Toll-like receptor 4 promotes migration and invasion abilities of human non-small-cell lung cancer cells

AIM:To evaluate the expression and biological role of Toll-like receptor 4 (TLR4) in human non-small-cell lung cancer (NSCLC) cells. METHODS:The mRNA and protein levels of TLR4 in NSCLC tissue were exa-mined by RT-qPCR, Western blot, and immunohistochemistry. After treating the A549 cells and SPC-A-1 cells with TLR4 stimulator lipopolysaccharide (LPS) and inhibitor TAK-242, RT-qPCR, Western blot and flow cytometry were performed to detect the expression of TLR4. The migration and invasion abilities were detected by Transwell assay, and the mRNA expression of matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor (VEGF) was also detected. RESULTS:The mRNA and protein levels of TLR4 were higher in the NSCLC tissue than those in the noncancerous tissue (P<0.01). LPS stimulation significantly increased the mRNA and protein expression levels of TLR4 in the NSCLC cell lines A549 and SPC-A-1 (P<0.01). The LPS-induced TLR4 activation enhanced the migration and invasion abilities of A549 cells and SPC-A-1 cells (P<0.01). LPS increased the expression levels of MMP-2, MMP-9 and VEGF in the A549 cells and SPC-A-1 cells (P<0.01). Moreover, the expression levels of TLR4, MMP-2, MMP-9 and VEGF, as well as the migration and invasion abilities of the cells were blocked by TAK-242 (P<0.01). CONCLUSION:TLR4 might be involved in the migration and invasion of NSCLC cells, and TLR4 inhibition might be considered as a therapeutic target for treatment of NSCLC.     

Effects of p21-activated kinase 6 on invasion and migration of human non-small-cell lung cancer A549 cells

AIM：To investigate the effects of p21-activated kinase 6 (PAK6) on the invasive and migratory abilities of human non-small-cell lung cancer A549 cells. METHODS：The expression of PAK6 mRNA in A549 cells, human bronchial epithelial (HBE) cells, non-small-cell lung cancer tissues and paired adjacent non-tumor tissues was measured by real-time PCR. After A549 cells were transfected with siRNA-PAK6 (siPAK6) or negative control (NC) for 48 h, the expression of PAK6 at mRNA and protein levels was measured by real-time PCR and Western blotting, respectively. The invasion and migration of A549 cells were detected by Matrigel invasion assay and Transwell migration assay. The cytoskeletal changes were observed with FITC-phalloidin staining under confocal microscope. RESULTS：The level of PAK6 mRNA in A549 cells was higher than that in HBE cells (3.50±1.16 vs 1.12±0.42, P<0.05). The level of PAK6 mRNA in non-small-cell lung cancer tissues was higher than that in paired adjacent non-tumor tissues (5.13±1.33 vs 1.08±0.37, P<0.05). The expression of PAK6 protein decreased by 72% in A549 cells transfected with siPAK6 (P<0.05), and the level of PAK6 mRNA significantly decreased in A549 cells transfected with siPAK6 (3.72±0.75 vs 0.69±0.21, P<0.05). Matrigel invasion assay and Transwell migration assay demonstrated that knockdown of PAK6 markedly attenuated the invasion and migration of A549 cells (P<0.05). The cytoskeletal actin remodeling and reduction of stress fibers in A549 cells transfected with siPAK6 were observed under confocal microscope. CONCLUSION：PAK6 may affect the invasive and migratory abilities of non-small-cell lung cancer cells by cytoskeletal actin remodeling.     

Effect of silencing hHBrk1 on proliferation and migration of lung carcinoma cells

AIM: To observe the effect of hHBrk1 gene on proliferation and migration of lung carcinoma cells. METHODS: Recombinant plasmids harboring 19-nt-long small interfering RNA (siRNA) were constructed and tested to selectively downregulate hHBrk1 gene in human lung cancer 95D cell line in vitro by stable transfection with Lipofectamine 2000. The mRNA level of the cells transfected with siRNA plasmids were monitored by Northern blotting and RT-PCR. Growth curve and flow cytometry were applied to determine the cell proliferation and cell cycle. Ability of cell migration was measured by Trans-well system. RESULTS: hHBrk1 gene was silenced by targeting siRNA, and stable silencing cell model was constructed. No difference in proliferation and clone formation between hHBrk1 silencing cells and control cells was observed. The ability of migration was decreased in hHBrk1 silencing cells as compared with control cells. CONCLUSION: hHBrk1 may play an important role in migration of the lung cancer cells.     

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

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

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.     

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.     

Effect of proline-spirooxindole on viability and apoptosis of non-small-cell lung cancer A549 cells

AIM:To investigate the effect of proline-spirooxindole on the viability and apoptosis of human non-small-cell lung cancer A549 cells. METHODS:The effect of proline-spirooxindole on the viability of A549 cells was determined by CCK-8 assay. The apoptosis was analyzed by flow cytometry. The effects of proline-spirooxindole on the expression of PARP and p53 and the phosphorylation of mTOR were determined by Western blot. RESULTS:After A549 cells were treated with proline-spirooxindole (25, 50 and 100 mg/L), the cell viability was decreased (P<0.01) compared with DMSO control group. The apoptotic rate was increased compared with DMSO control group (P<0.01). The protein expression of p53 was up-regulated, the increased apoptotic protein cleaved PARP was observed, and the phosphorylation of mTOR was inhibited (P<0.01). CONCLUSION:Proline-spirooxindole inhibits the viability of A549 cells and induces apoptosis, which may be related to the phosphorylation of mTOR.     

Effect of DEC1 gene over-expression on proliferation and invasion abilities of human esophageal cancer ECA109 cells

AIM: To investigate the effect of DEC1 gene over-expression on the proliferation and invasion abilities of human esophageal cancer ECA109 cells.METHODS: ECA109 cells were transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group). The mRNA and protein levels of DEC1, cyclin D1 and MMP-9 were evaluated by real-time PCR and Western blot, respectively. The effects of DEC1 over-expression on the proliferation and invasion abilities of the ECA109 cells were evaluated by CCK-8 assay, colony formation assay and Transwell test respectively.RESULTS: The DEC1 expression level in ECA109 cells in DEC1 group was significantly higher than that in vector group (P<0.01), but the levels of MMP9 and cyclin D1 expression were opposite (P<0.01). However, both the proliferation and invasion abilities of ECA109 cells in DEC1 groups decreased significantly as compared with those in vector group (P<0.05).CONCLUSION: The over-expression of DEC1 significantly inhibits the proliferation and invasion of ECA109 cells, which may be involved in the expression of cyclin D1 and MMP9.     

Baicalein inhibits proliferation and migration of gastric cancer MGC-803 cells

AIM: To investigate the effects of baicalein (BAI) on the proliferation and migration of gastric cancer MGC-803 cells and the mechanisms. METHODS: After MGC-803 cells were treated with BAI at different concentrations, the viability of the MGC-803 cells was tested by MTT assay. The cell colony formation ability were detected by plate colony formation assay. Wound-healing and Transwell cell migration assays were used to test the migration ability of the MGC-803 cells. The concentration of 12-hydroxyeicosatetraenoic acid (12-HETE) was measured by ELISA. The protein levels of platelet type 12-lipoxygenase (p12-LOX), vascular endothelial growth factor (VEGF), p-ezrin and epithelial-mesenchymal transition (EMT) markers in MGC-803 cells were determined by Western blot. RESULTS: BAI significantly inhibited the proliferation, plate colony formation and migration abilities of the MGC-803 cells (P<0.05 or P<0.01), down-regulated the concentration of p12-LOX metabolite 12-HETE significantly (P<0.05 or P<0.01), decreased the protein levels of p12-LOX, VEGF, p-ezrin, vimentin and Snail (P<0.05 or P<0.01), and increased the protein expression of E-cadherin (P<0.01). CONCLUSION: BAI suppresses the proliferation and migration abilities of gastric cancer MGC-803 cells effectively. These effects of BAI may be related to regulating the protein levels of p12-LOX, VEGF, p-ezrin and EMT-related proteins.     

Effect of di-indolyl thiozoline on proliferation of lung cancer A549 cells

AIM: To investigate the effect of di-indolyl thiozoline (DIIT) on the proliferation of human lung cancer A549 cells. METHODS: The effects of DIIT on the proliferation of human lung cancer A549 cell line were determined by CCK-8 assay and EdU assay. The effects of DIIT on the expression of cyclin-dependent kinase 4 (CDK4), cyclin D1, and the phosphorylation of Akt and mTOR were determined by Western blot. RESULTS: After the A549 cells were treated with DIIT at 12.5, 25, 50 and 100 mg/L, the cell viability detected by CCK-8 assay was decreased by 12%, 27% (P<0.01), 33% (P<0.01) and 52% (P<0.01), respectively, compared with DMSO control group. The EdU positive cell number determined by EdU assay was decreased by 10%, 21% (P<0.05), 26% (P<0.05) and 34% (P<0.01), respectively, compared with DMSO control group. Compared with DMSO control group, DIIT inhibited the phosphorylation of Akt and mTOR and the expression of cyclin CDK4 and cyclin D1 (P<0.05). CONCLUSION: Di-indolyl thiozoline inhibits the proliferation of A549 cells, which may be related to the decreases in phosphorylation levels of Akt and mTOR and the inhibition of cell cycle-related protein expression.     

Role of BAG2 gene in A549 cell proliferation and its clinical implications

AIM: To investigate the role of Bcl-2-associated athanogene 2 (BAG2) in the proliferation of human lung adenocarcinoma A549 cells and its clinical implications. METHODS: The abundance of BAG2 protein in A549 and lung bronchial epithelium (HBE) cells were measured by Western blot. After down-regulation of BAG2 by transfection of siRNA in A549 cells, the expression of cell proliferation and cell cycle related proteins were detected by CFSE assay, WST-1 assay and Western blot, respectively. Moreover, the expression of BAG2 in cDNA array which contained 10 pairs of lung cancer and adjacent tissue was verified. Meanwhile, BAG2 expression in GEO database, which included the human lung cancer and adjacent tissue microarray data was analyzed. The prognosis power of BAG2 was evaluated by the Kaplan-Meier survival curve analysis. RESULTS: BAG2 had remarkably higher expression level in A549 cells than that in HBE cells. Knockdown of BAG2 resulted in significantly inhibition of proliferation in A549 cells, accompany with the significantly down-regulation of cyclin B1 and cyclin E1. BAG2 was over-expressed in the lung cancer tissues, as compared with the adjacent normal tissues. Kaplan-Meier plotter and cDNA microarray results showed that patients with higher BAG2 expression were significantly associated with poorer survival. CONCLUSION: The BAG2 gene tends to regulate A549 cells proliferation via cyclin B1 and cyclin E1. BAG2 has significantly prognostic power on the survival of lung cancer patients.     

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.     

Effect of PDK1 on biological characteristics of lung cancer A549 cells

AIM: To investigate the effects of 3-phosphoinositide-dependent protein-1 (PDK1) on the biological characteristics of non-small-cell lung cancer cell line A549 and the underlying mechanisms.METHODS: The expression levels of PDK1 in lung normal epithelial cell line BEAS-2B and different lung cancer cell lines H460, SPCA1 and A549 were determined by Western blot and real-time PCR. Small interfering RNA was used to down-regulated PDK1 expression in the A549 cells, and then cell viability and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The expression of cell cycle-and apoptosis-related molecules at protein level and the activation of Akt/FoxO1 pathway were measured by Western blot. Insulin-like growth factor-1 (IGF-1, one of the most potent Akt activators) was used to evaluate the interaction between PDK1 and Akt/FoxO1 pathway.RESULTS: Compared with lung normal epithelial cell line BEAS-2B, PDK1 expression in the lung cancer cell lines was obviously increased (P<0.05). Knockdown of PDK1 suppressed cell viability and cell cycle, but promoted the apoptosis of the A549 cells. The results of Western blot showed that the protein levels of cyclin D1, CDK4, p-Rb, Bcl-2, p-Akt and cytoplasmic p-FoxO1 were significantly decreased after knockdown of PDK1, with increases in the protein levels of P27, cleaved caspase-3 and nuclear FoxO1. Pre-incubation with IGF-1 partly reversed the effect of PDK1 knockdown on Akt/FoxO1 pathway and increased the viability of A549 cells.CONCLUSION: In human non-small-cell lung cancer A549 cells, knockdown of PDK1 suppresses cell viability and promotes cell apoptosis by regulating the expression of cell cycle-and apoptosis-related molecules via Akt/FoxO1 pathway, suggesting that PDK1 may be a potential target for diagnosis and theatment of lung cancer.     

Effect of annexin A2 on proliferation,migration and apoptosis of human cervical cancer HeLa cells

AIM：To explore the effect of annexin A2 (ANXA2) on the proliferation, migration and apoptosis abilities of human cervical cancer HeLa cells. METHODS：Overexpression vectors and siRNA of ANXA2 were constructed, and then transfected into HeLa cells. The HeLa cells were divided into 4 groups:control group, scramble group, ANXA2 overexpression group and ANXA2-siRNA group. The expression of ANXA2 at mRNA and protein levels was examined by real-time PCR and Western blotting. MTT assay, Boyden chamber and flow cytometry were used to determine the effect of ANXA2 on the proliferation, migration and apoptosis abilities of the HeLa cells. RESULTS：The proliferation and migration of HeLa cells were obviously promoted by ANXA2 overexpression. The proliferation and migration of HeLa cells were remarkably inhibited by the transfection of ANXA2-siRNA. ANXA2 had no effect on apoptosis of HeLa cells. CONCLUSION:Silencing of ANXA2 effectively inhibits the proliferation and migration of cervical cancer cells, but has little effect on apoptosis. ANXA2 may play a pivotal role in the occurrence and development of cervical cancer, and may be used as a molecular target for the treatment of cervical cancer.     

miR-221 promotes proliferation of lung cancer A549 cells by down-regulating PTEN

AIM: To explore the effect of microRNA-221 (miR-221) on the proliferation of lung cancer A549 cells, and to investigate its mechanism. METHODS: The A549 cells were transfected with miR-221 mimics by Lipofectamine 2000. The expression of miR-221 was detected by RT-qPCR. The expression of PTEN at mRNA and protein le-vels was detected by RT-qPCR and Western blot, respectively. The cell proliferation was examined by CCK-8 assay and colony formation assay. The 3'-UTR of PTEN was cloned into luciferase reporter vector and its enzymatic activity was detected to verify whether miR-221 targeted to PTEN. RESULTS: The expression level of miR-221 in the A549 cells was significantly increased after transfection with miR-221 mimics as compared with negative control group and blank group (P<0.01). The mRNA and protein levels of PTEN were significantly down-regulated compared with control group and blank group (P<0.05). In addition, miR-221 over-expression significantly promoted the proliferation of A549 cells (P<0.05). Moreover, miR-221 inhibited the enzymatic activity of luciferase reporter vector of PTEN. CONCLUSION: Over-expression of miR-221 significantly promotes the proliferation ability of human lung cancer A549 cells by down-regulation of PTEN.     

Effect of PAK4-LIMK1-cofilin signaling on non-small-cell lung cancer migration and invasion

AIM: To explore the mechanism of p21-activated kinase 4 (PAK4) on non-small-cell lung cancer (NSCLC) migration and invasion.METHODS: After A549 and NCI-H520 cell lines were transfected with PAK4-siRNA or negative control, the expression of PAK4 at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The invasion and migration of A549 cells and NCI-H520 cells were measured by Matrigel invasion assay and Transwell migration assay. LIMK1, cofilin, and their respective phosphorylation were examined by Western blot. The interaction of PAK4 and LIMK1 was investigated by co-immunoprecipitation assay. The relationship between PAK4 and LIMK1 phosphorylation was examined by a protein kinase assay in the A549 cells and NCI-H520 cells. The expression of PAK4 and p-LIMK1 in 10 human NSCLC tissues was examined by Western blot. A549 cells and NCI-H520 cells were individually or commonly transfected with PAK4-siRNA or LIMK1 plasmid in order to observe the cell migration and invasion. RESULTS: After A549 cells and NCI-H520 cells were transfected with PAK4-siRNA for 48 h, the expression of PAK4 at mRNA and protein levels, and the numbers of invasion and migration cells in PAK4-siRNA group were lower than those in control group. Compared with control group, the phosphorylation of LIMK1 and cofilin was lower in PAK4-siRNA group, whereas the total expression levels of LIMK1 and cofilin did not change. The results of co-immunoprecipitation assays showed that PAK4 specifically interacted with LIMK1 in A549 and NCI-H520 cells. LIMK1 phosphorylation in the presence of PAK4 (K350M) was significantly lower than that in the presence of PAK4 (WT) or PAK4 (S445N) in the protein kinase assay. The PAK4 upregulation was positively correlated with the level of p-LIMK1 (P<0.05). After A549 cells and NCI-H520 cells were co-transfected with PAK4-siRNA and LIMK1 plasmid, the migration and invasion cell numbers in co-transfection group were higher than those in PAK4-siRNA transfection group. CONCLUSION: PAK4 promotes the invasive and migratory abilities of NSCLC, which is mediated by LIMK1 phosphorylation.     

Effect of hirsutine on hypoxia-induced migration and invasion abilities in human breast cancer MCF-7 cells

AIM: To investigate the effect of hirsutine on hypoxia-induced migration and invasion abilities of human breast cancer MCF-7 cells and its possible mechanism. METHODS: CCK-8 assay was employed to detect the cytotoxic effect of hirsutine on the MCF-7 cells. Cell migration was observed by wound healing assay, and cell invasion ability was measured by Transwell invasion assay. Western blot was used to analyze the protein levels of hypoxia-inducible factor-1α (HIF-1α), Snail, E-cadherin and matrix metalloproteinase-9 (MMP-9). The mRNA levels of HIF-1α was detected by RT-PCR. RESULTS: Hirsutine remarkably reduced the cell viability from 32 μmol/L (P<0.05), and the IC₅₀ value was 62.82 μmol/L. In hypoxia state, MCF-7 cells showed more powerful capabilities of migration and invasion (P<0.05), higher protein levels of HIF-1α, Snail and MMP-9 (P<0.05), lower protein level of E-cadherin (P<0.05), and higher mRNA level of HIF-1α (P<0.05). These hypoxia-induced effects were all inhibited by hirsutine at 16 μmol/L (P<0.05), apart from the mRNA level of HIF-1α. CONCLUSION: Hirsutine inhibits hypoxia-induced migration and invasion in human breast cancer MCF-7 cells most likely via down-regulation of the protein levels of HIF-1α, Snail and MMP-9, and up-regulation of the protein level of E-cadherin.     

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.     

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.