Downregulation of DEK induces cell apoptosis via inhibition of NF-κB signaling pathway in gastric carcinoma SGC-7901 cells

AIM: To investigate the effect of DEK downregulation on the apoptosis of gastric carcinoma SGC-7901 cells, and to explore its associations with NF-κB signaling pathway and apoptosis related proteins. METHODS: SGC-7901 cells with different treatments were divided into 3 groups including untreated group, control siRNA group and DEK siRNA group. The expression of DEK at mRNA and protein levels in the SGC-7901 cells was detected by real-time PCR and Western blot. The cell apoptosis was examined by flow cytometry. Furthermore, the activities of caspase-3 and caspase-9 in the SGC-7901 cells were investigated by Caspase-Glo^®-3/9 kit. Finally, the expression of key regulatory protein p65 of NF-κB signaling pathway and apoptosis-related proteins Bcl-2 and Bax in the SGC-7901 cells was investigated by Western blot. RESULTS: Compared with untreated group and control siRNA group, the expression of DEK at mRNA and protein levels was significantly downregulated in DEK siRNA group (P<0.05). In addition, the ratios of early phase apoptosis and total apoptosis in DEK siRNA group were markedly higher than those in untreated group and control siRNA group (P<0.05). Most notably, the decrease in p65 and Bcl-2 proteins, increase in Bax protein and the increases of caspase-3 and caspase-9 activities were observed in DEK siRNA group. CONCLUSION: Downregulation of DEK mediates cell apoptosis of gastric carcinoma may be tightly associated with NF-κB signaling pathway.     

MicroRNA-9 inhibits epithelial-mesenchymal transition in gastric cancer SGC-7901 cells by NRP1

AIM:To investigate the inhibitory effect of microRNA-9(miR-9) on epithelial-mesenchymal transition (EMT) in the gastric cancer SGC-7901 cells and its mechanism.METHODS:The gastric cancer cell line SGC-7901 was transfected with miR-9 mimics or negative control mimic (NCM),as miR-9 or NCM group,respectively.The SGC-7901 cells without transfection were used as control group.The expression level of miR-9 in each group was detected by RT-qPCR.The migration and invasion abilities of the SGC-7901 cells in the 3 groups were detected by Transwell assay.The protein expression of N-cadherin,E-cadherin,α-catenin and neuropilin-1(NRP1) was determined by Western blot.Antagonistic effect of NRP1 over-expression on miR-9 inhibition of EMT was detected by Western blot.The relationship between miR-9 and NRP1 was analyzed by dual luciferase assay.RESULTS:The expression level of miR-9 in miR-9 group was significantly up-regulated,which was 538 times higher than that in control group (P<0.05).The number of migratory cells in miR-9 group was significantly lower than that in control group (P<0.05).Compared with control group,the protein expression of N-cadherin and NRP1 in miR-9 group was significantly decreased,while the protein expression of E-cadherin and α-catenin protein was significantly increased.Over-expression of NRP1 resulted in the increase in the protein expression of N-cadherin in the gastric cancer cells of miR-9 group,and the decrease in the protein expression of E-cadherin and α-catenin significantly.The result of dual luciferase assay showed that NRP1 was a downstream target gene of miR-9(P<0.05).CONCLUSION:miR-9 may inhibit the expression of EMT-related proteins through the downstream target gene NRP1,thus inhibiting the EMT of gastric cancer SGC-7901 cells.     

Effects of all-trans retinoic acid on sensitivity of gastric cancer cells to radiotherapy

AIM: To investigate the effect of all-trans retinoic acid (ATRA) on the viability of gastric cancer cell SGC-7901 and the sensitivity to radiotherapy. METHODS: MTT assay was used to examine the cell viability. Radio-sensitivity and cell cycle were determined by colony formation assay and flow cytometry, respectively. The mRNA levels of Bax, Bcl-2, survivin and NF-κB in the cells were measured by RT-qPCR. RESULTS: ATRA inhibited the viability of SGC-7901 cells in a concentration-dependent manner. The maximal inhibition was at concentration of 8 μmol/L. Colony formation assay revealed that the combination of ATRA with X-ray treatment significantly reduced the values of D0 and Dq, and shifted down the fitting survival curve, as compared with radiotherapy alone. Moreover, ATAR markedly decreased the percentage of G₂/M phase in the SGC-7901 cells (P<0.05). In addition, following ATRA treatment, the mRNA levels of Bcl-2 and survivin were decreased (P<0.05), whereas the mRNA levels of Bax and NF-κB were increased (P<0.05). CONCLUSION: ATRA enhances the sensitivity of SGC-7901 cells to radiotherapy, inhibits G₂/M arrest and regulates the mRNA expression of Bax, Bcl-2, survivin and NF-κB.     

miR-130b reverses temozolomide resistance in glioma

AIM:To investigate the expression level of microRNA-130b (miR-130b) and the molecular me-chanisms of miR-130b in temozolomide (TMZ)-resistant glioma. METHODS:The relative levels of miR-130b in 3 glioma cell lines (U251, SHG-44 and U87) were assessed by RT-qPCR. The half maximal inhibitory concentration (IC₅₀) of TMZ for the glioma cell lines was analyzed. To establish the TMZ-resistant glioma cell line, U251 cells were exposed to gradually increasing concentrations of TMZ. The IC₅₀ and resistance index (RF) were calculated with GraphPad Prism software. miR-130b-overexpressing U251/TR cells and miR-130b-knockdown U251 cells were established by transient transfection with miR-130b mimics and miR-130b inhibitor, respectively. The viability of the glioma cells was measured by CCK-8 assay. The apoptosis of glioma cells was analyzed by Annexin V/PI apoptosis assay. Bioinformatics software was used to predict the potential target gene of miR-130b, and such prediction was validated by luciferase reporter assay. Electrophoretic mobility shift assay was performed to detect the DNA binding ability of NF-κB. Western blot was used to determine the protein levels of tumor necrosis factor-α (TNF-α), Bcl-2, X-linked inhibitor of apoptosis protein (XIAP) and survivin in the glioma cells. RESULTS:The IC₅₀ values of TMZ for the giloma cell lines U251, SHG-44 and U87 were 54.8, 94.8 and 149.6 μmol/L, respectively. U251/TR cells were approximately 8.1 times resistant to TMZ as compared with its parental cells. Up-regulation of miR-130b significantly reduced the resistance of U251/TR cells to TMZ. On the contrary, down-regulation of miR-130b dramatically increased the tolerance of U251 cells to TMZ. The overexpression of miR-130b promoted apoptosis induced by TMZ in the U251/TR cells. However, the knockdown of miR-130b expression decreased the percentage of apoptotic cells in the U251 cells induced by TMZ (P<0.05). Luciferase reporter assay confirmed that TNF-α was a direct target gene of miR-130b. Knockdown of miR-130b in the U251 cells significantly promoted, while overexpression of miR-130b in the U251/TR cells reduced the DNA binding ability of NF-κB as well as the levels of TNF-α, Bcl-2, XIAP and survivin. Furthermore, NF-κB inhibitor Bay 11-7082 enhanced TMZ-induced apoptosis in the U251/TR cells. CONCLUSION:The expression of miR-130b is significantly decreased in TMZ-resistant glioma cells. miR-130b inhibits resistance of glioma to TMZ by targeting TNF-α/NF-κB pathway.     

Expression of microRNA-1284 in gastric cancer and underlying mechanism

AIM: To evaluate the correlation between microRNA-1284 (miR-1284) and gastric cancer, and to investigate the underlying mechanism. METHODS: The expression of miR-1284 was examined by real-time PCR in 63 gastric cancer (GC) tissue samples and 63 non-malignant adjacent tissue samples. The correlation between miR-1284 and the clinicopathological feature of GC was analyzed. Lentiviral vector containing miR-1284 was constructed and transfected into GC SGC-7901 cells. After transfection, the expression of miR-1284 was examined by real-time PCR. The cell activity was evaluated by CCK-8 assay. The cell cycle and apoptosis were determined by flow cytometry. The ability of cell migration was measured by wound-healing assay. The potential target gene of miR-1284 was predicted by online bioinformatic softwares. The expression of JAG1 mRNA was examined by real-time PCR. The protein levels of JAG1, Notch1 and NF-κB were analyzed by Western blotting. RESULTS: Compared with non-malignant adjacent tissue samples, the results of real-time PCR showed significant downregulation of miR-1284 in 42 GC tissue samples (P<0.05). The expression level of miR-1284 was not significantly associated with age and gender of the patients, tumor size, TNM staging and lymph node metastases (P>0.05), but significantly associated with histologic grading (P<0.05). Compared with LV-NC-GFP group and control group, after transfection of miR-1284 in LV-miR-1284 group, the expression of miR-1284 was significantly increased (P<0.05), the percentages of apoptotic cells and the cells in G₀/G₁ phase were significantly increased (P<0.05), the cells activity and ability of migration were significantly decreased (P<0.05), and the expression of JAG1, Notch1 and NF-κB was significantly decreased (P<0.05). CONCLUSION: The inhibitory effect of miR-1284 on gastric cancer may be associated with the regulation of its targeting gene JAG1.     

MicroRNA-126 enhances radiosensitivity of SGC-7901 cells via targeting inhibition of EZH2

AIM: To investigate the molecular biological mechanisms by which microRNA-126 (miR-126) enhances the radiosensitivity of gastric cancer cells. METHODS: SGC-7901 cells were cultured in vitro. In order to over-express miR-126 in SGC-7901 cells, miR-126 mimic was transfected. The mRNA and protein levels of enhancer of zeste ho-molog 2 (EZH2) were detected by RT-qPCR and Western blot, respectively. The targeting relationship between miR-126 and EZH2 was determined by dual-luciferase reporter assay. To estimate the effect of EZH2 on miR-126-enhanced radiosensitivity of the SGC-7901 cells, the pcDNA3.1-EZH2 vector was also co-transfected with miR-126 mimic, and then CCK-8 assay and flow cytometry were used to detect the viability and apoptotic rate of the cells after radiation. RESULTS: Over-expression of miR-126 significantly inhibited the expression of EZH2 in SGC-7901 cells both at protein and mRNA levels (P<0.05). A direct targeting relationship between miR-126 and EZH2 was confirmed by dual-luciferase reporter assay. Compared with the cells only transfected with miR-126 mimic, co-transfection of pcDNA3.1-EZH2 with miR-126 mimic increased the viability but reduced the apoptosis of the cells treated by radiation (P<0.05). CONCLUSION: Targeting inhibition of EZH2 may be one of the mechanisms by which miR-126 enhances the radiosensitivity of gastric cancer cells.     

Effect of ixazomib on apoptosis and NF-κB signaling pathway in pancreatic cancer cells

AIM:To investigate the effects of ixazomib on the apoptosis and NF-κB signaling pathway in pancreatic cancer cells. METHODS:Human pancreatic cancer cell lines CFPAC-1 and PANC-1 were cultured, and the cells were treated with ixazomib at 0, 10, 20, 30 and 40 nmol/L for 12, 18, 24 and 48 h. The expression of NF-κB p65, IκB kinase (IKK), Bax and caspase-3 in the cells at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by CCK-8 assay. The apoptosis was analyzed by flow cytometry. RESULTS:Treatment with ixazomib at 10~40 nmol/L inhibited the viability of PANC-1 cells and CFPAC-1 cells, and the inhibitory rate was increased significantly with the increases in the concentration and time (P<0.05). Compared with the control cells, treatment with ixazomib significantly increased the apoptotic rates of PANC-1 cells and CFPAC-1 cells in a dose- dependent manner (P<0.05), and significantly decreased the mRNA expression levels of NF-κB p65 and IKK in the PANC-1 cells and CFPAC-1 cells (P<0.05), while the mRNA expression levels of apoptotic factors Bax and caspase-3 in the PANC-1 cells and CFPAC-1 cells were significantly increased (P<0.05). The results of Western blot showed that treatment with ixazomib significantly decreased the protein levels of NF-κB p65 and IKK in the PANC-1 cells and CFPAC-1 cells (P<0.05), which was consistent with the results of mRNA expression. The protein levels of apoptosis factors Bax and caspase-3 in the CFPAC-1 cells were significantly increased (P<0.05), and the protein level of caspase-3 in the PANC-1 cells was increased significantly (P<0.05). However, Bax protein did not increase significantly in 10 nmol/L ixazomib group. CONCLUSION:Ixazomib, a proteasome inhibitor, inhibits the viability of pancreatic cancer cells and promotes apoptosis by inhibiting the activation of NF-κB signaling pathway in a time- and dose-dependent manner.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells

AIM:To explore the effect of pyrrolidine dithiocarbamate (PDTC),an NF-κB inhibitor,on the proliferation and apoptosis of human multiple myeloma U266 cells and its mechanisms.METHODS:The U266 cells were treated with PDTC at different concentrations (0,25,50,100 and 200 μmol/L)in vitro.The growth inhibitory rate of the U266 cells was detected by CCK-8 assay and cell counting.The cell cycle of the U266 cells was determined by flow cyto-metry,and the apoptosis was examined by flow cytometry with Annexin V-FITC/PI staining.The effect of PDTC on the expression of DNA methyltransferase 1(DNMT1) at mRNA and protein levels was measured by RT-qPCR and Western blot,respectively.The effects of PDTC on the protein levels of NF-κB (P65),DNMT1,Bcl-2,cyclin D1,cleaved caspase-3 and cleaved caspase-8 were determined by Western blot.RESULTS:The protein level of NF-κB (P65) was decreased after treatment with PDTC for 48 h or 72 h.PDTC inhibited the proliferation of U266 cells in both dose-and time-dependent manners.After treatment with PDTC for 48 h,the percentage of U266 cells in G₂ phase increased compared with control group (P<0.05).PDTC induced the apoptosis of U266 cells in a dose-dependent manner.The expression of DNMT1 at mRNA and protein levels decreased (P<0.05).The results of Western blot showed that the expression of Bcl-2 in PDTC groups decreased,while the protein levels of cyclin D1,cleaved caspase-3 and cleaved caspase-8 were higher than those in control group (P<0.05).CONCLUSION:The NF-κB inhibitor PDTC inhibits the proliferation of U266 cells by inducing cell apoptosis.It may be related to the down-regulated expression of DNMT1,cell cycle arrest and activation of the apoptotic pathways.     

Inhibition of HMGB1 expression promotes apoptosis of hemangioma endothelial cells

AIM: To investigate the effect of inhibiting high-mobility group box protein 1 (HMGB1) expression on the viability and apoptosis of hemangioma endothelial cells (HemECs). METHODS: Human HemECs were isolated and cultured, and HMGB1 small interfering RNA (HMGB1-siRNA) was transfected into the cells. The cell viability was detected by CCK-8 assay. The apoptosis and reactive oxygen species (ROS) content were analyzed by flow cytometry. The protein levels of HMGB1, NF-κB p65, p-IκBα, cyclin D1 and survivin were determined by Western blot. RESULTS: The protein expression of HMGB1 in the HemECs transfected with HMGB1-siRNA was significantly lower than that in blank control group (P<0.05). Compared with NC group, the cell viability was decreased significantly in the HemECs transfected with HMGB1-siRNA, the apoptotic rate was significantly increased, the content of ROS increased significantly, and the protein levels of NF-κB p65, p-IκBα, cyclin D1 and survivin were significantly decreased (P<0.05). After exposure to NF-κB signaling pathway inhibitor PDTC, the cell viability was inhibited, the apoptosis was increased, ROS content, and the protein levels of NF-κB p65, p-IκBα, cyclin D1 and survivin were down-regulated significantly, as compared with si-HMGB1 group (P<0.05). CONCLUSION: Inhibition of HMGB1 reduces the viability of HemECs and induces apoptosis by increasing the content of ROS and down-regulating the activity of NF-κB signaling pathway.     

Effect of integrin β1 on multidrug resistance in gastric cancer SGC-7901 cells

AIM: To study the effect of integrin β1 on multidrug resistance in gastric cancer and its possible mechanisms. METHODS: The expression of integrin β1 at mRNA and protein levels in the SGC-7901 cells and SGC-7901/DDP cells was determined by qPCR and Western blot. The expression of integrin β1 in the SGC-7901/DDP cells was silenced by antisense oligodeoxynucleotide. The cell viability was detected by the CCK-8 assay, the cell apoptosis were analyzed by flow cytometry, and the protein levels of integrin β1, Bcl-2/Bax, cleaved caspase-3/caspase-3, cytochrome C (Cyt-C) and p-AKT/AKT were determined by Western blot.RESULTS: The expression of integrin β1 at both mRNA and protein levels was significantly upregulated in SGC-7901/DDP cells. The expression of integrin β1 was increased in SGC-7901 cells treated with chemotherapeutic agents such as cisplatin, paclitaxel and 5-fluorouracil. Knockdown of integrin β1 induced apoptosis of SGC-7901/DDP cells with an increased sensitivity to the chemotherapeutic agents. Meanwhile, knockdown of integrin β1 downregulated the protein levels of Bcl-2/Bax, p-AKT^Ser473 and p-AKT^Thr308, while promoted the release of Cyt-C and upregulated the protein level of cleaved caspase-3. CONCLUSION: Knockdown of integrin β1 increases the sensitivity of SGC-7901/DDP cells to the chemotherapeutic agents, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of AKT pathway by inhibiting phosphorylations of AKT at Ser473 and Thr308.     

lncRNA-MALAT1 targets and downregulates miR-570-3p expression to promote proliferation of gastric cancer cells

AIM:To investigate whether long non-coding RNA MALAT1 (lncRNA-MALAT1) targets and down-regulates microRNA-570-3p (miR-570-3p) expression to further promote the proliferation of gastric cancer cells. METHODS:Gastric cancer cell line SGC7901 was cultured in vitro and divided into 3 groups:blank control, si-MALAT1 and si-MALAT1 NC. The si-MALAT1 and si-MALAT1 NC groups were transfected with MALAT1 siRNA and its negative control, respectively. The cell proliferation was evaluated by MTS assay. The expression of miR-570-3p was detected at different time points in the pure SGC7901 gastric cancer cell line, and the expression of lncRNA-MALAT1 and miR-570-3p in different groups was detected by RT-qPCR. The potential complementary binding sites of lncRNA-MALAT1 and miR-570-3p were predicted by RegRNA. The MALAT1 gene and its mutant fragment were cloned into luciferase reporter vector psiCHECK-2. Restriction enzyme analysis and sequencing were used to identify whether the recombinant plasmids carrying MALAT1 or MALAT1-Mut were successfully constructed. miR-570-3p mimic, miR-570-3p inhibitor, miR-570-3p mimic negative control and miR-570-3p inhibitor negative control were co-transfected into the 293T cells with the luciferase repor-ters containing MALAT1 or MALAT1-Mut. Dual-luciferase reporter assay was performed to detect luciferase activity in different groups in order to verify the relationship between lncRNA-MALAT1 and miR-570-3p. RESULTS:Compared with blank control group and si-MALAT1 NC group, the A₄₉₀ value in si-MALAT1 group was significantly decreased (P<0.01). The expression of miR-570-3p presented an obvious declining trend over time. The expression of lncRNA-MALAT1 in si-MALAT1 group was remarkably decreased, whereas the expression of miR-570-3p was obviously increased. The dual-luciferase reporter assay indicated that the MALAT1 reporter luciferase activity decreased significantly in miR-570-3p mimic group compared with mimic negative control (P<0.01), and the luciferase activity of MALAT1 reporter was obviously up-regulated in miR-570-3p inhibitor group compared with miR-570-3p mimic group (P<0.01). However, miR-570-3p mi-mic, miR-570-3p inhibitor, miR-570-3p mimic negative control and miR-570-3p inhibitor negative control showed no effect on the luciferase activity of MALAT1-Mut reporter. CONCLUSION:lncRNA-MALAT1 targets and down-regulates miR-570-3p expression to further promote the proliferation of gastric cancer cells.     

Up-regulation of miR-181a attenuates releases of CSE-induced pro-inflammatory factors and expression of collagen IV,fibronectin and α-SMA in human bronchial epithelial cells

AIM: To investigate the effect and potential mechanism of microRNA-181a (miR-181a) on cigarette smoke extract (CSE)-induced the productions of pro-inflammatory factors and the expression of collagen IV, fibronectin and α-smooth muscle actin (α-SMA) in human bronchial epithelial cells (HBECs). METHODS: CSE-induced miR-181a expression was detected by RT-qPCR in the HBECs. After tansfected with miR-181a mimic, the releases of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and transforming growth factor-β1 (TGF-β1) were measured by ELISA, the protein expression of collagen IV, fibronectin and α-SMA was determined by Western blot. The activation of NF-κB/TGF-β1/Smad3 pathway was also evaluated by Western blot. RESULTS: CSE increased the levels of TNF-α, IL-1β, IL-6 and TGF-β1 and the expression of collagen IV, fibronectin and α-SMA, and decreased the expression of miR-181a in the HBECs (P<0.05). However, transfected with miR-181a mimic partially prevented the releases of TNF-α, IL-1β, IL-6 and TGF-β1, and inhibited the expression of collagen IV, fibronectin and α-SMA (P<0.05). Additionally, the activation of NF-κB/TGF-β1/Smad3 evoked by CSE was attenuated after transfected with miR-181a mimic. CONCLUSION: Up-regulation of miR-181a prevents the releases of CSE-induced pro-inflammatory factors and expression of collagen IV, fibronectin and α-SMA in the HBECs, and its mechanism may be related to the inhibition of NF-κB/TGF-β1/Smad3 pathway.     

Effect of ruthenium-polypyridine complex on apoptosis of gastric cancer SGC-7901 cells

AIM:To study the effect of ruthenium-pyridine complex Ru1 on apoptosis of gastric cancer SGC-7901 cells. METHODS:MTT assay and crystal violet staining method were used to detect the viability and cell number of SGC-7901 cells treatment with Ru1. Annexin V-FITC and PI staining was performed to test the apoptosis rate of SGC-7901 cells. The protein expression of Bax and Bcl-2 was detected by Western blot. RESULTS:The results of MTT assay and crystal violet staining showed that the ruthenium-pyridine complexes significantly reduced the viability and cell number of SGC-7901 cells. Treatment with Ru1 for 24 h significantly increased the apoptotic rate of SGC-7901 cells (P<0.05). Ru1 up-regulated the expression of Bax protein and down-regulated the expression of Bcl-2 protein in the SGC-7901 cells (P<0.05). CONCLUSION:Ru1 induces apoptosis of SGC-7901 cells by affecting the expression of Bax and Bcl-2 proteins.     

miR-105 inhibits cell proliferation,migration and invasion abilities in non-small-cell lung cancer cells by targeting and negative regulation of KIFC1

AIM:To study the effects of microRNA-105(miR-105) on the cell proliferation, migration and invasion abilities of non-small-cell lung cancer (NSCLC) H460 cells, and further to explore its mechanism. METHODS:The expression of miR-105 and kinesin family member C1 (KIFC1) mRNA in the NSCLC tissues and adjacent tissues and cells was detected by RT-qPCR. The protein expression of KIFC1 in the NSCLC tissues, adjacent normal tissues and cells was determined by Western blot. The H460 cells were divided into miR-105 group (transfection with miR-105 mimics), miR-negative control (NC) group (transfection with miR-NC), inhibitor-NC group (transfection with NC of inhibitor), inhibitor-miR-105 group (transfection with miR-105 inhibitor), si-NC group (transfection with NC siRNA), si-KIFC1 group (transfection with KIFC1 siRNA), miR-105+vector group (miR-105 mimics and pcDNA 3.1 co-transfection) and miR-105+KIFC1 group (miR-105 mimics and pcDNA 3.1-KIFC1 co-transfection). The cell proliferation was measured by MTT assay and colony formation assay. The migration and invasion abilities were detected by Transwell methods. The relative luciferase acitivity was evaluated by double luciferase reporter assay. RESULTS:Compared with the adjacent tissues, the expression of miR-105 was significantly decreased and the expression of KIFC1 was significantly increased in NSCLC tissues (P<0.05). Compared with human normal embryonic lung fibroblasts MRC-5, the expression of miR-105 in the H460 cells was significantly decreased, and the expression of KIFC1 was significantly increased (P<0.05). miR-105 inhibited the relative luciferase activity of H460 cells with wild-type KIFC1 and negatively regulated the protein expression of KIFC1. Over-expression of miR-105 and knockdown of KIFC1 expression significantly inhibited the proliferation, migration and invasion abilities of H460 cells. Over-expression of KIFC1 reversed the inhibitory effect of miR-105 on the cell proliferation, migration and invasion abilities of H460 cells. CONCLUSION:miR-105 inhibits the proliferation, migration and invasion abilities of NSCLC cells. The mechanism may be related to targeting and negatively regulating expression of KIFC1.     

Regulation of ectopic trypsin and proinflammatory cytokine expression by NF-κB and AP-1 in influenza A virus induced myocarditis

AIM: To investigate the regulatory effects of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) on the expression of ectopic trypsin and proinflammatory cytokines in influenza A virus (IAV)-induced myocarditis. METHODS: Male BALB/c mice of 8 weeks old (n=40) were randomly divided into 4 groups: normal control group (NC), infection control group (IC), NF-κB inhibitor group (NI) and AP-1 inhibitor group (AI). The mice in NC group and IC group were instilled intranasally with 15 μL saline and 40 plaque forming units (PFU) IAV, respectively. The mice in NI group and AI group were infected intranasally with 40 PFU IAV and injected intraperitoneally with 10 mg/kg NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) or 2.5 mg/kg AP-1 inhibitor nordihydroguaiaretic acid (NDGA) once daily. The mice were euthanized at day 9 after instillation, and the hearts were removed for pathological and biochemical analysis. RESULTS: IAV infection induced significant up-regulation of ectopic trypsin, and proinflammatory cytokines interleukin 6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in the myocardium, and triggered acute myocarditis. PDTC significantly inhibited NF-κB activation and up-regulation of ectopic trypsin and proinflammatory cytokines, and effectively suppressed IAV replication and myocardial inflammatory response (P<0.01). NDGA effectively inhibited AP-1 activity (P<0.01) and mildly suppressed up-regulation of proinflammatory cytokines (P<0.05), but had no effects on the expression of ectopic trypsin, IAV replication and the extent of myocarditis (P>0.05). CONCLUSION: IAV infection induces up-regulation of ectopic trypsin and proinflammatory cytokines in myocardium predominantly by the activation of NF-κB. AP-1 signaling pathway might be only partially involved in the regulation of proinflammatory cytokines.     

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.     

Effects of baicalin on glial fibrillary acidic protein and nuclear factor-κB expression in juvenile rat hippocampus after status convulsion

AIM: To study the effects of baicalin (BC) on glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) expression and neuronal apoptosis in juvenile rat hippocampus after status convulsion (SC). METHODS: One hundred and ninety five juvenile male Sprague-Dawley rats were randomly divided into 3 groups: normal saline pretreatment group (NS group), SC group and SC with BC pretreatment group (BC group). Each of these 3 groups would be subdivided into 5 subgroups sacrificed at 4 h, 12 h, 24 h, 48 h and 72 h after SC. The rat SC model was prepared by lithium-pilocarpine chemical method. The protein expression of GFAP and NF-κB was detected by the method of immunohistochemistry. The mRNA expression of GFAP was detected by RT-PCR. The neuronal apoptosis was observed by TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: Compared with NS group, the GFAP positive cells was increased in SC group (P<0.05). Compared with SC group, the expression of GFAP was significantly reduced in BC group (P<0.05). Compared with NS group, the NF-κB positive cells was increased in SC group (P<0.05). Compared with SC group, the expression of NF-κB was significantly reduced in BC group. RT-PCR showed that the expression trend of GFAP mRNA was similar to that of the protein. Compared with NS group, the TUNEL positive cells in the hippocampus CA1 area in SC group increased significantly 12 h after SC (P<0.01), and reached a peak at 48 h. After the intervention with BC, the TUNEL positive cells decreased significantly between 12~48 h after SC (P<0.05 or P<0.01), but the number of TUNEL positive cells remained significantly greater than that in NS group (P<0.05). CONCLUSION: The expression of GFAP and NF-κB in the hippocampus increased after SC in rats. Baicalin decreases the expression of GFAP and NF-κB in hippocampus of rats with pilocarpine-induced seizures, and reduces the number of neuronal apoptosis, suggesting that baicalin may protect against the brain damage caused by status convulsion.     

Trx-1 over-expression attenuates oxidative stress injury in MPP+ -induced PC12 cells by regulating NF-κB signaling pathway

AIM:To investigate the inhibitory effect of thioredoxin 1 (Trx-1) over-expression on oxidative stress injury in 1-methyl-4-phenylpyridinium (MPP⁺)-induced rat pheochromocytoma PC12 cells by regulating NF-κB signaling pathway.METHODS:The PC12 cells were damaged by treatment with MPP⁺ at 1, 3 and 5 mmol/L, and the optimal concentration of 3 mmol/L was selected. The cell viability was measured by MTT assay. The oxidative stress indexes lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the cell culture supernatant were detected, and the protein expression of Trx-1 was determined by Western blot. Lentiviral infection with Ad-Trx-1-GFP sequence was used to establish a model of MPP⁺-treated PC12 cells with Trx-1 over-expression. The effects of Trx-1 over-expression on the cell viability, oxidative stress responses and NF-κB signaling pathway were determined by MTT assay, commercial kits and Western blot. The effects of phorbol 12-myristate 13-acetate (PMA), an activator of NF-κB signaling pathway, on the viability and oxidative stress of PC12 cells were observed. The NF-κB signaling pathway inhibitor pyrrolidine dithiocarbamate (PDTC) was used in MPP⁺-treated PC12 cells with Trx-1 over-expression, and the cell viability and oxidative stress responses were measured. RESULTS:The viability of PC12 cells, SOD activity in the supernatant and protein expression of Trx-1 were decreased, while LDH activity and MDA content in the supernatant were increased significantly by treatment with MPP⁺ at 1, 3 and 5 mmol/L. The effect of MPP⁺ at 3 mmol/L and 5 mmol/L was significantly greater than that at 1 mmol/L (P<0.05), and no significant difference between 3 mmol/L and 5 mmol/L was observed (P>0.05). The inhibitory effect of MPP⁺ on the viability of PC12 cells, and the oxidative stress injury and activation of NF-κB signaling pathway induced by MPP⁺ were significantly attenuated by over-expression of Trx-1. The inhibitory effect of MPP⁺ on the viability of PC12 cells and the oxidative stress injury induced by MPP⁺ were promoted by the activation of NF-κB signaling pathway, while the protective effects of Trx-1 over-expression on the MPP⁺-treated PC12 cells were enhanced by the inhibition of NF-κB signaling pathway. CONCLUSION:Over-expression of Trx-1 protects MPP⁺-treated PC12 cells from oxidative stress injury by regulating NF-κB signaling pathway.     

Role of Fas overexperssion in reversing cisplatin resistance in stomach cancer cells

AIM: To study the effect of Fas on cisplatin resistance in stomach cancer cells and its possible mechanisms.METHODS: The expression of Fas at mRMA and protein levels in SGC-7901 cells and SGC-7901/DDP cells was determined by RT-qPCR and Western blot. Fas-containing adenovirus vector was transfected into the SGC-7901/DDP cells to upregulate Fas expression. The cell viability was detected by CCK-8 assay. The cell cycle and cell apoptosis were analyzed by flow cytometry. The protein levels of Fas, P38/p-P38, JNK/p-JNK, cleaved caspase-8/caspase-8 and cleaved caspase-3/caspase-3 were detected by Western blot.RESULTS: The expression of Fas at both mRNA and protein levels was significantly downregulated in the SGC-7901/DDP cells. Fas expression was decreased by cisplatin in a dose-dependent manner in the SGC-7901 cells. Overexpression of Fas suppressed the viability and induced apoptosis in the SGC-7901/DDP cells, and upregulated the protein levels of p-P38, p-JNK, cleaved caspase-8 and cleaved caspase-3.CONCLUSION: Overexpression of Fas increases the sensitivity of the SGC-7901/DDP cells to cisplatin, and inhibits the cell growth and promotes cell apoptosis. The mechanism may be related to the activation of JNK and P38 pathway.