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

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

Effects of marrow stromal cell-mediated microenvironment on human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the effects of marrow stromal HS-5 cells on hepatocellular carcinoma SMMC-7721 cells in the tumor microenvironment. METHODS: The effects of HS-5 cell-conditioned medium (HS-5-CM) on the proliferation, migration and invasion abilities of SMMC-7721 cells were detected by MTT, wound-healing and Transwell assays. After co-culture of SMMC-7721 cells with HS-5 cells in the Transwell chamber, the expression of chemokine CCL5 and its receptor CCR5 at mRNA and protein levels in SMMC-7721 cells was examined by quantitative real-time PCR (qRT-PCR), ELISA or Western blotting. Akt and p-Akt473 protein levels in SMMC-7721 cells treated with PI3K inhibitor LY294002 were observed by Western blotting. RESULTS: HS-5-CM promoted the proliferation, migration and invasion abilities of SMMC-7721 cells. The expression of CCL5 and CCR5 at mRNA and protein levels in SMMC-7721 cells was increased after co-cultured with HS-5 cells. PI3K inhibitor LY294002 inhibited the activation of PI3K-Akt signaling pathway and the secretion of CCL5 in SMMC-7721 cells after co-cultured with HS-5 cells. CONCLUSION: HS-5 cells significantly promote the proliferation, migration and invasion abilities of SMMC-7721 cells. Co-culture of SMMC-7721 cells with HS-5 cells activates PI3K-Akt signaling pathway to increase the secretion of CCL5 in SMMC-7721 cells.     

p38 MAPK-Pim-3 signal pathway may be involved in cardiomyocyte anoxia preconditioning against anoxia/reoxygenation injury

AIM: To investigate the role of mitogen-activated protein kinases (MAPKs) pathways and the molecular mechanism by which the proto-oncogene Pim-3 protects cardiomyocyte against anoxia/reoxygenation (A/R) injury. METHODS: The primarily cultured neonatal rat ventricular cardiomyocytes were randomly divided into 4 groups: control group; A/R group; APC+A/R group; SB203850, U0126 or SP600125+APC+A/R group. The cells were pre-incubated with U0126 (ERK1/2 inhibitor), SP600125 (SAPK/JNK inhibitor), or SB203850 (p38 MAPK inhibitor) at concentration of 10 μmol/L for 30 min before the APC. The activities of p38 MAPK, JNK and ERK1/2 were detected by Western blotting. The viability of cardiomyocytes was assayed by MTT and the apoptosis of cardiomyocyte was detected by TUNEL. RESULTS: U0126, SB203850, and SP600125 abolished the increased expression of ERK1/2, p38-MAPK, and JNK proteins induced by APC+A/R or A/R, respectively. The expression level of Pim-3 protein significantly decreased when the p38 MAPK signal pathway was inhibited. Meanwhile, the activity of LDH and the apoptosis index increased, and the viability of cardiomyocytes decreased. CONCLUSION: Pim-3 expression through a p38 MAPK signaling pathway may protect cardiomyocytes from A/R injury.     

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

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

Effect of HC-1119 on biological behaviors of triple-negative breast cancer BT549 cells

AIM: To explore the effect of new artificially synthesized androgen receptor (AR) antagonist HC-1119 on the biological function of triple-negative breast cancer (TNBC) BT549 cells and the molecular mechanism. METHODS: The AR expression was assessed in different human breast cancer cell lines MDA-MB-231, T47D, MCF-7, SKBR3 and BT549 by Western blot. The TNBC BT549 cells with AR positive expression were treated with HC-1119. The cell viability was measured by CCK-8 assay. The apoptosis rate and cell cycle distribution were analyzed by flow cytometry. The migration and invasion abilities were detected by Transwell assay in vitro. The protein expression of E-cadherin, vimentin and P21 was determined by Western blot. RESULTS: AR was positively expressed in BT549 cells. HC-1119 inhibited the cell viability in a time-and dose-dependent manner (P<0.05), increased the percentage of apoptotic cells and the percentage of S-phase cells significantly, repressed the migration and invasion abilities (P<0.05), and decreased P21 expression at protein level (P<0.01). No influence on the expression of E-cadherin and vimentin in the BT549 cells was observed. CONCLUSION: AR antagonist HC-1119 decreases the viability, migration ability and invasion ability, enhances the apoptosis, and arrests the cell cycle distribution of TNBC BT549 cells. HC-1119 represses the viability of BT549 cells by down-regulating P21 expression, while the process of epithelial-mesenchymal transition is not involved in the inhibition of cell migration.     

miR-140-3p inhibits viability,invasion and migration of non-small-cell lung cancer A549 cells by targeting PD-L1

AIM: To explore the target relationship between microRNA-140-3p (miR-140-3p) and programmed cell death ligand 1 (PD-L1) and their effect on the viability, migration and invasion of non-small-cell lung cancer A549 cells.METHODS: RT-qPCR was used to detect the miR-140-3p expression in HLF-1, A549 and H1299 cells, and then the A549 cells with the most significant difference were selected as the subsequent research object. TargetScan software and dual-luciferase reporter assay were performed to predict and confirm the target relationship between miR-140-3p and PD-L1. RT-qPCR and Western blot were used to determine the effects of miR-140-3p mimic and inhibitor on PD-L1 expression level. MTT assay was used to detect the viability of A549 cells. Transwell assay was performed to detect the migration and invasion abilities of the A549 cells.RESULTS: miR-140-3p was significantly down-regulated in the A549 cells and H1299 cells (P<0.05). Transfection with miR-140-3p mimic decreased the expression of PD-L1 and inhibited the viability, migration and invasion of the A549 cells. Transfection with pcDNA3.0-PD-L1 reversed the inhibitory effect of miR-140-3p on the viability, migration and invasion of the A549 cells.CONCLUSION: miR-140-3p inhibits the viability, migration and invasion of A549 cells by targeting PD-L1.     

Effect of luteolin on TGF-β1-induced epithelial-mesenchymal transition in lung cancer A549 cells

AIM:To investigate the effects of luteolin on the invasion and epithelial-mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1) in lung cancer A549 cells. METHODS:The effect of luteolin at 5, 10, 20, 40, 80 and 160 μmol/L on the viability of A549 cells was measured by MTT assay. The invasion ability was analyzed by Transwell method. The morphological changes of the A549 cells were observed under microscope.The protein expression of E-cadherin and vimentin in the A549 cells were determined by Western blot. RESULTS:The viability of the A549 cells was significantly inhibited by luteolin in a dose-time dependent manner (P<0.05). The IC₅₀ of luteolin for the A549 cells (24 h) was 68.79 μmol/L, while that (48 h) was 47.86 μmol/L. TGF-β1 induced morphological alteration of the A549 cells from epithelial to mesenchymal forms. Luteolin significantly inhibited TGF-β1-induced invasion of the A549 cells (P<0.01). The protein expression of E-cadherin was significantly down-regulated and the protein expression of vimentin was significantly up-regulated in the presence of TGF-β1 at 5 μg/L (P<0.01). However, luteolin reversed TGF-β1-induced EMT, up-regulation of E-cadherin and down-regulation of vimentin (P<0.01). CONCLUSION:Lu-teolin reverses TGF-β1-induced EMT in the lung cancer A549 cells.     

Tripchlorolide activates p-ERK and induces autophagy in lung cancer A549 cells

AIM: To investigate the effects of tripchlorolide (TP) on proliferation and autophagy of human lung cancer A549 cells, and explore its mechanism. METHODS: MTT assay was performed to analyze the effect of TP on the viability of human lung cancer A549 cells. The A549 cells were treated with TP, and their autophagy was observed under the fluorescence microscope through acridine orange staining. Green fluorescence spots were observed by fluorescence microscopy through GFP-LC3 plasmid transfection experiment. The levels of LC3 and p-ERK in the A549 cells after TP treatment were determined by Western blot. RESULTS: The viability of human lung cancer A549 cells was significantly inhibited by TP in a dose-time dependent manner (P<0.05). The number of the intracellular acidic follicles dyed with bright red fluorescence was significantly increased after TP treatment in A549 cells. The number of green dot-like congregate autophagosomes in cell cytoplasm was significantly increased after TP treatment in the A549 cells transfected with GFP-LC3 plasmid, while the normal treatment only induced a few cells with autophagosome formation. At the same time, we did not observe the dot-like congregate autophagosomes after TP treatment in the A549 cells transfected with GFP-control plasmid. Compared with control group, the expression of LC3-Ⅱ protein was up-regulated in A549 cells after TP treatment (P<0.01). Furthermore, treatment with TP in A549 cells for 48 h also led to a significant upregulation of phosphorylated form of ERK (P<0.01). In contrast, no significant change in the levels of total ERK protein was observed. Compared with 100 nmol/L TP group, TP+3-MA group down-regulated the protein levels of LC3-Ⅱ (P<0.01) and p-ERK (P<0.01) in the A549 cells. CONCLUSION: TP significantly inhibits the growth of A549 lung cancer cells and induces the autophagy, which may be correlated with upregulation of p-ERK protein.     

27-Hydroxycholesterol modulates lung cancer cell proliferation by activation of LXR signaling pathway

AIM:To investigate the effect of cholesterol metabolite 27-hydroxycholesterol (27-OHC) on the proliferation of lung cancer cells. METHODS:Human lung cancer A549 cells were treated with 27-OHC at different concentrations (0, 0.3125, 0.625, 1.25, 2.5, 5 and 10 μmol/L) for 24~48 h. The cell viability, cell cycle, cell prolife-ration, the intracellular cholesterol levels and cholesterol metabolism-related molecule expression were subsequently assessed by CCK-8 assay, flow cytometry, EdU staining, tissue total cholesterol detection kit, real-time PCR and Western blot. RESULTS:27-OHC decreased the viability of the A549 cells in a dose-and time-dependent manner (P<0.01) and inhibited the cell proliferation (P<0.05). The expression of typical liver X receptor (LXR) downstream target proteins including ATP-binding cassette transporter A1 (ABCA1), low-density lipoprotein receptor (LDLR), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CR) were modulated, which promoted the efflux of intracellular cholesterol, and reduced cholesterol influx and de novo synthesis, resulting in decreased intracellular cholesterol levels and cell viability. Furthermore, the inhibitory effect of 27-OHC on A549 cell viability was significantly attenuated after the LXR pathway was partially blocked by 5 μmol/L GSK2033 treatment (P<0.05). CONCLUSION:27-OHC inhibits A549 cell prolife-ration via activation of LXR signaling pathway.     

Ursolic acid inhibits migration and invasion of human lung cancer A549 cells by targeting miRNA-133a

AIM: To investigate the effects of ursolic acid (UA) on the migration and invasion of human lung cancer cell line A549, and to explore its mechanism. METHODS: The cell viability was detected by MTT assay. The expression of miRNA-133a was detected in the A549 cells treated with UA by real-time PCR. The miRNA-133a mimics and inhibitor were transfected into the A549 cells, and the transfection efficiency was analyzed by real-time PCR. The cell migratory and invasive abilities were determined by wound healing and Transwell methods, respectively. RESULTS: The viability of the human lung cancer A549 cells was significantly inhibited by UA in a dose-dependent manner (P<0.05). IC₅₀ of UA (24 h) for lung cancer A549 cells was 31.04 μmol/L. UA treatment significantly inhibited the migratory and invasive abilities of A549 cells in a concentration-dependent manner, accompanied by significantly elevation of miRNA-133a expression. The mimics and inhibitor of miRNA-133a significantly upregulated and downregulated the expression of miRNA-133a in the transfected A549 cells, respectively. In addition, the viability of the A549 cells was decreased extremely after tansfected with the miRNA-133a mimics (P<0.01), so did the results of the cell migration and invasion test. The A549 cells tansfected with the miRNA-133a inhibitor showed an opposite changes of the cell viability, migration and invasion. CONCLUSION: UA inhibited the viability, migration and invasion of lung cancer A549 cells by elevating the expression of miRNA-133a.     

Expression of fractalkine and CX3CR1 in renal tissues of patients with WHO class IV lupus nephritis

AIM: To observe the expression of fractalkine, and its receptor, CX3CR1, in renal tissues of patients with diffuse proliferative lupus glomerulonephritis (WHO class IV), minimal glomerular abnormalities, and normal kidney. Meanwhile, the correlation among the expression of fractalkine, CX3CR1 and CD68-positive macrophages was investigated, and the role of fractalkine and CX3CR1 in the pathogenesis of lupus nephritis was discussed. METHODS: The expressions of fractalkine, CX3CR1 and CD68 were detected immunohistochemically in kidney tissue sections obtained from twenty-one patients with WHO class IV lupus nephritis, eighteen cases with minimal glomerular abnormalities, and eight normal kidneys which were no abnormality under light microscope. RESULTS: (1) Fractalkine was generally indistinguishable in tissue sections from normal kidney and minimal glomerular abnormalities. CX3CR1-positive cells and CD68-positive macrophages were sparsely detected in the glomeruli and in the cortical interstitium. (2) There were considerable CX3CR1-positive cells and macrophages in both the glomeruli and the interstitium in sections from class IV lupus nephritis. The number of CX3CR1-positive cells significantly correlated with the number of macrophages in the glomeruli and in the interstitium respectively (r=0.956, P<0.01 and r=0.965, P<0.01). (3) Significant expression of fractalkine was seen in the cortical renal tubules from class IV lupus nephritis. The percentage of fractalkine-positive tubules significantly correlated with the number of CX3CR1-positive cells and macrophages in the interstitium respectively (r=0.720, P<0.01 and r=0.770, P<0.01). CONCLUSION: These expression patterns show that fractalkine and CX3CR1 may play an important role in the pathogenesis of class IV lupus nephritis.     

Regulatory effects of miR-195 on biological behaviors of lung cancer A549 cells

AIM: To investigate the regulatory effects of microRNA (miR)-195 on the biological behaviors, such as viability, apoptosis and migration, of lung cancer A549 cells, and to explore the related mechanisms. METHODS: After miR-195 mimics were transfected into the A549 cells, the cell viability, cell cycle distribution and apoptosis were measured by CCK-8 assay and flow cytometry. Transwell assay was used to detect cell migration ability. Furthermore, the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb/Rb were determined by Western blot. Dual-luciferase reporter assay was used to screen and identify the possible target genes of miR-195. RESULTS: Over-expression of miR-195 in the A549 cells inhibited the cell viability and induced cell cycle arrest, accompanied with the decrease in the cell migration ability and the increase in the apoptotic rate (P<0.05). Furthermore, the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb were significantly decreased (P<0.05). Dual-luciferase reporter assay demonstrated that MYB was a potential target gene of miR-195. Over-expression of MYB in the A549 cells partially reversed the effects of miR-195 on the cell viability, apoptosis and migration. CONCLUSION: miR-195 inhibits lung cancer A549 cell growth and migration, and promotes cell apoptosis by targeting MYB gene.     

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

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

Effect of serum amyloid A on invasion,migration and MAPK signaling pathway in osteosarcoma cells

AIM: To investigate the effect of silencing of serum amyloid A (SAA) on the viability, apoptosis, migration and mitogen-activated protein kinase (MAPK) signaling pathway in osteosarcoma U2OS cells. METHODS: Small interfering RNA (siRNA) targeting SAA was transfected into U2OS cells to silence the expression of SAA gene. The U2OS cells were divided into blank control group, negative control group, and experimental group. The cells in negative control group and experimental group were transfected into negative control siRNA and SAA-siRNA, respectively. The cells in blank control group were without any treatment. The viability of the cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry with Annexin V-FITC/PI double staining. The migration and invasion abilities of the cells were detected by Transwell chamber assay. The protein levels of SAA, phosphorylated p38 MAPK (p-p38 MAPK) and phosphorylated c-Jun N-terminal kinase (p-JNK) in the cells were determined by Western blot. RESULTS: The protein expression of SAA in SAA-siRNA group was significantly lower than that in blank control group (P<0.05). Compared with blank control group, the cell viability in SAA-siRNA group was significantly decreased (P<0.05), the apoptotic rate was significantly increased (P<0.05), and the invasion and migration abilities were significantly decreased (P<0.05). The protein levels of p-p38 MAPK and p-JNK in SAA-siRNA group were significantly lower than those in blank control group (P<0.05), and no significant difference of total JNK and p38 protein levels was observed. CONCLUSION: Silencing of SAA expression inhibits the viability of osteosarcoma cells, induces apoptosis and decreases the migration of osteosarcoma cells, which may be related to the activation of MAPK signaling pathway.     

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.     

Celastrol blocks cell cycle of A549 cells by regulating miR-17-5p/miR-155-5p and targeting cyclin D1

AIM: To investigate the effect of celastrol on the cell cycle of human lung adenocarcinoma A549 cells and to probe into its mechanisms.METHODS: A549 cells were exposed to celastrol at gradient concentrations. The cell viability and apoptosis were detected by MTT assay and flow cytometry, respectively, and the median lethal concentration (LC₅₀) of celastrol was screened. The A549 cells were treated with celastrol at LC₅₀, and the cell cycle was detected by flow cytometry. The expression of cyclin D1 was determined by Western blot, and the expression of microRNA (miR)-17-5p and miR-155-5p was detected by real-time PCR. The correlation between cyclin D1 and miR-17-5p or miR-155-5p was predicted by bioinformatics software. After miR-17-5p mimics/miR-155-5p mimics/mutant-miR-17-5p/mutant-miR-155-5p and pcDNA-GFP-cyclin D1-3'UTR were cotransfected into the A549 cells, the changes of GFP expression were evaluated by fluorescence microscopy and flow cytometry. Finally, after miR-17-5p mimics or miR-155-5p mimics were transfeced into the A549 cells, the expression of miR-17-5p and miR-155-5p was detected by real-time PCR, and the protein level of cyclin D1 was determined by Western blot. RESULTS: With the increasing concentration of celastrol, the viability inhibition rate and apoptotic rate of the A549 cells were increased, indicating that celastrol effectively inhibited the growth of A549 cells and induced apoptosis. The LC₅₀ of celastrol was almost 3 μmol/L. After treatment with celastrol at LC₅₀, the A549 cell cycle was arrested at G₁ phase, the protein expression of cyclin D1 was down-regulated (P<0.01), and the expression levels of miR-17-5p and miR-155-5p were significantly increased (P<0.01). The results of bioinformatics software prediction indicated that there were binding sites for miR-17-5p and miR-155-5p in the 3'-UTR of cyclin D1. After cotransfected with miR-17-5p or miR-155-5p and pcDNA-GFP-cyclin D1-3'UTR into the A549 cells, the expression of GFP declined (P<0.05). After miR-17-5p or miR-155-5p mimics were transfected into A549 cells, the results of real-time PCR showed this treatment significantly increased the miRNA expression (P<0.01), and the results of Western blot showed the transfection inhibited cyclin D1 expression (P<0.01).CONCLUSION: Celastrol blocks the A549 cells at G₁ phase, inhibits the viability and induces apoptosis, which may be caused by up-regulating the expression of miR-17-5p and miR-155-5p, and then down-regulating cyclin D1 expression. This study provides a new theoretical basis for the treatment of non-small-cell lung cancer with celastrol.     

Effects of SphK1 and FAK on epithelial-mesenchymal transition in colon cancer HCT116 cells

AIM: To investigate the effects of sphingosine kinase l(SphK1) and focal adhesion kinase(FAK) on the epithelial-mesenchymal transition(EMT) of human colon cancer HCT116 cells. METHODS: Human colon cancer HCT116 cells were divided into 3 groups. N, N-dimethylsphingosine(DMS) was used to suppress the activity of SphK1. PF573228 was used to suppress the activation of FAK. The cells treated with equal volume of culture medium severed as control group. The cell viability was measured by MTT assay. The protein expression of SphK1, FAK and the EMT relative protein E-cadherin, N-cadherin, vimentin and matrix metalloproteinase(MMP) 2 was analyzed by Western blot. The mRNA expression of SphK1, sphingosine-1-phosphate(S1P), FAK, E-cadherin and vimentin was detected by real-time PCR. The ability of tumor cell migration was measured by wound-healing assay. RESULTS: The cell viability of HCT116 cells was suppressed by DMS and PF573228 in dose and time dependent manners. DMS significantly suppressed the expression of SphK1, FAK, N-cadherin, vimentin and MMP2, meanwhile enhanced the expression of E-cadherin. PF573228 reduced the expression of FAK, SphK1, N-cadherin, vimentin and MMP2, meanwhile increased the expression of E-cadherin(P<0.01). In addition, the migration ability of HCT116 cells was significantly decreased by treating with DMS and PF573228(P<0.01). Compared with control group, the mRNA expression of FAK, SphK1, S1P and vimentin was decreased, while the expression of E-cadherin was increased significantly in PF573228 group and DMS group(P<0.05). CONCLUSION: SphK1 and FAK signaling pathways may play an important role in the occurrence of EMT in the colon cancer HCT116 cells.     

miRNA-126 inhibits proliferation,migration and invasion of human lung cancer A549 cells via EGFR/AKT/mTOR pathway

AIM: To investigate the effects of microRNA(miRNA)-126 on the proliferation, migration and invasion of human lung cancer cell lines, and to explore its mechanism. METHODS: The A549 cells were transfected with miRNA-126 agomir by Lipofectamine 2000. The expression of miRNA-126 was detected by real-time PCR. The cell activity was detected by MTT assay. The number of viable A549 cells was counted by the method of Trypan blue exclusion. The cell colony-forming capability was determined by cell colony formation test. The cell migration and invasion abilities were assayed by wound healing and Transwell methods, respectively. The protein levels of p-EGFR, EGFR, p-AKT, AKT, p-mTOR and mTOR were determined by Western blot. RESULTS: The expression level of miRNA-126 was significantly increased in the A549 cells compared with negative control(NC) group and control group(P<0.01). The proliferation of A549 cells was decreased extremely after transfected with the miRNA-126 agomir(P<0.01), so did the result of the cell colony-formation test. The migration and invasion abilities of the lung cancer cells were also significantly inhibited. The protein levels of p-EGFR, p-AKT and p-mTOR were significantly down-regulated compared with NC group and control group(P<0.01). CONCLUSION: Over-expression of miRNA-126 significantly inhibits the proliferation, migration and invasion ability of human lung cancer A549 cells by down-regulation of EGFR/AKT/mTOR pathway.