Sphingosine kinase 1 enhances invasion and migration of non-small-cell lung cancer cells by ERK1/2 signaling pathway

AIM To explore the effects of sphingosine kinase 1 (SphK1) on the migration and invasion of non-small-cell lung cancer (NSCLC) cells and its mechanism. METHODS Thirty-one tumor specimens, which were surgically resected and routinely histologically confirmed as NSCLC, and matched adjacent lung tissues were selected. Immunohistochemical staining and RT-qPCR were used to detect the expression of SphK1. The pcDNA3.1-SphK1 vector (SphK1 group), empty pcDNA3.1 vector control (NC group), SphK1 siRNA (siSphK1 group) or control siRNA (siNC group) was transfected into human lung adenocarcinoma A549 cells, and the protein levels of SphK1, E-cadherin, fibronectin and p-ERK1/2 were determined by Western blot. The effects of over-expression of SphK1 and inhibition of ERK1/2 on migration and invasion of A549 cells were evaluated by Transwell assays. RESULTS SphK1 was highly expressed in the NSCLC tissues and was associated with tumor stage. SphK1 over-expression significantly promoted the migration and invasion of A549 cells, increased the protein levels of p-ERK1/2 and fibronectin, and decreased the protein expression of E-cadherin (P<0.05), but the opposite result was observed after SphK1 interference. The ERK1/2 inhibitor U0126 significantly inhibited the up-regulation of p-ERK1/2 and fibronectin levels and the down-regulation of E-cadherin expression induced by SphK1 over-expression, and also inhibited the invasion and migration of A549 cells promoted by SphK1 over-expression (P<0.05). CONCLUSION SphK1 may reduce E-cadherin protein levels, increase fibronectin protein levels, and promote the invasion and migration of NSCLC cells through ERK1/2 signaling pathway.     

Aβ1-40 induces inflammation and phenotypic switching via activation of MAPKs signaling pathway in vascular smooth muscle cells

AIM To investigate the effects of amyolid β-protein 1-40 （Aβ1-40） on inflammation, viability, migration and phenotypic switching in vascular smooth muscle cells （VSMCs）, and to analyze the underlying mechanisms. METHODS The VSMCs were treated with Aβ1-40 at different concentration gradients for appropriate time. CCK-8 and Transwell assays were performed to evaluate the viability and migration ability of VSMCs. The levels of inflammatory factors including interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α）, phenotypic switching-related proteins including α?smooth muscle actin （α?SMA）, osteopontin （OPN） and Krüppel?like factor 4 （KLF4）, and mitogen-activated protein kinases （MAPKs） signaling pathway-related proteins including p-p38 MAPK, p-ERK1/2 and p-JNK were determined by Western bolt. RESULTS After Aβ1-40 treatment, the levels of inflammatory factors IL-1β and TNF-α in the VSMCs were significantly increased （P<0.05）, and the expression of phenotypic switching-related proteins was altered, as indicated by down-regulation of α?SMA and up-regulation of OPN and KLF4 （P<0.05）. Treatment with Aβ1-40 within a certain concentration range promoted the viability and migration of the VSMCs. In addition, the protein levels of p-p38 MAPK, p-ERK1/2 and p-JNK were significantly increased by Aβ1-40 treatment （P<0.05）. Furthermore, pretreatment with specific inhibitors of MAPKs pathway significantly reduced the levels of IL-1β and TNF-α （P<0.05）, and inhibited the phenotypic switching, as indicated by up-regulation of α?SMA and down-regulation of OPN and KLF4 （P<0.05）. CONCLUSION Treatment with Aβ1-40 induces the inflammation and phenotypic switching in VSMCs via activation of MAPKs signaling pathway.     

Procaine regulates CXCR7 and affects AKT/STAT3 signaling pathway to inhibit viability,migration and invasion of bladder cancer cells

AIM To investigate the effects of procaine (PCA) and CXC chemokine receptor 7 (CXCR7) on the viability, migration and invasion of bladder cancer cells and its potential mechanism. METHODS Human bladder cancer RT4 cells were treated with PCA at different concentrations, and were divided into PBS group (without PCA treatment), PCA group (treated with 4 mmol/L PCA), siRNA negative control (si-Con) group (transfected with si-Con), CX?CR7 siRNA (si-CXCR7) group (transfected with si-CXCR7), PCA+pcDNA group (treated with 4 mmol/L PCA and transfected with pcDNA) and PCA+pcDNA-CXCR7 group (treated with 4 mmol/L PCA and transfected with pcDNA-CX?CR7). The siRNA and pcDNA were transfected into the RT4 cells by liposome method. The mRNA expression of CX?CR7 in the RT4 cells was detected by RT-qPCR. The cell viability was measured by CCK-8 assay. The invasion and migration abilities of the cells were detected by Transwell assays. The protein levels of CXCR7, AKT, STAT3, p-AKT and p-STAT3 were determined by Western blot . RESULTS Compared with PBS group, the viability, migration ability and invasion ability of the RT4 cells treated with PCA at different concentrations were significantly decreased (P<0.05), and the expression of CXCR7 at mRNA and protein levels in PCA group was also significantly decreased (P<0.05). Compared with si-Con group, the expression of CXCR7 at mRNA and protein levels in si-CXCR7 group was significantly decreased, and the viability, migration ability and invasion ability of the cells were also significantly decreased (P<0.05). Compared with PCA+pcDNA group, the expression of CXCR7 at mRNA and protein levels in PCA+pcDNA-CXCR7 group was significantly increased, and the viability, migration ability and invasion ability of the cells were also significantly increased (P<0.05). Compared with PBS group, the protein levels of p-AKT and p-STAT3 in PCA group were significantly decreased(P<0.05). Compared with PCA+pcDNA group, the protein levels of p-AKT and p-STAT3 in PCA+pcDNA-CX?CR7 group were significantly increased (P<0.05). No significant difference in the protein levels of AKT and STAT3 among the groups was observed. CONCLUSION Treatment with PCA inhibits the viability, migration and invasion of bladder cancer cells by inhibiting the expression of CXCR7. Over-expression of CXCR7 reverses this effect of PCA. Its mechanism may be related to AKT/STAT3 signaling pathway.     

Effect of formononetin on viability,migration and invasion of ovarian cancer cells in vitro

AIM To observe the effect of formononetin on the viability, migration and invasion of ovarian cancer cells, and to explore its mechanism. METHODS Human ovarian serous cystadenocarcinoma SKOV-3 cells were cultured in vitro. The cells were treated with formononetin at 0, 25, 50 and 100 μmol/L for 48 h. The cell viability was measured by MTS assay. The migration and invasion abilities of the SKOV-3 cells were detected by scratch wound assay and Transwell assay. RT-qPCR and Western blot were used to detect the mRNA and protein levels of E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS The viability of SKOV-3 cells was decreased with the increase in the formononetin concentration compared with control group (P<0.01). The wound migration distance of the cells in 50 μmol/L formononetin group was less than that in control group (P<0.01). The number of invasive SKOV-3 cells across the Transwell sub-compartment was significantly decreased in 50 μmol/L formononetin group compared with control group (P<0.01). The mRNA and protein levels of E-cadherin in 50 μmol/L formononetin group were significantly higher than those in control group (P<0.01), while the mRNA and protein levels of MMP-9 in 50 μmol/L formononetin group were significantly lower than those in control group (P<0.01). CONCLUSION Formononetin inhibits the migration and invasion abilities of ovarian cancer SKOV-3 cells by increasing expression of E-cadherin and decreasing expression of MMP-9.     

Niflumic acid inhibits the proliferation,migration and invasion of glioma U87 cells

AIM To investigate the effect of niflumic acid （NFA） on human glioma U87 cells and to clarify the potential mechanism. METHODS The U87 cells were cultured in vitro and divided into blank control group, and 50, 100 and 200 μmol/L NFA groups. MTT assay was performed to determine the viability of cells in various groups. Migration and invasion abilities were measured by real-time cell analysis （RTCA）. RESULTS The results of MTT assay showed that compared with blank control group, the viability of U87 cells was increased after treatment with NFA for 12 h （P<0.05 or P<0.01）, while the viability was significantly decreased after treatment with NFA for 24 and 48 h （P<0.05 or P<0.01） in a concentration-dependent manner. The results of RTCA showed that compared with control group, the cell migration and invasion abilities were inhibited in 100 and 200 μmol/L NFA groups （P<0.05 or P<0.01） and the inhibitory effects were more obvious in 200 μmol/L NFA group （P<0.01）. CONCLUSION NFA inhibits the viability, migration and invasion of human glioma U87 cells.     

Ligustilide inhibits angiogenesis and epithelial-mesenchymal transition in human hemangioendothelial cells by reducing VEGF levels

AIM To investigate the effect of ligustilide on human hemangioendothelial cells (HemECs) and to analyze its mechanism. METHODS The effect of ligustilide at different concentrations on the viability of HemECs was measured by CCK-8 assay. The HemECs were divided into control group and ligustilide （10, 25 and 50 μmol/L） treatment groups, and the proliferation of HemECs was detected by EdU staining. The effects of ligustilide on the angiogenesis of HemECs was tested by microtubule formation experiment. The protein expression of vascular endothelial growth factor (VEGF) and epithelial-mesenchymal transition (EMT)-related markers in HemECs cells was determined by Western blot. RESULTS There was no significant difference in the viability of the cells treated with ligustilide at the concentrations between 0.1~50 μmol/L compared with control cells. Compared with control group, ligustilide at 25 and 50 μmol/L significantly reduced the number of EdU-positive cells and microtubule-like structures (P<0.05), reduced the protein expression level of VEGF (P<0.05), increased the protein expression of E-cadherin, and decreased the protein expression of vimentin and β-catenin (P<0.05). Compared with control group, the expression of VEGF and vimentin was significantly up-regulated, and the protein expression of E-cadherin was significantly down-regulated in VEGF overexpression group (P<0.05). Compared with VEGF overexpression group, the expression of VEGF and vimentin in 50 μmol/L ligustilide-treated VEGF-overexpressing cells were significantly reduced (P<0.05), and the protein expression of E-cadherin was significantly increased (P<0.05). CONCLUSION Ligulide inhibits the proliferation of HemECs, and also inhibits the angiogenesis and EMT process of HemECs by reducing the level of VEGF.     

Expression of histone chaperone ASF1B in prostate cancer cells and its effect on cell viability in vitro

AIM To investigate the expression of histone chaperone anti-silencing function 1B （ASF1B） in prostate cancer cells and its effect on cell viability in vitro. METHODS Human prostate cancer PC-3 cells were used, and knockdown of ASF1B was conducted by small interfering RNA （siRNA） transfection into the cells. The cells were divided into control group, siRNA negative control vector （mock） group and siRNA-ASF1B group. The viability of the PC-3 cells treated with ASF1B-siRNA for 12, 24 and 48 h was measured by MTT assay. Flow cytometry was used to analyze cell apoptosis and cell cycle distribution. The mRNA expression of apoptosis-related molecules was detected by RT-qPCR, and the expression levels of MAPK/JNK/ERK signaling pathway-related proteins were determined by Western blot. RESULTS The protein level of ASF1B in the normal cells （benign prostatic hyperplasia） was significantly lower than that in the PC-3 cells （P<0.01）. Compared with control group and mock group, the protein expression level of ASF1B in the PC-3 cells transfected with siRNA-ASF1B plasmid and the viability of the PC-3 cells were significantly decreased （P<0.01）. The apoptotic rate of the PC-3 cells transfected with siRNA-ASF1B plasmid was significantly higher than that in control group （P<0.01）, and the cell cycle was arrested at G₁ phase. The mRNA levels of p53, caspase-3, Bax and PARP-1 in the PC-3 cells transfected with siRNA-ASF1B plasmid were up-regulated compared with those in control group and Mack group （P<0.01）. In addition, the protein levels of MAP2K4 and p-JNK in the PC-3 cells in siRNA-ASF1B group were significantly higher than those in mock group （P<0.01）, while the protein level of p-ERK was significantly lower than that in mock group （P<0.01）. CONCLUSION ASF1B silencing induces G₁ arrest and promotes apoptosis of PC-3 cells. Activating MAPK/JNK/ERK signaling pathway may be a possible contributor to the anti-prostate cancer effect of siRNA-ASF1B.     

Rab11-FIP4 regulates migration and invasion of colorectal cancer cells through IGF1R

AIM To investigate the role, clinical implications and the underlying mechanisms of Rab11-family interacting protein 4 (Rab11-FIP4) in colorectal cancer (CRC). METHODS The expression levels of Rab11-FIP4 in CRC tissues and corresponding paracancerous tissues were compared by immunohistochemistry, Western blot and RT-qPCR. The above methods were also used to detect the expression levels of Rab11-FIP4 in CRC cells under normal environment and hypoxia. The patiens were divided into Rab11-FIP4 high expression group (n=61) and Rab11-FIP4 low expression group (n=39) according to the immunohistochemical staining score.The overall survival and recurrence time of the 2 groups were compared by Kaplan-Meier survival analysis. HCT116 and LoVo cells with stable over-expression of Rab11-FIP4 were constructed using a lentiviral system. The cytological characteristics effects of Rab11-FIP4 over-expression in CRC cells were examined by CCK-8 assay, clonogenic assay and the Transwell assay. The co-immunoprecipitation was used to detect the correlation between Rab11-FIP4 and insulin-like growth factor 1 receptor (IGF1R). Human phosphokinase array was performed to investigate the signaling pathhway affected by IGF1R in CRC cells with increased expression of Rab11-FIP4. The relationship between Rab11-FIP4 and hypoxia-inducible factor 1α (HIF-1α) was analyzed by tissue microarrays and dual-luciferase reporter assay. RESULTS Rab11-FIP4 expression was up-regulated in CRC tissues and high expression of Rab11-FIP4 was associated with poor prognosis of the patients with CRC (P<0.05). Over-expression of Rab11-FIP4 promoted the viability, migration and invasion of CRC cells (P<0.05). High expression of Rab11-FIP4 regulated ERK1/2 and AKT signaling pathway via IGF1R (P<0.05). Hypoxia promoted the activation of HIF-1α on the Rab11-FIP4 promoter, thereby up-regulating the expression of Rab11-FIP4 (P<0.05). CONCLUSION Rab11-FIP4 may act as an oncogene to regulate migration and invasion of colorectal cancer cells through IGF1R.     

Paeonol affects viability and migration ability of hepatocellular carcinoma cells by up-regulating miR-424-3p and inhibiting PI3K/AKT signaling pathway

AIM To investigate the effect of paeonol on the viability and migration ability of hepatocellular carcinoma cells and its molecular mechanism. METHODS Human hepatocellular carcinoma Hep3B cells was treated with paeonol at different concentrations (50, 100, 200 and 400 mg/L). The cell viability was measured by CCK-8 assay to determine the optimal drug concentration. The Hep3B cells were divided into normal control (NC) group, paeonol group, miR-NC group, miR-424-3p group, paeonol+anti-miR-NC and paeonol+anti-miR-424-3p group. The expression level of miR-424-3p was detected by RT-qPCR. The migration ability was detected by Transwell assay. The protein levels of cyclin D1, matrix metalloproteinase 2 (MMP2), MMP9 and PI3K/AKT signaling pathway-related molecules were determined by Western blot. RESULTS Paeonol intervention inhibited the viability of Hep3B cells in a concentration-dependent manner (P<0.05). The concentration of paeonol at 200 mg/L was selected for the following study. Paeonol intervention inhibited the protein expression of MMP2 and MMP9 in the Hep3B cells, and inhibited the migration ability of the Hep3B cells. Paeonol intervention promoted the expression of miR-424-3p in the Hep3B cells (P<0.05). Over-expression of miR-424-3p inhibited the expression of cyclin D1, MMP2 and MMP9 in the Hep3B cells and inhibited cell viability and migration ability (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on the viability and migration ability of the Hep3B cells (P<0.05). Paeonol inhibited phosphorylation levels of PI3K and AKT in the Hep3B cells and inhibited the activation of PI3K/AKT signaling pathway (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on PI3K/AKT signaling pathway in the Hep3B cells (P<0.05). CONCLUSION Paeonol inhibits the viability and migration ability of hepatocellular carcinoma cells by up-regulating miR-424-3p and inhibiting PI3K/AKT signaling pathway.     

Effect of inhibiting lncRNA LINC01503 on viability,migration and invasion of lung cancer cells by targeted regulation of miR-335-5p

AIM To investigate the effects of long non-coding RNA (lncRNA) LINC01503 on the viability, migration and invasion of lung cancer cells and its mechanism. METHODS Human lung carcinoma H1299 cells were divided into si-NC group (transfected with si-NC), si-LINC01503 group (transfected with si-LINC01503), pcDNA group (transfected with pcDNA), pcDNA-LINC01503 group (transfected with pcDNA-LINC01503), miR-NC group (transfected with miR-NC), miR-335-5p group (transfected with miR-335-5p mimics), si-LINC01503+anti-miR-NC group (co-transfected with si-LINC01503 and anti-miR-NC), si-LINC01503+anti-miR-335-5p group (co-transfected with si-LINC01503 and anti-miR-335-5p), miR-NC+WT-LINC01503 group (co-transfected with miR-NC and WT-LINC01503), miR-NC+MUT-LINC01503 group (co-transfected with miR-NC and MUT-LINC01503), miR-335-5p+WT-LINC01503 group (co-transfected with miR-335-5p and WT-LINC01503) and miR-335-5p+MUT-LINC01503 group (co-transfected with miR-335-5p and MUT-LINC01503). The expression of miR-335-5p and LINC01503 was detected by RT-qPCR. Western blot was used to detect protein expression. MTT assay was used to detect cell viability. Transwell assay was used to detect the migration and invasion abilities. Dual-luciferase reporter assay was used to confirm the targeted relationship between LINC01503 and miR-335-5p. RESULTS Compared with normal tissues, the expression of LINC01503 was significantly increased in the lung cancer tissues, and the expression of miR-335-5p was significantly decreased (P<0.05). Compared with stage I/II , the expression level of LINC01503 in the lung cancer tissues of stage III/IV was significantly increased, and the expression of miR-335-5p was significantly decreased (P<0.05). The patients with high expression of LINC01503 had lower short-term survival rates than those with low expression of LINC01503 (P<0.05). Compared with normal human bronchial epithelial cell line BEAS-2B, the expression of miR-335-5p in lung cancer cell lines H1299, A549 and SPC-A-1 were significantly decreased, and the expression of LINC01503 was significantly increased (P<0.05). Over-expression of miR-335-5p and inhibition of LINC01503 expression inhibited the viability, migration and invasion of H1299 cells, and inhibited the protein expression of cyclin D1, matrix metalloproteinase-2 （MMP-2） and MMP-9 (P<0.05). LINC01503 targeted and regulated miR-335-5p expression, and interfering with miR-335-5p expression reversed the inhibitory effect of inhibiting LINC01503 expression on the viability, migration and invasion of H1299 cells. CONCLUSION Inhibition of lncRNA LINC01503 inhibits the viability, migration and invasion of lung cancer cells. The mechanism may be related to the targeted regulation of miR-335-5p.     

miR-485-5p inhibits viability and migration and invasion abilities of hepatocellular carcinoma Hep3B cells by targeting SOX5

AIM: To investigate the effects of microRNA-485-5p (miR-485-5p) on the viability, migration and invasion abilities of hepatocellular carcinoma cells and the underlying mechanism. METHODS: The expression levels of sex determining region Y-box 5 (SOX5) mRNA and miR-485-5p in the hepatocellular carcinoma Hep3B cells were detected by RT-qPCR with normal hepatocyte THLE-3 as control. Western blot was used to measure the expression levels of SOX5, proliferating cell nuclear antigen (PCNA), Ki67, cyclin D1 and matrix metalloproteinase-2 (MMP-2). The viability of Hep3B cells was measured by MTT assay. The migration and invasion abilities of the Hep3B cells were detected by Transwell assay. Dual-luciferase reporter assay system was applied to verify the relationship between miR-485-5p and SOX5. RESULTS: Compared with the control cells, the expression level of miR-485-5p was decreased in hepatocellular carcinoma cells Hep3B, Huh7 and HCCLM3 (P<0.05), while the expression of SOX5 at mRNA and protein levels were significantly increased (P<0.05). Over-expression of miR-485-5p inhibited the viability, migration and invasion of Hep3B cells. miR-485-5p targeted the 3′-UTR of SOX5 and negatively regulated the expression of SOX5. Knocking-down of SOX5 expression inhibited the viability, migration and invasion of Hep3B cells. Over-expression of SOX5 partially reversed the inhibitory effect of miR-485-5p over-expression on the viability, migration and invasion of Hep3B cells. CONCLUSION: miR-485-5p inhibits the viability, migration and invasion of Hep3B cells by targeting SOX5 gene. miR-485-5p is a potential molecular target for hepatocellular carcinoma.     

Expression of TSTA3 in esophageal squamous-cell carcinoma and its significance

AIM To investigate the expression of TSTA3 in esophageal squamous cell carcinoma (ESCC) and the effects of TSTA3 on the proliferation, invasion and migration abilities of human ESCC cell lines KYSE150 and KYSE450. METHODS The expression of TSTA3 was detected by immunohistochemistry in 45 cases of ESCC and matched adjacent tissues. The mRNA expression levels of TSTA3 in ESCC cells were detected by qPCR. Over-expression of TSTA3 in KYSE150 cells and KYSE450 cells was carried out by lentivirus infection. The effects of TSTA3 on the viability and colony formation ability of ESCC cells were examined by MTT assay and colony formation assay. Transwell assays were performed to detect the effects of TSTA3 on migration and invasion abilities of ESCC cells. The effect of TSTA3 on core fucosylation modification of ESCC cells was analyzed by flow cytometry. RESULTS The expression of TSTA3 in the ESCC tissues was significantly higher than that in matched adjacent tissues (P<0.01). Over-expression of TSTA3 had no effect on the proliferation of ESCC cells (P>0.05), but promoted the migration and invasion of ESCC cells (P<0.01). Moreover, over-expression of TSTA3 increased the core fucosylation modification level of ESCC cells (P<0.01). CONCLUSION TSTA3 may promote the development and progression of ESCC via regualting fucosylation modification level, and serve as a biomarker for early diagnosis and treatment of ESCC.     

Preliminary exploration for effect of EphA2 on drug resistance of colorectal carcinoma cells

AIM: To investigate the effect of Eph receptor A2 (EphA2) on drug resistance of colorectal carcinoma cells and its possible mechanisms. METHODS: Real-time PCR and Western blot were used to detect the expression of EphA2 at mRNA and protein levels in LoVo and LoVo/5-FU cells. EphA2 siRNA was transfected to down-regulate the EphA2 expression in LoVo/5-FU cells, and the drug sensitivity was calculated by CCK-8 assay. Meanwhile, cell migration and invasion were measured by wound healing assay and Transwell assay, and the protein levels of E-cadherin, β-catenin, N-cadherin, vimentin, Notch and Snail were determined by Western blot. RESULTS: The expression of EphA2 at both mRNA and protein levels was significantly up-regulated in LoVo/5-FU cells (P<0.05). Knockdown of EphA2 suppressed the cell viability, and migration and invasion abilities, but promoted drug sensitivity of LoVo/5-FU cells. Up-regulation of E-cadherin and β-catenin, and down-regulation of N-cadherin and vimentin were observed, indicating that the epithelial-mesenchymal transition (EMT) process was suppressed. Knockdown of EphA2 decreased the expression levels of Notch and Snail. CONCLUSION: Down-regulation of EphA2 partly reverses drug resistance of LoVo/5-FU cells. The mechanism may be related to suppressing cell growth, migration, invasion and EMT process via Notch/Snail signaling pathway.     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）, and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）, the apoptotic rate was significantly increased （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）, and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

Dasatinib inhibits the viability and migration of renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and down-regulating AKT phosphorylation in vitro

AIM To explore the effect of dasatinib on the viability, apoptosis and migration of human renal carcinoma cell lines 786-O and 769-P, as well as the molecular mechanism in vitro. METHODS 786-O cells and 769-P cells were treated with different concentrations （0~2 μmol/L） of dasatinib, and 0 μmol/L dasatinib was used as blank control group. MTT method was used to detect cell viability. Wound healing assay was used to detect the effect of dasatinib on migration. Hoechst 33258 staining was used to observe the effect of dasatinib on apoptosis. Flow cytometry was used to detect the effect of dasatinib on cell cycle. Western blot method was used to detected cell cycle- and apoptosis-related protein levels. RESULTS Dasatinib inhibited viability and migration of 786-O and 769-P cells, and the inhibitory effect of dasatinib increased with the concentration of dasatinib （P<0.05）. The IC₅₀ values of dasatinib against 786-O and 769-P cell lines were （0.958 7±0.028 8） μmol/L and （0.784 3±0.066 0） μmol/L, respectively. After treatment with dasatinib for 24 h, the expression of pro-apoptotic proteins cleaved caspase-3 and cleaved caspase-9 increased significantly （P<0.01）, while the expression of cyclin D1 decreased （P<0.05）. The cycle-related pathway proteins p53 and p21 increased （P<0.05）, while the level of p-AKT was decreased （P<0.05）. CONCLUSION Dasatinib impaired the viability and migration ability of human renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and down-regulating AKT phosphorylation.     

mTOR kinase inhibitor CC-223 suppresses human breast cancer cell viability

AIM: To investigate the inhibitory effect of CC-223, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on the viability of human breast cancer cells and its mechanism. METHODS: The inhibitory effect of CC-223 on the viability of MCF-7 cells and MDA-MB-231 cells was measured by CCK-8 assay. The cell cycle distribution of breast cancer cells was examined by flow cytometry. The expression of cell cycle-related proteins and oncoproteins c-Myc and survivin was analyzed by Western blot. RESULTS: CC-223 significantly inhibited the viability of both MCF-7 and MDA-MB-231 cells (P<0.05). CC-223 induced cell cycle arrest in both G₁ phase and G₂/M phase in the MCF-7 cells (P<0.05). However, low concentration of CC-223 treatment resulted in the accumulation of MDA-MB-231 cell cycle in the G₂/M phase, and the cell number in G₁ phase was unaffected. Treatment with CC-223 for 24 h clearly inhibited the protein levels of cyclin B1, cyclin D1 and phosphorylated cell division cycle protein 2 in the breast cancer cells (P<0.05). CC-223 suppressed the expression of c-Myc and survivin in both MCF-7 cells and MDA-MB-231 cells (P<0.05). CONCLUSION: CC-223 inhibits cell viability by blocking cell cycle progression and down-regulating expression of c-Myc and survivin in both MCF-7 and MDA-MB-231 cells.     

Panax notoginseng saponins suppress oxygen-glucose deprivation/reoxygenation-induced pyroptosis of SH-SY5Y cells

AIM To investigate the effect of Panax notoginseng saponins (PNS) on pyroptosis of SH-SY5Y cells induced by oxygen-glucose deprivation/reoxygenation (OGD/R). METHODS The OGD/R was conducted to induce ischemia/reperfusion injury in SH-SY5Y cells. The effects of PNS on the viability (detected by CCK-8 assay) and membrane permeability [indicated by lactate dehydrogenase (LDH) leakage and propidium iodide (PI) staining positive cell proportion] of OGD/R-induced SH-SY5Y cells were observed. The protein levels of gasdermin D (GSDMD), GSDMD N-terminal fragment (GSDMD-N), caspase-1 and caspase-4, and the release of interleukin-1β (IL-1β) and IL-18 in the cells were also determined. RESULTS After exposure to OGD/R, the viability of SH-SY5Y cells dramatically decreased (P<0.01), while the LDH leakage, the PI staining positive cell proportion, the protein levels of GSDMD, GSDMD-N, caspase-1 and caspase-4, and the release of IL-1β and IL-18 were significantly increased (P<0.01). However, PNS treatment enhanced the viability of SH-SY5Y cells inhibited by OGD/R (P<0.01), but reduced the leakage of LDH and the percentage of PI staining positive cells (P<0.05 or P<0.01). Moreover, PNS reversed the increases in the protein levels of GSDMD, GSDMD-N, caspase-1 and caspase-4 and the release of IL-1β and IL-18 in OGD/R-induced SH-SY5Y cells (P<0.05 or P<0.01). CONCLUSION Treatment with PNS alleviates OGD/R-induced injury in SH-SY5Y cells. Its mechanism may be related to inhibition of SH-SY5Y cell pyroptosis induced by OGD/R.     

miR-556-3p regulates viability,migration and invasion of endometrial cancer cells by targeting SASH1

AIM To investigate whether microRNA-556-3p （miR-556-3p） regulates the viability, migration and invasion of endometrial cancer cells by targeting SASH1 gene. METHODS The expression of miR-556-3p, and the mRNA and protein levels of SASH1 in endometrial cancer tissues were detected by RT-qPCR and Western blot. Anti-miR-556-3p or pcDNA-SASH1 was transfected into endometrial cancer Ishikawa cells. The cell viability was detected by MTT assay, the migration and invasion abilities of the cells were detected by Transwell chamber method, and the protein expression levels of cyclin D1, p21, matrix metalloproteinase-2 （MMP-2） and MMP-9 were detected by Western blot. StarBase prediction and dual-luciferase reporter experiments were used to analyze the targeting relationship between miR-556-3p and SASH1. Anti-miR-556-3p and si-SASH1 were co-transfected into the Ishikawa cells, and their effects on cell viability, migration and invasion were examined by the methods described above. RESULTS Compared with adjacent tissues, the expression of miR-556-3p in endometrial cancer tissues was increased significantly, and the expression of SASH1 at mRNA and protein levels was decreased significantly （P<0.05）. Inhibition of miR-556-3p expression or induction of SASH1 over-expression obviously reduced the viability of Ishikawa cells, the number of migratory cells, the number of invasive cells and the protein levels of cyclin D1, MMP-2 and MMP-9, and dramatically increased the protein level of p21 （P<0.05）. miR-556-3p targeted SASH1 and negatively regulated its expression. Knock-down of SASH1 expression reversed the inhibitory effect of miR-556-3p expression inhibition on the viability, migration and invasion of Ishikawa cells. CONCLUSION Inhibition of miR-556-3p expression suppresses the viability, migration and invasion of endometrial cancer cells. The mechanism is related to the regulation of its target gene SASH1.     

Effects of marrow stromal cell-mediated microenvironment on human lung adenocarcinoma A549 cells

AIM: To investigate the effects of marrow stromal cell line HS-5 on human lung adenocarcinoma A549 cells in the tumor microenvironment. METHODS: The effects of HS-5 cell-conditioned medium (HS-5-CM) on the viability and migration ability of A549 cells were detected by MTT assay and wound-healing assay. After treatment with HS-5-CM, the expression of CX3C chemokine receptor 1 (CX3CR1) at mRNA level in the A549 cells was examined by qPCR. The protein levels of p-ERK and ERK in the A549 cells treated with MAPK/ERK pathway inhibitor U0126 were observed by Western blot, the migration ability of the A549 cells was measured by wound-healing assay, and the protein expression of CX3CR1 was determined by Western blot. RESULTS: HS-5-CM promoted the viability and migration ability of the A549 cells (P<0.01). The expression of CX3CR1 at mRNA level in the A549 cells was increased after treatment with HS-5-CM. MAPK/ERK inhibitor U0126 inhibited the activation of MAPK/ERK signaling pathway (P<0.01), and reduced the migration ability (P<0.01) and the expression of CX3CR1 (P<0.05) in the A549 cells. CONCLUSION: HS-5-CM significantly promotes the A549 cell viability and migration ability. Activation of MAPK/ERK signaling pathway and the expression of CX3CR1 may play a important role in this process.