PGRN gene silencing on proliferation and migration abilities of human non-small-cell lung cancer A549 cells

AIM: To investigate the effect of small interfering RNA (siRNA)-mediated progranulin (PGRN) gene silencing on the proliferation and migration abilities of human non-small-cell lung cancer A549 cells and its mechanism. METHODS: The mRNA and protein expression levels of PGRN in the A549 cells and human bronchial epithelial (HBE) cells were detected by qPCR and Western blot. A549 cells were transfected with PGRN-siRNA by liposome method. The expression of PGRN at mRNA and protein levels in the A549 cells transfected with PGRN-siRNA was detected by qPCR and Western blot, respectively. The cell viability was measured by MTT assay. The cell proliferation ability was measured by living cells counting and crystal violet staining assays. The cell migration ability was measured by wound-healing and Transwell assays. The protein levels of proliferating cell nuclear antigen (PCNA), cyclin D1, Bcl-2 and Bax were determined by Western blot. The protein levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated protein kinase B (p-Akt) were also determined by Western blot. RESULTS: The expression of PGRN at mRNA and protein levels was higher in the A549 cells than that in the HBE cells (P<0.05). The expression of PGRN at mRNA and protein levels in the A549 cells transfected with PGRN-siRNA was significantly decreased, and the cell proliferation and migration abilities were significantly decreased. The protein expression levels of PCNA, cyclin D1 and Bcl-2 were significantly reduced and the protein expression level of Bax was significantly increased (P<0.05). Meanwhile, the protein levels of p-ERK1/2 and p-Akt were down-regulated (P<0.05). CONCLUSION: PGRN gene silencing obviously inhibits the proliferation and migration abilities of human non-small-cell lung cancer A549 cells. The PI3K/Akt and MAPK/ERK signaling pathways may play an important role in these processes.     

Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1

AIM: To observe the expression of Akt/GSK-3β/Snail signaling pathway-related molecules in cisplatin-resistant cell line A549/DDP mediated by transforming growth factor-β₁ (TGF-β₁), and to explore the association of Akt/GSK-3β/Snail signaling pathway with epithelial-mesenchymal transition (EMT). METHODS: The A549/DDP cells were divided into TGF-β₁ (+) group, TGF-β₁ (-) group and LY294002 group. The morphological changes of A549/DDP cells treated with TGF-β₁ were observed under microscope. The protein expression of E-cadherin and N-cadherin was determined by the methods of immumofluorescence and Western blot. The protein levels of Akt, p-Akt, GSK-3β, p-GSK-3β^Ser9 and Snail were also detected by Western blot. RESULTS: The A549/DDP cells in TGF-β₁ (+) group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers. Compared with TGF-β₁ (-) group, the protein expression of E-cadherin in TGF-β₁ (+) group was significantly decreased (P<0.05), and N-cadherin was significantly increased (P<0.05). The protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were also significantly increased (P<0.05). Compared with TGF-β₁ (+) group, the protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were significantly decreased in LY294002 group (P<0.05). No difference of Akt and GSK-3β expression between TGF-β₁ (-) group and TGF-β₁ (+) group was observed. CONCLUSION: The mechanism of EMT in A549/DDP cells might be related to Akt/GSK-3β/Snail signaling pathway activated by TGF-β₁.     

Anticancer function of Shp2 in lung adenocarcinoma A549 cells and rela-ted molecular mechanisms

AIM: To explore the anticancer function of Shp2 in lung adenocarcinoma A549 cells and the related molecular mechanisms. METHODS: The viability and proliferation of A549 cells treated with Shp2 specific inhibitor Phps-1 or cisplatin (DDP) were measured by CCK-8 assay and EdU assay. Annexin V-FITC/PI double staining was applied to detect apoptotic rate of A549 cells with different interventions. The protein levels of caspase-3-17p, Bcl-2, Bax, p-STAT3/STAT3 and p-ERK/ERK were determined by Western blot. RESULTS: Compared with control group, Phps-1 at the concentration of 20 μmol/L significantly increased the viability of A549 cells after 24 h of treatment (P<0.05). Meanwhile, the proliferation rate of A549 cells in Phps-1 20 μmol/L group was significant increased compared with control group (P<0.05). The apoptotic rate of A549 cells in DDP treatment group decreased from 13.01%±2.62% to 3.67%±0.93% after adding Phps-1 (P<0.05). Phps-1 down-regulated the protein levels of caspase-3-17p, Bax and p-ERK, but up-regulated p-STAT3.CONCLUSION: Shp2 is a tumor suppressor in A549 cells, which may be associated with the activation of STAT3 signal pathway.     

Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells via PI3K/Akt signaling pathway

AIM:To investigate the effect of apelin-13 on nicotine-induced apoptosis of cardiomyocytes and its potential molecular mechanism. METHODS:Rat H9c2 cells were treated with nicotine (10 μmol/L) to induced apoptosis. Flow cytometry was used to detect apoptotic rate. Western blot was used to determined the expression of related proteins. RESULTS:Compared with control group, nicotine treatment significantly increased the apoptotic rate of the H9c2 cells (P<0.01), and the protein levels of apoptosis-related proteins Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with nicotine group, apelin-13+nicotine significantly decreased the apoptotic rate of the H9c2 cells (P<0.01) and the the protein levels of Bax and cleaved caspase-3, but markedly increased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with apelin-13+nicotine group, apelin-13+nicotine+PI3K/Akt inhibitor LY294002 significantly increased the apoptotic rate of the H9c2 cells (P<0.01) and the protein levels of Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt and p-PI3K (P<0.05). CONCLUSION:Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells through PI3K/Akt signaling pathway.     

Effect of miR-24 on chemotherapy sensitivity in lung cancer A549 cells

AIM: To study the effect of microRNA (miR)-24 on chemotherapy sensitivity and its possible mechanisms in human lung adenocarcinoma A549 cells. METHODS: The expression of miR-24 in the A549 cells and A549/DDP cells was determined by real-time PCR. Transfection of miR-24 inhibitor was used to down-regulate the miR-24 level in the A549/DDP cells. The viability and apoptosis rate were measured by CCK-8 assay and flow cytometry, respectively. The protein levels of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, cytochrome C (Cyt C), phosphorylated extracellular signal regulated kinase (p-ERK) and P53 were detected by Western blot. Luciferase reporter assay was used to predict and identify the target genes of miR-24. RESULTS: The expression of miR-24 was significantly higher in the A549/DDP cells than that in the A549 cells (P<0.05). miR-24 inhibitor induced cell apoptosis and increased the sensitivity of the A549/DDP cells to cisplatin. Furthermore, miR-24 inhibitor down-regulated the ratio of Bcl-2/Bax, while up-regulated the protein levels of P53, p-ERK, cleaved caspase-9, cleaved caspase-3 and Cyt C. Incubation with U0126, a specific ERK inhibitor, partly reversed the viability of miR-24 inhibitor transfected A549/DDP cells. Bioinformatics analysis demonstrated that p53 was a potential target gene of miR-24. Co-teansfection of miR-24 inhibitor and P53 siRNA in A549/DDP cells partially reversed the effect of miR-24 inhibitor on cell viabiltiy. CONCLUSION: Down-regulation of miR-24 increases the sensitivity of A549/DDP cells to cisplatin. The mechanism may be related to directly targeting p53 gene and over-activation of ERK/P53 signaling pathway, thus promoting apoptosis via mitochondrial apoptosis pathway.     

Expression and significance of miR-193b in cervical cancer

AIM: To investigate the expression of microRNA-193b(miR-193b) in the cervical tissues, and further to explore the effect of silencing miR-193b on diamminedichloroplatinum(DDP)-treated HeLa cell viability. METHODS: The expression levels of miR-193b in different cervical tissues were examined by qPCR. After transfection of miR-193b-inhibitor, the cell migration was determined by Transwell assay, the sensitivity of HeLa cells to DDP was measured by MTT assay, the protein levels of phosphate and tension homology deleted on chromsome ten(PTEN), protein kinase B(Akt), p-Akt and p-glycoprotein(P-gp) were determined by Western blot. RESULTS: The mRNA level of miR-193b was significantly increased in the cervical cancer tissues compared with normal cervical tissues(P<0.05). Knockdown of miR-193b obviously inhibited migration and enhanced sensitivity to DDP of HeLa cells(P<0.05). Additionally, after transfection of miR-193b-inhibitor, the expression of PTEN was increased, whereas the protein levels of p-Akt and P-gp were decreased(P<0.05). CONCLUSION: miR-193b is highly expressed in the cervical cancer tissues. Inhibition of miR-193b augments the sensitivity to DDP of HeLa cells, at least in part, through PTEN-PI3K/Akt signaling pathway.     

Salinomycin inhibited proliferation and induced apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP

AIM: To investigate the effect of salinomycin on the proliferation and apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP. METHODS: The inhibitory effect of salinomycin on the growth of A549/DDP cells was tested by MTT method in vitro . The apoptosis and mitochondrial membrane potential (ΔΨ_m) of A549/DDP cells were assayed by flow cytometry. The activity of caspase-3, 8 and 9 was determined by the method of colorimetry. The levels of cytochrome C, Bcl- 2, Bax, β-catenin, and phosphorylated low-density lipoprotein receptor-related protein 6(p-LRP6) were measured by Western blotting. RESULTS: Salinomycin inhibited the growth of A549/DDP cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L decreased ΔΨ_m level, and increased reactive oxygen species (ROS), cytochrome C and cytosolic Ca²⁺ release in the cells. Salinomycin also increased the acti-vity of caspase-3, 8, and 9 in the cells, reduced the ratio of Bcl-2/Bax, and decreased the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin depresses the cell growth by inhibiting Wnt signaling, and induces the apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP via mitochondria-dependent and Bcl-2/Bax pathways.     

Stable reversal of multidrug resistance in A549/DDP cells by shRNA targeting MRP1 gene

AIM: To reverse multidrug resistance (MDR) of A549/DDP cells with short hairpin RNA (shRNA) expression vectors. METHODS: Two multidrug resistance-associated protein 1( MRP1 ) gene-specific shRNA expression plasmids pSilencer 2.1-U6 neo-MRP1 were constructed and introduced into A549/DDP cells. MRP1 mRNA was assayed by real-time fluorescent quantitative PCR. The MRP1 function was determined by rhodamine 123(Rho123) retention and the protein expression of MRP1 was detected by immunofluorescent staining. The viability of A549/DDP cells was evaluated by MTT method. RESULTS: MRP1 shRNA expression plasmids were successfully constructed. The expression of MRP1 at mRNA and protein levels was significantly decreased after sh-MRP1-2.1-1 and sh-MRP1-2.1-2 were transfected into A549/DDP cells. The intracellular accumulation of Rho123 significantly increased from(16.93±0.58)% to (89.02±0.59)% and (82.56±1.37)%. IC₅₀ of cisplatin were decreased from (101.45±0.64) μmol/L to (38.06±0.05) μmol/L and (53.72±0.36) μmol/L. IC₅₀ of 5-fluorouracil were decreased from (263.20±2.00) μmol/L to (98.82±1.16) μmol/L and (141.81±0.49) μmol/L. CONCLUSION: The shRNA expression plasmid pSilencer 2.1-U6 neo-MRP1 can stably and permanently inhibit MRP1 gene. The sensitivity of A549/DDP cells to drug is reversed.     

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.     

H IF-1αm ediates effect of interm ittent hypoxia on biological behavior of A 549 cells in vitro

ATM: To investigate whether hypoxia-inducible factor 1α (HIF-1α) mediates the effect of intermittent hypoxia on A549 cell viability, apoptosis and invasive ability METHODS: A549 cells were transfected with HIF-1α-siRNA and cultured under intermittent hypoxia. The expression of HIF-1α and its downstream genes, such as Bcl-2, Bax, P53, P21 and VEGF at mRNA and protein levels was determined by real-time PCR and Western blot. The viability of the A549 cells was measured by MTT assay. The apoptosis and cell cycle distribution of the A549 cells were examined by flow cytometry. The invasive ability of the A549 cells was detected by transwell test. RESULTS: The expression levels of HIF-1α, Bcl-2 and VEGF in non-HIF-1α-siRNA transfected A549 cells cultured in intermittent hypoxia environment[blank controlgroup(IH C),empty vector control group (IH E) and negative control group (IH N)] were higher than those in the A549 cells in normoxia group (RA), but the expression levels of Bax and P21 were lower than those in RA group (P<0.05). The siRNA-mediated HIF-1α gene silencing[intermittent hypoxia silenced group (IHS)] resulted in obvious down-regulation of HIF-1α, Bcl-2 and VEGF, and significant increase in the protein expression of P21 and Bax(P<0.05). The expression level of P53 in intermittent hypoxia groups was significantly higher than that in RA group, and no significant difference of P53 expression in different intermittent hypoxia groups was observed. Compared with normoxia, intermittent hypoxia resulted in significantly enhanced cell viability, decreased apoptosis, and enhanced invasive ability of non-HIF-1α-siRNA transfected A549 cells (P<0.05). The siRNA-mediated HIF-1α gene silencing resulted in significant cell viability inhibition, elevated apoptotic rate and decreased invasive ability under hypoxic condition (P<0.05).CONCLUSION: Intermittent hypoxia promotes the viability and invasion of A549 cells by HIF-1α-mediated downstream gene expression. HIF-1α gene silencing inhibits A549 cell growth and invasion under intermittent hypoxia by inhibition of HIF-1α signal pathways in vitro.     

Expression of miRNA-181a in human lung adenocarcinoma cells and its effect on cell function

AIM: To study the expression of microRNA (miRNA)-181a in different human lung adenocarcinoma cell lines, and to investigate the effect of miRNA-181a on cell function and its mechanism in human lung adenocarcinoma drug resistant cell A549/DDP. METHODS: Real-time PCR was used to detect the expression of miRNA-181a in BEAS-2B cells, A549 cells and A549/DDP cells. The A549/DDP cells were transfected with pGenesil-miRNA-181a eukaryotic expression plasmid. At the same time, the untransfection group and negative transfection group were also set up. The expression of miRNA-181a, cell viability, cell growth inhibition and apoptosis rate during cis-diamminedichloroplatinum (DDP) treatment, cell cycle, cell invasion, the protein expression of miRNA-181a target genes bcl-2 and p53 in the A549/DDP cells were determined by real-time fluorescence quantitative PCR, MTT assay, flow cytometry, Transwell method and Western blot, respectivly. RESULTS: The expression of miRNA-181a in A549 cells and A549/DDP cells was significantly lower than that in BEAS-2B cells, and the lowest expression level was observed in A549/DDP cells (P<0.05). The expression of miRNA-181a in A549/DDP cells was significantly increased after transfection with pGenesil-miRNA-181a (P<0.05). The cell viability, cell cycle and invasion ability of the A549/DDP cells were inhibited after miRNA-181a transfection (P<0.05). The cell growth inhibition rate and apoptotic rate of the A549/DDP cells were increased (P<0.05). The expression of Bcl-2 was reduced, but the expression of P53 was increased after transfection with miRNA-181a in A549/DDP cells (P<0.05). CONCLUSION: miRNA-181a may be correlated with the development of human lung adenocarcinoma. miRNA-181a can serve as a new target for treatment of lung cancer.     

Effect of Vaccinium vitis procyanidin on regulation of glioma cell growth

AIM: To explore the effect of Vaccinium vitis procyanidin on the growth of glioma cells. METHODS: Glioma C6 cells were cultured and divided into control and 10, 20 and 40 μg/L Vaccinium vitis procyanidin groups. The influence of Vaccinium vitis procyanidin on the growth of C6 cells was measured by MTT assay and the observation under inverted microscope. The apoptotic rate was detected by Annexin V/PI staining. The protein expression of Bcl-2 and Bax was determined by immunocytochemistry. The protein levels of Bcl-2, Bax and caspase-3 were also examined by Western blotting. RESULTS: The growth of C6 glioma cells was inhibited by Vaccinium vitis procyanidin at concentrations of 10, 20 and 40 μg/L. The growth was significantly inhibited in 40 μg/L Vaccinium vitis procyanidin group at 24 h and 48 h, and in 20 and 40 μg/L Vaccinium vitis procyanidin groups at 72 h (P<0.01). The density of the cells was decreased when the concentration of Vaccinium vitis procyanidin increased. The apoptotic rate was increased when the concentration of Vaccinium vitis procyanidin increased either. The expression of Bcl-2 was decreased and Bax was increased after 10, 20 and 40 μg/L Vaccinium vitis procyanidin treatments. The ratio of Bax/Bcl-2 was increased when the dose of Vaccinium vitis procyanidin increased (P<0.05 or P<0.01). The expression of Bcl-2 was decreased (P<0.01), and Bax and caspase-3 were increased after 10, 20 and 40 μg/L Vaccinium vitis procyanidin treatments. The ratio of Bax/Bcl-2 was increased when the dose of Vaccinium vitis procyanidin increased (P<0.01). CONCLUSION: Vaccinium vitis procyanidin inhibits the growth of glioma cells by down-regulating Bcl-2 protein and up-regulating Bax protein to activate caspase-3, thus inducing apoptosis.     

Effects of adenovirus-mediated shRNA targeting PTEN on proliferation and apoptosis of activated hepatic stellate cells in vitro

AIM:To investigate the down-regulation of phosphatase and tensin homolog deleted on chromosome 10(PTEN) gene by adenovirus-mediated short hairpin RNA(shRNA) on proliferation and apoptosis of activated hepatic stellate cells(HSCs) in vitro and the related signaling transduction pathways. METHODS:The activated HSCs were cultured in vitro and transfected with recombinant adenovirus expressing shRNA targeting PTEN. The proliferation of HSCs was measured by MTT assay and the apoptosis was assessed by TUNEL and flow cytometry. Western blotting was used to detect the protein levels of PTEN, Bax, Bcl-2, Akt, p-Akt, ERK1/2 and p-ERK1/2 in HSCs, and real-time fluorescent quantitative PCR was applied to detect the mRNA expression of Akt and ERK1. RESULTS:The recombinant adenovirus expressing shRNA targeting PTEN was successfully transfected into activated HSCs in vitro, and significantly promoted the proliferation of HSCs in a time-dependent manner within a certain extent. The apoptotic rate of HSCs was significantly decreased 72 h after transfection(P<0.05). Meanwhile, reduced expression of Bax and elevated expression of Bcl-2 were induced 72 h after transfection(P<0.05). Furthermore, the expression of p-Akt and p-ERK1/2 were increased significantly(P<0.05), while no significant difference in the expression of Akt and ERK1 at mRNA and protein levels was observed(P>0.05). CONCLUSION:Down-regulation of PTEN by adenovirus-mediated shRNA dramatically promotes the proliferation of activated HSCs, and inhibits the apoptosis through Bcl-2/Bax pathway. In addition, the phosphorylation of Akt and ERK1/2 is increased, indicating that PI3K/Akt and ERK1/2 signal transduction pathways may play an important role in the regulation of proliferation and apoptosis of HSCs.     

Protective effect of paeoniflorin on nerve cells in APP/PS1 mice and its mechanism

AIM: To investigate the therapeutic and preventive effects of paeoniflorin (PF) on APP/PS1 mice, and to explore the possible mechanism. METHODS: Fifteen male 5-month-old APP/PS1 non-dominant mice were chosen as normal control group, 15 male 5-month-old APP/PS1 double transgenic mice were used as model group, and 15 male 5-month-old APP/PS1 double transgenic mice treated with 5 mg/kg PF by intraperitoneal injection were allocated in administation group. The learning and memory ability of the mice in each group was detected by Morris water maze. The apoptosis was assessed by TUNEL fluorescence staining. The protein expression of PI3K, Akt, p-PI3K, p-Akt, caspase-3, caspase-9, Bcl-2 and Bax in cerebral cortex and hippocampus was detected by Western Blot. The protein expression levels and distribution of caspase-3 and caspase-9 were detected by immunohistochemistry. RESULTS: (1) Compared with normal control group, the learning and memory ability declined in APP/PS1 model group. Compared with APP/PS1 model group, PF obviously improve the ability of learning and memory in mice. (2) Compared with normal control group, the apoptosis of nerve cells in APP/PS1 model group significantly increased and distributed in wider areas, while that in PF group was reduced (P<0.05). (3) Compared with APP/PS1 model group, PF could significantly lower pro-apoptotic factors, caspase-3, caspase-9 and Bax (P<0.05), and increase the expression of anti-apoptotic factors, p-PI3K, p-Akt and Bcl-2 (P<0.05). CONCLUSION: PF can up-regulate the expression of Bcl-2 and down-regulate the expression levels of caspase-9, caspase-3 and Bax via the activation of PI3K/Akt pathway, thereby inhibiting the nerve cell apoptosis and protecting the nerve cells, so as to treat neurodegenerative diseases.     

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.     

PQS attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibiting endoplasmic reticulum stress

AIM:To study the effect of Panax quinquefoliumsaponin (PQS) on cardiomyocyte apoptosis induced by thapsigargin (TG). METHODS:Primary cultured cardiomyocytes from neonatal SD rats were divided into control group, TG group, PQS (40 mg/L, 80 mg/L and 160 mg/L)+TG group, si-PERK+TG group, and mock+TG group. The cells were treated with 1 μmol/L TG for 24 h to induce apoptosis. The PERKgene in the cardiomyocytes was knocked down by RNAi. The cell viability was detected by CCK-8 assay. Apoptosis was analyzed by flow cytometry. Wes-tern blotting was used to determine the expression of ERS molecules GRP78, CRT, ATF4 and CHOP, anti-apoptosis protein Bcl-2 and pro-apoptosis protein Bax. RESULTS:Compared with control group, TG significantly and the apoptosis, reduced the cell viability (P<0.05), increased the phosphorylation of PERK and eIF2α, increased the expression of GRP78, CRT, ATF4, CHOP and pro-apoptosis protein Bax, and decreased the expression of anti-apoptosis protein Bcl-2 (P<0.05). Compared with TG group, PQS treatment (160 mg/L) significantly reduced the apoptosis and increased the cell viability (P<0.05). All the 3 different concentrations of PQS significantly increased the expression of anti-apoptosis protein Bcl-2 and reduced the expression of pro-apoptosis protein Bax (P<0.05) in a dose-dependent manner. PQS pretreatment and knockdown of PERK both reduced the protein levels of GRP78, CRT, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, CHOP and pro-apoptosis protein Bax, and increased the expression of anti-apoptosis protein Bcl-2 (P<0.05). CONCLUSION: PQS at concentration of 160 mg/L attenuated cardiomyocyte apoptosis induced by TG. PQS had the similar effect as PERKknockdown on cardiomyocyte apoptosis. The mechanism may be associated with inhibiting PERK-eIF2α-ATF4-CHOP pathway of ERS-related apoptosis.     

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.     

miR-125a-5p reverses cisplatin resistance of non-small-cell lung cancer A549/DDP cells by targeting LIMK1

AIM:To explore the effect of microRNA-125a-5p (miR-125a-5p) on cisplatin (DDP) resistance of non-small-cell lung cancer A549/DDP cells and its related mechanisms. METHODS:The expression levels of miR-125a-5p and LIM kinase 1 (LIMK1) in non-small-cell lung cancer tissues, A549 cells and A549/DDP cells were detected by RT-qPCR. The A549/DDP cell viability, apoptotic rate and expression of drug resistance-related proteins after over-expression or knockdown of miR-125a-5p and/or LIMK1 expression were detected by MTT assay, flow cytometry and Western blot, respectively. The targeting relationship between miR-125a-5p and LIMK1 was verified by TargetScan online prediction and dual-luciferase reporter system. The cell viability, apoptotic rate and expression of drug resistance-related proteins after co-expression of miR-125a-5p and LIMK1 were also determined. RESULTS:The expression level of miR-125a-5p was down-regulated and LIMK1 expression was up-regulated in non-small-cell lung cancer tissues and cell lines (P<0.05). The results of dual-luciferase assay indicated that miR-125a-5p negatively regulated the expression of LIMK1. The expression of drug resistance-related proteins and the viability of A549/DDP cells were inhibited after over-expression of miR-125a-5p or knockdown of LIMK1, while the apoptosis was enhanced. Over-expression of LIMK1 attenuated the inhibitory effect of miR-125a-5p on A549/DDP cell viability and drug resistance-related protein expression (P<0.05). CONCLUSION:miR-125a-5p reverses the resistance of A549/DDP cells to DDP by inhibiting the expression of LIMK1 and drug resistance-related proteins.     

Emodin reduces hypoglycemia/hypoxia-induced injury of microglia by TLR4/NF-κB signaling pathway

AIM: To investigate the mechanism of emodin on the protection of glucose-deficient/anoxic microglia. METHODS: A microglia BV2 cell model induced by hypoglycemia/hypoxia (HH) was established. The glucose-deficient/anoxic cells treated with emodin were labeled as HH+emodin (20, 40 and 80 μmol/L) groups. The BV2 cells with TLR4 over-expression treated with emodin under hypoglycemia/hypoxia condition was labeled as HH+pcDNA-TLR4+ emodin (40 μmol/L) group. The cell viability was measured by MTT assay. Lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels were detected by ELISA. The apoptosis was analyzed by flow cytometry. The protein levels of Bax, Bcl-2, TLR4, p-IκB and IκB were determined by Western blot. RESULTS: Compared with HH+DMSO group, the viability was significantly increased, the levels of LDH and TNF-α and apoptotic rate were significantly decreased, the protein levels of Bax, TLR4 and p-IκB were significantly decreased, the protein level of Bcl-2 was significantly increased in HH+emodin groups (P<0.05). Over-expression of TLR4 reversed the effect of emodin on promoting the viability and inhibiting apoptosis in the BV2 cells. CONCLUSION: Emodin has a protective effect on hypoglycemia/hypoxia induced microglia, and its mechanism may be related to the inactivation of TLR4/NF-κB signaling pathway.     

Effects of RUNX3 siRNA on the growth and drug sensitivity of SH-SY5Y cells

AIM: To investigate the effects of RUNX3 gene on the growth and drug sensitivity of SH-SY5Y cells.METHODS: The siRNA plasmid of RUNX3 was constructed and transfected into SH-SY5Y cells. Stable transfectants were identified by RT-PCR and Western blotting. The growth curve, cell cycle distribution, drug sensitivity assay and accumulation of adriamycin in cells were detected by MTT assay and flow cytometry. The expressions of cyclin D1, CDK4, CDK6, p21, p27, Bcl-2, Bax, P-gp and MRP were analyzed by Western blotting. RESULTS: mU6pro-RUNX3 siRNA was successfully constructed and transfected into SH-SY5Y cells. Down-regulation of RUNX3 significantly promoted the cellular proliferation, inhibit the drug sensitivity and intracellular adriamycin accumulation of cells, compared with that in the controls (P<0.05). The expressions of P-gp, Bcl-2 and cyclin D1 in transfected cells were increased, while p21 decreased.CONCLUSION: RUNX3 might play important roles in the development of neuroblastoma.