Mechanism of AKT1 negatively regulating breast cancer cell migration

AIM: To investigate the molecular mechanism and downstream signaling pathway by which AKT1 inhibition regulates breast cancer cell migration. METHODS: RNA interference was used to knockdown the expression of AKT1. Western blot assay was performed to examine the expression of AKT1 total protein, β-catenin total protein and β-catenin nuclear protein. Immunofluorescence was used to examine the cellular localization of β-catenin. Transwell assay was used to investigate whether β-catenin nuclear accumulation as an alternative pathway was responsible for breast cancer metastasis induced by AKT1 inhibition. RESULTS: The total protein expression of AKT1 was decreased in MCF-7 and MDA-MB-231 cells treated with AKT1 siRNA. A significant increase in the protein expression of β-catenin was observed in MCF-7 cells and MDA-MB-231 cells treated with AKT1 siRNA. Immunofluorescence staining showed that MCF-7 cells and MDA-MB-231 cells displayed strong β-catenin staining in the cytoplasm and nucleus after knockdown of AKT1 expression. The ability of tumor cell migration increased dramatically after treated with AKT1 specific siRNA in the breast cancer MCF-7 cells and MDA-MB-231 cells in Transwell assay. XAV-939 reversed breast cancer cell migration induced by knockdown of AKT1 expression. CONCLUSION: β-catenin nuclear accumulation contributes to AKT1 inhibition-mediated breast cancer cell migration.     

Effect of HMGB1 expression knockdown on invasion ability of breast cancer cells induced by TNF-α

AIM:To investigate the effect of high-mobility group box-1 (HMGB1) expression knockdown on the invasion ability of breast cancer cells induced by tumor necrosis factor-α (TNF-α). METHODS:HMGB1 siRNA was used to transfect into the breast cancer MDA-MB-231 cells. The expression of HMGB1 at mRNA and protein levels was determined by RT-qPCR and Western blot. After the MDA-MB-231 cells with HMGB1 expression knockdown were treated with TNF-α, the apoptosis rate was analyzed by flow cytometry, the cell invasion ability was measured by Transwell assay, and the cell migration ability was detected by cell scratch test. The protein expression of E-cadherin, MMP-2, N-cadherin, MMP-9 and Bax was determined by Western blot. RESULTS:The expression of HMGB1 at mRNA and protein levels in the MDA-MB-231 cells transfected with HMGB1 siRNA was significantly lower than that in the non-transfected cells (P<0.05). The apoptosis rate in the cells was increased after TNF-α treatment, and the cell invasion and migration abilities were also increased. The protein level of E-cadherin in the cells was decreased, the protein level of N-cadherin was increased, and the protein levels of MMP-2, MMP-9 and Bax were also increased (P<0.05). After the MDA-MB-231 cells with HMGB1 expression knockdown were induced by TNF-α, the apoptotic rate was increased, the invasion and migration abilities were decreased, the protein levels of E-cadherin and Bax were increased, and the protein levels of N-cadherin, MMP-2 and MMP-9 were decreased, as compared with the cells only induced by TNF-α without knockdown of HMGB1 expression (P<0.05). CONCLUSION:Knockdown of HMGB1 expression enhances the apoptosis of breast cancer cells induced by TNF-α, and inhibited the cell invasion, migration and epithelial-mesenchymal transition induced by TNF-α. The mechanism may be related with the changes of protein expression of MMP-2, MMP-9 and Bax.     

Effect of Wnt/β-catenin signaling pathway regulated by HDAC1 on apoptosis of breast cancer cells

AIM: To study the effect of histone deacetylase 1 (HDAC1) on the apoptosis of breast cancer cells.METHODS: The expression of HDAC1 at mRNA and protein levels in normal mammary epithelial cell line MCF-10A and breast cancer cell lines BT549, MCF-7 and MDA-MB-231 was measured by RT-qPCR and Western blot. HDAC1 siRNA was transfected into MDA-MB-231 cells, and then RT-qPCR and Western blot were used to determine the expression level of HDAC1. The cell viability was measured by MTT assay, and apoptosis was analyzed by flow cytometry. The protein levels of β-catenin, c-Myc, cyclin D1 and cleaved caspase-3 were determined by Western blot. Breast cancer cells with HDAC1 knockdown were treated with Wnt/β-catenin signaling pathway activator, and then the cell viability and apoptosis were measured.RESULTS: The expression of HDAC1 at mRNA and protein levels in BT549, MCF-7 and MDA-MB-231 cells was significantly higher than that in normal mammary epithelial cell line MCF-10A, and the highest expression level of HDAC1 was observed in MDA-MB-231 cells (P<0.05). HDAC1 siRNA reduced the expression of HDAC1 at mRNA and protein levels in the breast cancer cells. The viability of MDA-MB-231 cells was decreased after knockdown of HDAC1 expression, the apoptotic rate was increased, the protein level of cleaved caspase-3 in the cells was elevated, and the protein levels of β-catenin, c-Myc and cyclin D1 were decreased (P<0.05). Wnt/β-catenin signaling pathway activator reversed HDAC1 knockdown-induced apoptosis and decrease in viability of MDA-MB-231 cells, and reduced the protein level of cleaved caspase-3.CONCLUSION: Knockdown of HDAC1 expression induces apoptosis of breast cancer cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Atorvastatin inhibits IL-1β and IL-18 releases via lowering the expression of NLRP1 inflammasome

AIM: To explore the role of nucleotide-binding oligomerization domain-like receptor protein 1 (NLRP1) inflammasome in atorvastatin-induced reduction of interleukin-1β (IL-1β) and interleukin-18 (IL-18) releases from the THP-1 macrophages. METHODS: Lipopolysaccharide (LPS, 10 μg/L) was used to trigger the secretion of IL-1β and IL-18 in the THP-1 macrophages. The cells were incubated with different concentrations of atorvastatin (1, 10 and 20 μmol/L) for 24 h, or treated with 10 μmol/L atorvastatin for different time (12 h, 24 h and 48 h). NLRP1 siRNA was transfected into the THP-1 cells. The mRNA expression of NLRP1 inflammasome was detected by RT-PCR. The protein expression of NLRP1 inflammasome was determined by Western blot. The secretion of proinflammatory cytokines IL-1β and IL-18 was quantified by ELISA. RESULTS: Atorvastatin inhibited the mRNA and protein expression of NLRP1 inflammasome in the THP-1 macrophages in a dose- and time-dependent manner. Transfection of NLRP1 siRNA significantly decreased the protein expression of NLRP1 and promoted the suppressive effect of atorvastatin on IL-1β and IL-18 secretion in the THP-1 macrophages. CONCLUSION: Atorvastatin inhibits the production of IL-1β and IL-18 in the macrophages through decreasing NLRP1 inflammasome expression, possibly contributing to the anti-inflammatory effect of atorvastatin on atherosclerosis.     

Using folic acid-modified polyethylenimine-SPIO nanoparticles for PD-L1 siRNA delivery to target gastric cancer

AIM: To synthesis and characterize a multi-functional siRNA delivery agent with effective therapeutic effects and MR-tracing ability for programmed death ligand-1 (PD-L1) positive gastric cancer SGC-7901 cell line. METHODS: The characterization, binding ability, cytotoxicity, transfection efficiency and cellular internalization of the polyplex were determined. The PD-L1 knockdown effect was analyzed, and cytokines secreted by cocultured T cells were measured.RESULTS: We developed folic acid (FA)-PEG-SS-PEI-SPION as siRNA delivery agent for PD-L1 knockdown. At N/P ratio of 10, the FA-PEG-SS-PEI-SPION bound PD-L1 siRNA to form polyplex in a diameter of (116.7±2.5) nm with zeta potential of (9.14±0.80) mV. Transfection efficiency of the targeted polyplex was (95.06±0.44)%, compared with (93.87±1.05)% of the untargeted polyplex. Mean fluorescence intensity of the targeted polyplex was 1 892.67±81.51, significantly higher than 1 324.33±186.58 of the untargeted. The cellular magnetic resonance (MR) imaging showed the polyplex also acted as T₂ weighted contrast agent for cancer MR imaging. The relative mRNA level of PD-L1 in polymer/siRNA-2 treatment group was (9.07±0.79)%. Decreased protein expression of PD-L1 was showed by Western blot. The secretion levels of IFN-γ and TNF-α in cocultured T cells increased, while that of IL-10 decreased. CONCLUSION: Our findings highlighted the potential of the multifunctional theranostic nanoparticles for effective targeting PD-L1 knockdown therapy and MR imaging diagnosis in gastric cancers.     

Role of NOX1 in TNF-α-induced oxidative damage and inflammation in alveolar epithelial cells

AIM: To explore the role of NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced oxidative damage and inflammation in alveolar epithelial cells.METHODS: The mRNA and protein expression levels of NOX1 in alveolar epithelial cells after TNF-α treatment were determined by real-time PCR and Western blot. NOX1 siRNA and its negative control were transfected into the alveolar epithelial cells. After the induction of TNF-α, NOX1 levels in the cells were measured by real-time PCR and Western blot, and the content of malondialdehyde (MDA) in the cells was detected by thiobarbituric acid method. Xanthine oxidation assay was used to detect the activity of superoxide dismutase (SOD) in the cells. The contents of interleukin-4 (IL-4), IL-6 and IL-1β in cell culture medium were examined by ELISA. The rate of apoptosis was analyzed by flow cytometry. Western blot was used to detect the level of apoptotic protein cleaved caspase-3.RESULTS: The expression of NOX1 at mRNA and protein levels in TNF-α-induced cells was increased after induction (P<0.05). After transfection of NOX1 siRNA, the expression of NOX1 at mRNA and protein levels in the cell was downregulated (P<0.05). Transfection of siRNA negative control had no effect on the expression level of NOX1 in the cells. The content of MDA in the cells after TNF-α treatment was increased, the activity of SOD was reduced, the releases of IL-4, IL-6 and IL-1β by the cells were increased, and the apoptotic rate and the level of apoptotic protein cleaved caspase-3 were increased as compared with the cells that were not treated with TNF-α (P<0.05). The content of MDA in the cells with NOX1 knockdown induced by TNF-α was reduced, the activity of SOD elevated, and the releases IL-4, IL-6 and IL-1β, the apoptotic rate and the level of apoptotic protein cleaved caspase-3 decreased, as compared with the cells only treated with TNF-α induction (P<0.05).CONCLUSION: TNF-α induces the expression of NOX1 in the alveolar epithelial cells. Knockdown of NOX1 expression reduces cellular oxidative damage, releases of inflammatory factors, and cell apoptosis.     

Knockdown of IK1 potassium channel inhibits expression and function of ClC-3 chloride channel

AIM: To investigate whether the ClC-3 chloride channel is an acting target of the IK1 potassium channel, and to study the action of IK1 potassium channel on the functional activities and expression of ClC-3 chloride channels. METHODS: IK1 gene was silenced by IK1 siRNA in poorly-differentiated nasopharyngeal carcinoma cells (CNE-2Z). Real-time PCR and Western blot were used to detect the expression of ClC-3 at mRNA and protein levels. The distribution of ClC-3 protein in the cells was observed under confocal immunofluorescence microscope. The chloride current was recorded by the patch-clamp technique. RESULTS: IK1 siRNA was successfully transfected into the CNE-2Z cells and knocked down the expression of IK1 potassium. The mRNA expression of ClC-3 was increased by the IK1 siRNA. IK1 siRNA inhibited the expression of ClC-3 protein. A chloride current was activated by hypotonic challenges, and the hypotonicity-induced current was reduced in the cells which successfully transfected with IK1 siRNA. CONCLUSION: The knockdown of IK1 potassium channels inhibits the expression and function of ClC-3 chloride channel.     

Effect of ABCE 1 gene silencing on proliferation and migration of human bladder cancer cell line T24

AIM: To investigate the effect of small interfering RNA (siRNA)-mediated ABCE1 knockdown on the survival, cell cycle and invasion of human bladder cancer cell line T24. METHODS: The siRNA against ABCE1 was constructed and transfected into the T24 cells with Lipofectamine^TM 2000. The expression of ABCE1 was detected by RT-PCR and Western blot. Flow cytometry was used to detect the cell cycle. The effects of ABCE1 gene silencing on proliferation, migration and invasion of T24 cells were evaluated by CCK-8 assay, wound-healing assay and Transwell invasion assay, respectively. RESULTS: Compared with control group and blank group, the mRNA and protein levels of ABCE1 were significantly decreased in experimental group after transfected with ABCE1 siRNA. The cell cycle was arrested at G₀/G₁ phase and the cell number in S phase was decreased in the T24 cells. Compared with control group and blank group, the proliferation of T24 cells in experimental group was inhibited significantly, and the migration and invasion abilities of T24 cells in experimental group decreased significantly. CONCLUSION: Knockdown of ABCE1 gene may decrease migration, invasion and proliferation abilities in T24 cells.     

Effects of GSK-3β knockdown by RNA interference on formation of keloid in vitro

AIM: To study the suppressive effect of glycogen synthase kinase-3β (GSK-3β) knockdown by RNA interference on the formation of keloid. METHODS: Human keloid fibroblasts (KFB) in vitro were transfected with 3 pairs of specific GSK-3β small interfering RNA (siRNA). The best siRNA to inhibit the GSK-3β expression in human KFB was screen by RT-PCR and Western blot. The expression of GSK-3β and related proteins at mRNA and protein levels in the KFB was determined by RT-PCR and Western blot.RESULTS: The GSK-3β siRNA1434 remarkably inhibited the expression of GSK-3β at mRNA and proteins levels in the human KFB. After transfection with GSK-3β siRNA, the protein levels of β-catenin, p-GSK-3β, Wnt2 and cyclin D1 were all decreased. KFB growth became slow. With the extension of time, the inhibition of cell growth increased, and the cell doubling time was significantly delayed. CONCLUSION: siRNA targeting GSK-3β efficiently knocks down the expression of GSK-3β in the human KFB, and inhibits the activation of Wnt signaling pathway, thus inhibiting the growth of keloid. GSK-3β may be a potential therapeutic target for keloid.     

TPX2 induces apoptosis of rectal cancer HR-8348 cells through p38 MAPK signaling pathway

AIM: To study the effect of targeting protein for Xenopus kinesin-like protein 2 (TPX2) expression knockdown on the apoptosis of rectal cancer HR-8348 cells.METHODS: The HR-8348 cells transfected with TPX2 small interfering RNA (siRNA) served as TPX2 siRNA group. The non-transfected cells were used as control group. The cells transfected with siRNA negative control (siRNA-NC) were used as siRNA-NC group. The TPX2 siRNA-transfected cells exposed to p38 MAPK inhibitor SB203580 served as TPX2 siRNA+SB203580 group. The expression of TPX2 at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry. The protein levels of p38 MAPK, p-p38 MAPK, cleaved caspase-3 and Bcl-2 in the HR-8348 cells were determined by Western blot.RESULTS: After transfection, the expression of TPX2 at mRNA and protein levels was decreased in TPX2 siRNA-transfected cells (P<0.05). Transfection with siRNA-NC had no effect on TPX2 mRNA and protein levels in the cells. After knockdown of TPX2 expression, the viability of rectal cancer HR-8348 cells and the expression of Bcl-2 were decreased, while the apoptotic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK were increased significantly reduced (P<0.05). Compared with TPX2 siRNA group, the apopto-tic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK in TPX2 siRNA+SB203580 group were significantly decreased, while the viability was significantly increased (P<0.05).CONCLUSION: Knockdown of TPX2 expression promotes apoptosis of rectal cancer HR-8348 cells by activating p38 MAPK signaling pathway.     

Effects of TRPC1 on TGF-β1-induced migration of human bronchial epithelial cells

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the migration of human bronchial epithelial cells (16HBE) induced by transforming growth factor-β1 (TGF-β1). METHODS: Silencing of TRPC1 gene expression was performed by siRNA. The cell activity and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The migration and invasion abilities of the 16HBE cells were detected by wound- healing assay and Transwell assay. The protein expression of E-cadherin and vimentin was determined by Western blot. RESULTS: TGF-β1 treatment significantly enhanced the cell migration distance compared with control groups (P < 0.01). The results of CCK-8 assay and flow cytometry indicated that there were no significant difference in proliferation and apoptosis among TRPC1 siRNA group, TGF-β1 group and control group (P > 0.05). The results of wound-healing and Transwell assays showed that migration and invasion abilities in TRPC1 siRNA + TGF-β1 group were markedly suppressed compared with TGF-β1 group (P < 0.01). The protein expression of E-cadherin and vimentin induced by TGF-β1 was inhibited by TRPC1 silencing compared with TGF-β1 group (P < 0.05). CONCLUSION: TRPC1 is involved in the migration of human bronchial epithelial cells induced by TGF-β1 through regulating the protein expression of E-cadherin and vimentin.     

Effects of autophagy inhibitor bafilomycin A1 on macrophages polarization

AIM:To investigate the effect of bafilomycin A1 (Baf A1) on polarization in mouse macrophages RAW264.7. METHODS:The macrophages RAW264.7 were treated with Baf A1, the concentration of M1/M2 polarization related cytokines were determined by ELISA. The markers of M1/M2 polarization in the macrophages were determined by flow cytometry. The formation of autophagicbody was observed by immunofluorescence. Western blot was used to detect the expression of autophagy related protein levels. RESULTS:The concentration of M1 related proinflammatory cytokine tumor necrosis factor-α (TNF-α) was increased significantly after Baf A1 intervention (P<0.01). However, the concentration of M2 related anti-inflammatory cytokines IL-10 and IL-13 showed no significant difference. The double positive rate of CD197 and HLA-DR (M1 marker) positive cells in Baf A1 treated group were significant higher than that in control group (P<0.05), indicated that Baf A1 induced polarization of macrophage to M1. The results of immunofluoresence showed that the autophagosomes formation was notable increased in Baf A1 group (P<0.05), meanwhile Western blot results showed the expression of autophay related protein LC3-Ⅱ but not P62 was marked up-regulated (P<0.05). For autophagy activator rapamycin (Rapa) treated group, autophagosome formation was also significant increased (P<0.05), but the expression of P62 was notable down-regulated (P<0.05). CONCLUSION:Baf A1 induces polarization of mouse macrophage RAW264.7 to M1, which may be related to the inhibitory effect on the formation of autolysosome.     

Effect of HDAC1 silencing on apoptosis of squamous cell carcinoma of skin via regulating STAT3 signaling pathway

AIM: To investigate the effect of histone deacetylase 1 (HDAC1) silencing on apoptosis of squamous cell carcinoma of skin. METHODS: Skin squamous cell carcinoma A431 cells were transfected with HDAC1 small interfering RNA (HDAC1 siRNA) or small interfering RNA negative control (siRNA NC). The expression levels of HDAC1 in transfected cells were detected by RT-PCR and Western blot. The cell viability was measured by MTT assay, and the apoptosis was analyzed by flow cytometry. The protein levels of STAT3, p-STAT3 and cleaved caspase-3 were determined by Western blot. The inhibitor of STAT3 signaling pathway was used to treat the A431 cells transfected with HDAC1 siRNA. The cell viability was detected by MTT assay, the apoptosis was analyzed by flow cytometry, and the protein levels of STAT3, p-STAT3 and cleaved caspase-3 were determined by Western blot. RESULTS: HDAC1 siRNA inhibited the expression of HDAC1 at mRNA and protein levels in the A431 cells. After interfering with the expression of HDAC1, the cell viability and the protein level of p-STAT3 in the cells decreased, while the apoptotic rate and the protein level of cleaved caspase-3 in the cells were increased. After treatment with the inhibitor of STAT3 pathway, the viability of A431 cells transfected with siRNA and the protein level of p-STAT3 decreased, while the apoptotic rate and the protein le-vel of cleaved caspase-3 in the cells were increased. CONCLUSION: Interference with HDAC1 expression may regulate the STAT3 signaling pathway to inhibit the viability of skin squamous cell carcinoma cells, thus promoting the apoptosis of squamous cell carcinoma of skin.     

EZH2-mediated regulation of NF-κB target gene expression in gastric cancer

AIM: To explore the mechanism by which over-expression of enhancer of zeste homolog 2 (EZH2) in a panel of gastric cancer cell lines is involved in tumorigenesis of gastric cancer. METHODS: Real-time PCR and Western blot were employed to examine the mRNA and protein levels of EZH2, respectively. MTS assay, cell migration and soft agar assay were performed to investigate the role of EZH2 in the regulation of stomach cancer behaviors. The effect of EZH2 on NF-κB target gene expression was determined by Luciferase reporter and real-time PCR. Co-immunoprecipitation was used to analyze the interaction of EZH2 and p65 in HEK293T cells. RESULTS: The expression levels of EZH2 were significantly increased in the gastric cancer cells compared with normal gastric epithelial cells. Pharmacological inhibition by DZNep or knockdown of EZH2 significantly compromised AGS and SNU-16 cell activity, cell migration and anchorage-independent cell growth. Moreover, siRNA knockdown of EZH2 impaired NF-κB downstream targets, such as IL-8, CXCL5 and CCL20. In addition, the interaction of EZH2 and p65 was detected. CONCLUSION: EZH2 mediates the growth of gastric cancer cells through the regulation of NF-κB downstream gene expression.     

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.     

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.     

Effects of SRSF9/SRp30c on proliferation and migration abilities of glioma cells by regulating GRβ

AIM: To investigate the expression of serine-arginine-rich splicing factor 9/serine-arginine-rich protein 30c (SRSF9/SRp30c) and glucocorticoid receptor β (GRβ) in the glioma cells and the relationship of them. METHODS: Small interfering RNA (siRNA) was used to knock down the expression of SRSF9 in the U87 cells. Short hairpin RNA (shRNA) derived from lentivirus was used to establish U87 stable knockdown cell line. Fluorescence microscopy was used to observe and detect transfection efficiency. The expression of GRβ and SRSF9/SRp30c at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability, colony formation ability and migration ability were measured by CCK-8 assay, colony formation assay and wound healing experiment. RESULTS: The mRNA and protein levels of SRSF9/SRp30c and GRβ in the U87 cells were both down-regulated after knockdown of SRSF9 (P<0.05). Fluorescence microscopic observation showed that a stable cell line was constructed successfully, and the transfection efficiency exceeded 80%. After knockdown of SRSF9 expression in the U87 cells, the cell viability and colony formation ability were reduced (P<0.05). The migration ability was weakened significantly after SRSF9 was knocked down (P<0.05). CONCLUSION: SRSF9/SRp30c may promote the proliferation and migration of the glioma cells by regulating GRβ.     

Effects of lutein on viability of breast cancer cells

AIM:To investigate the effect of lutein on the viability of breast cancer cells and its possible mechanism. METHODS:The human breast cancer T47D cells were divided into control group and lutein (6.25, 12.5, 25, 50 mg/L) treatment groups. The effect of lutein on the viability of T47D cells was measured by MTT assay. The mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione peroxidase 1 (GPx1) and superoxide dismutase 2(SOD2) was detected by RT-qPCR. Fluorescent probes DCFH-DA was used to determine the production of reactive oxygen species (ROS). The protein expression of Nrf2 and p65 was determined by Western blot. RESULTS:The MTT results showed that lutein inhibited T47D breast cancer cell viability in a dose-and time-dependent manner. The RT-qPCR results showed that the mRNA levels of Nrf2, GPx1 and SOD2 were higher in lutein treatment groups than those in the control group (P<0.05), and with the increased concentrations and extension of intervention time of lutein, the relative mRNA levels were all increased. The ROS levels were significantly decreased in the lutein-treated groups (P<0.05). The results of Western blot demonstrated that the protein expression of Nrf2 was significantly increased (P<0.05), and p65 protein was decreased (P<0.05) in a dose-dependent manner with lutein treatment for 48 h. CONCLUSION:Lutein significantly inhibits the viability of breast cancer cells, and the inhibition roles may be related to up-regulation of the expression of Nrf2, antioxidant enzymes GPx1 and SOD2 mRNA expression and down-regulation of oxidative stress, thus blocking the NF-κB signaling pathway.     

Functional expression of B7H1 on pancreatic carcinoma panc-1 cells

AIM: To observe the effect of B7H1 expression in pancreatic carcinoma cells on the proliferation and activation of co-cultured T lymphocytes. METHODS: B7H1 expression in panc-1 cells before and after interferon-γ(IFN-γ) treatment or B7H1-siRNA transfection was evaluated by RT-PCR and flow cytometry. The influence of B7H1 expression on co-cultured PHA-activated T lymphocytes was determined by the methods of MTT and enzyme-linked immunosorbent assay (ELISA). RESULTS: B7H1 was highly expressed in panc-1 cells and up-regulated after IFN-γ stimulation. Such up-regulation led to the significant inhibition of T cell proliferation and secretion of cytokines such as IFN-γ and interleukin-2(IL-2). However, IL-10 production was enhanced. In contrast, knockdown of B7H1 expression in panc-1 cells by RNA interference resulted in increased T cell proliferation as well as IFN-γ and IL-2 production. Meanwhile, the IL-10 secretion decreased. CONCLUSION: B7H1-expressing panc-1 cells suppress T cell function by inhibiting T cell proliferation and production of Th1 cytokines. Suppression of B7H1 expression through siRNA restores T cell immune functions, indicating a potential strategy for immunotherapy against pancreatic cancer.