Akt, Akt and caspase-3/9 were determined by Western blotting. The generation of Aβ42 in the cells was measured by ELISA.RESULTS: The cell survival rate was remarkably increased after transfection with NGB compared with control group and empty plasmid group (P<0.05). The over-expression of NGB significantly inhibited the decrease in mitochondrial membrane potential induced by pAPPswe. The over-expression of NGB inhibited the apoptosis of the cells. Furthermore, over-expression of NGB not only inhibited the expression of caspase-3 and caspase-9, but also induced the production of p-Akt, which was prevented by LY294002, an inhibitor of PI3K/Akt. The generation of Aβ42 was inhibited in the cells with the over-expression of NGB. CONCLUSION: Over-expression of NGB significantly inhibits the SH-SY5Y cell injuries induced by pAPPswe and inhibits the expression of caspase-3/9, which is tightly related with cell apoptosis. Furthermore, the neuroprotective roles of NGB may be via activating PI3K/Akt signaling pathway.     

Protective effect of NAD(P)H:quinone oxidoreductase 1 on dopaminergic cells

AIM: To determine the influence of NAD(P)H:quinone oxidoreductase 1 (NQO1) on dopamine-induced toxicity in dopaminergic cells.METHODS: MTT assay was used to determine the toxic curve of dopamine in SH-SY5Y cells. Lipofection was applied to transfect SH-SY5Y cells with an NQO1 expression plasmid. The endogenous and transfected NQO1 expression was detected by immunofluorescence staining and Western blotting. The content of cellular quinone protein was measured by nitroblue tetrazolium (NBT) method. RESULTS: Dopamine reduced SH-SY5Y cell proliferation in a dose-dependent manner, which was correlated with an increase in the content of quinone protein. Increased expression of NQO1 by transient transfection or by phase II enzyme inducer sulforaphane treatment alleviated dopamine-induced toxicity and reduced the content of cellular quinone protein. CONCLUSION: Increased NQO1 expression protects SH-SY5Y cells against cytotoxicity caused by dopamine.     

Effect of down-regulation of X-box binding protein 1 gene expression on viability and apoptosis of glioma cells

AIM: To investigate the effect of down-regulation of X-box binding protein 1 (XBP1) expression on the viability and apoptosis of glioma cells. METHODS: The mRNA expression of XBP1 in the glioma tissues was detected by qPCR. Small interfering RNA (siRNA) interfering with XBP1 expression (XBP1-siRNA) was transfected into human brain glioma U251 cells. At the same time, control group (the cells without special treatment) and negative control (NC-siRNA) group (transfected with siRNA without any interference) were set up. The mRNA expression of XBP1 in the 3 groups 48 h after transfection was detected by qPCR. The protein levels of XBP1, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1 (cyclin D1), phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt (p-Akt) were determined by Western blot. The cell viability was measured by CCK-8 assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The expression level of XBP1 in the glioma tissues was significantly higher than that in the tumor adjacent tissues (P<0.05). The XBP1 expression at mRNA and protein levels was significantly decreased in the cells transfected with XBP1-siRNA (P<0.05). No statistically significant difference of the cell viability, cell cycle, apoptotic rate and the protein levels of PCNA, Bcl-2, Bax, cyclin D1, PI3K and p-Akt between NC-siRNA group and control group was observed. Compared with control group, the cell viability, S-phase cells and the protein levels of PCNA, Bcl-2, cyclin D1, PI3K, and p-Akt in XBP1-siRNA group were decreased significantly, and the apoptotic rate, G₀/G₁-phase cells and Bax protein expression were significantly increased (P<0.05). CONCLUSION: Down-regulation of XBP1 gene expression in brain glioma cells reduces the viability of cancer cells, blocks the cells in G₁ phase and promote apoptosis. The mechanism is related to the inhibition of PI3K/Akt signaling pathway.     

Protective effect of osthole on SH-SY5Y cells transfected with APP595/596 gene

AIM: To explore the protective effect of osthole on the SH-SY5Y cells transfected with APP595/596 gene, and to investigate the molecular mechanism. METHODS: The SH-SY5Y cells were transfected with APP595/596 gene in vitro for establishing a cell model to study the pathogenic role of amyloid β-protein (Aβ). The cell viability was detected by CCK-8 assay. The release of lactate dehydrogenase (LDH) was determined by the colour reaction of diaphorase-INT. The cell apoptotic rate was analyzed by flow cytometry. The expression of β-site APP cleaving enzyme 1(BACE1) at mRNA and protein levels was detected by RT-PCR and Western blot. The expression of Aβ was measured by the technique of immunofluorescence cytochemistry and Western blot. RESULTS: Treatment with osthole inhibited the LDH release, and increased the viability of the cells. The percentage of apoptotic cells was also significantly decreased. Osthole also inhibited the expression of BACE1 at mRNA and protein levels and the protein expression of Aβ. CONCLUSION: Osthole has protective effect on SH-SY5Y cells transfected with APP595/596 gene. The mechanism may be association with inhibiting the mRNA and protein expression of BACE1.