lncRNA TTN-AS1 regulates the viability and invasion of lung adenocarcinoma A549 cell via miR-519d-3p/MMP2 pathway

AIM To investigate the expression level of long noncoding RNA （lncRNA） TTN antisense RNA 1 （TTN-AS1） in lung adenocarcinoma tissues and the effects of TTN-AS1 silencing on the viability and invasion of lung adenocarcinoma A549 cells. METHODS RT-qPCR was used to detect the expression of TTN-AS1, microRNA-519d-3p （miR-519d-3p） and matrix metalloproteinase 2 （MMP2） mRNA in 32 cases of lung adenocarcinoma and adjacent normal tissues. The untransfected A549 cells were divided into blank group, si-NC group （with si-NC transfection） and si-lncRNA group （with silencing of lncRNA TTN-AS1 expression）, with n=5 in each group. The effects of TTN-AS1 silencing on the viability and invasion of A549 cells were detected by CCK8 and Transwell methods. The targeting regulatory effects of TTN-AS1 on miR-519d-3p and miR-519d-3p on MMP2 were determined by dual-luciferase reporter assay, RNA immunoprecipitation test, RT-qPCR and Western blot. RESULTS The expression level of TTN-AS1 in 32 cases of lung adenocarcinoma tissues is notably higher than that in the adjacent normal tissues （P<0.05）. Silencing of TTN-AS1 in A549 cells significantly suppressed the cell viability and invasion. TTN-AS1 negatively regulated the expression of miR-519d-3p via sponging and absorbing miR-519d-3p. MMP2 is the target gene of miR-519d-3p and can be negatively regulated by miR-519d-3p. Overexpression of MMP2 partially reversed the inhibitory effect of TTN-AS1 silencing and miR-519d-3p overexpression on the invasion of A549 cells. CONCLUSION The lncRNA TTN-AS1 is overexpressed in lung adenocarcinoma tissues, and it regulates lung adenocarcinoma A549 cell viability and invasion via miR-519d-3p/MMP2 pathway.     

Screening of differentially expressed genes in colorectal cancer based on TCGA database and verification of novel gene CCDC78

AIM To explore novel genes closely related to the prognosis of colorectal cancer through bioinformatics analysis of RNA sequencing (RNA-Seq） data of gene expression profile in large samples of colorectal cancer tissues from The Cancer Genome Atlas (TCGA) database, and to validate the new genes and study their functions. METHODS The RNA-Seq data were downloaded from TCGA database to screen differentially expressed genes, R-survival package was used to carry out survival analysis of differentially expressed genes to screened out genes related to prognosis of colorectal cancer, and the most significantly up-regulated genes that have not been reported in cancer studies were selected for further verification. RT-qPCR was employed to detect mRNA expression of these novel genes in human colon cancer cell lines and human colorectal cancer tissues. The colon cancer cell line with stable silencing of the novel gene expression was constructed by transfection of lentivirus vector, and its viability, migration and invasion were detected by CCK-8 assay and Transwell assay. RESULTS (1) A total of 4 017 differentially expressed genes were found in the gene expression profile of the colorectal cancer samples. Kaplan-Meier survival analysis showed that 69 genes were closely related to poor prognosis in patients with colorectal cancer, 36 of which were up-regulated, and 11 of these 36 genes have not been reported in cancer studies. (2) The mRNA expression of the top 3 up-regulated genes CCDC78, PGGHG and TSPEAR of the 11 genes was significantly increased in colon cancer cell lines, and the expression of CCDC78 mRNA was also up-regulated in colorectal cancer tissues. (3) CCDC78 gene silencing significantly suppressed the viability, migration and invasion of colon cancer cells (P<0.01). CONCLUSION Bioinformatics analysis based on TCGA database is helpful to discover nov?el genes related to prognosis of colorectal cancer, and CCDC78 may be a novel oncogene associated with poor prognosis of colorectal cancer.     

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.     

Significance of TRPM8 protein expression in lung adenocarcinoma

AIM To investigate the significance of transient receptor potential cation channel subfamily M member 8 （TRPM8） protein expression in lung adenocarcinoma. METHODS The tumor samples from 112 cases of patients with lung adenocarcinoma were collected in our hospital, and 4~5 years of follow-up was conducted. The protein expression of TRPM8 was analyzed by immunohistochemical staining, and the correlations between the TRPM8 protein expression and the clinical characteristics including prognosis of the patients with lung adenocarcinoma were investigated. After TRPM8 protein expression was up-regulated in A549 lung adenocarcinoma cells by lentiviral infection, the proliferation of A549 cells was analyzed by CCK-8 assay and colony formation assay, the cell cycle and apoptosis were analyzed by flow cytometry, and the migration and invasion abilities of the cells were measured by scratch experiment and Transwell assay. The TRPM8 protein expression was stably up-regulated in H1299 cells by lentiviral infection, and then the left and right buttocks of the immunodeficient mice were subcutaneously injected with empty vector control cells and TRPM8-overexpressing cells, respectively. The effects of TRPM8 on the growth of H1299 cell-derived xenograft tumor in immunodeficient mice were evaluated. RESULTS The 4~5-year survival rate in the patients with high TRPM8 protein expression was significantly higher than that in the patients with low expression of TRPM8 protein （P=0.017）. The tumor maximum diameter in the patients with high TRPM8 protein expression was significantly smaller than that in the patients with low TRPM8 protein expression （P=0.028）. The viability, the number of colonies and the migration and invasion abilities of TRPM8-overexpressing A549 cells were significantly decreased as compared with empty vector and parental cells （P<0.01）. The results of flow cytometry analysis showed that the proportion of A549 cells at S stage was significantly increased in TRPM8 overexpression group as compared with empty vector group （P<0.01）. The growth rate and the weight of TRPM8-overexpressing H1299 cell-derived xenograft tumor in immunodeficient mice were significantly lower than those in empty vector group （P<0.01）. CONCLUSION TPRM8 is a tumor suppressor in lung adenocarcinoma, and low expression of TRPM8 protein was a poor prognositic indicator of patients with lung adenocarcinoma.     

Knock-down of NEDD1 expression inhibits lung adenocarcinoma cell proliferation and migration

AIM: To explore the role of neural precursor cell expression developmentally down-regulated protein 1 (NEDD1) in the development and progression of lung cancer. METHODS: The differences of NEDD1 expression levels between lung cancer tissues and tumor-adjacent tissues were analyzed by the method of immunohistochemistry and TCGA database. Kaplan-Meier curve was used to analyze the correlation between lung cancer prognosis and the expression level of NEDD1. The proliferation of A549 cells was tested by plate colony formation experiment after knock-down of NEDD1 expression. The apoptosis rate and cell cycle distribution were examined by flow cytometry. The migration ability of the A549 cells was detected by Transwell assay. The protein levels of cell cycle-related molecules were determined by Western blot. Database analysis was performed to evaluate the relationship between the expression of NEDD1 and cyclin-dependent kinases 2 (CDK2). RESULTS: Compared with the tumor-adjacent tissues, the expression level of NEDD1 in the lung cancer tissues was increased, so as the database analysis, and the higher expression of NEDD1 showed a poorer prognosis. Under light microscope, the A549 cells showed a low proliferation rate after silencing the NEDD1 expression, and the colony formation ability of the cells was also reduced; knock-down of NEDD1 expression induced the apoptosis and inhibited the cell migration; knock-down of NEDD1 expression blocked the cells in G₁/S phase, and the protein levels of p-Rb and cyclinD1 were decreased, while the protein levels of p-Chk1, p-Chk2 and p-p53 were increased (P<0.05). A positive correlation between the expression of NEDD1 and CDK2 was noted by database analysis. CONCLUSION: NEDD1 plays an crucial role in promoting cell proliferation via inhibiting apoptosis and accelerating cell cycle, high expression of NEDD1 in lung adenocarcinoma tissue is related to poor prognosis, thus NEDD1 may be used as a candidate marker molecule for the diagnosis and prognosis of lung cancer.     

Effect of HMGB2 on cell cycle and proliferation of lung adenocarcinoma

AIM: To investigate the effect of high-mobility group protein B2 (HMGB2) on cell cycle and proliferation of lung adenocarcinoma cells. METHODS: Cancer RNA-Seq Nexus (CRN) was used to analyze HMGB2 expression in lung adenocarcinoma tissues. OncoLnc was used to analyze the correlation between HMGB2 and prognosis of lung adenocarcinoma patients. Cancer Single-cell State Atlas (CancerSEA) was used to analyze the correlation between HMGB2 and 14 kinds of functional states of lung adenocarcinoma. siRNA was used to inhibit HMGB2 expression in human lung adenocarcinoma A549 cells. The silencing effects were verified by real-time PCR and Western blot, and the cell proliferation was detected by CCK8 and EdU assays. RESULTS: HMGB2 was over-expressed in the lung adenocarcinoma tissues. The overall survival of the patients with lung adenocarcinoma in HMGB2 high expression group was significantly lower than that of the patients with low expression of HMGB2 (log-rank test P=0.017 3). HMGB2 expression was positively correlated with cell cycle and proliferation of lung adenocarcinoma cells. The viability and proliferation ability of A549 cells after HMGB2 expression knock-down were significantly reduced (P < 0.05). CONCLUSION: The expression of HMGB2 is positively correlated with the cell cycle and proliferation of lung adenocarcinoma, and it can be used as a potential marker for evaluating the prognosis and therapeutic target of patients with lung adenocarcinoma.     

苹果MLP家族基因鉴定及非生物胁迫响应分析

在苹果中鉴定了13个Major Latex Protein(MLP)家族基因Md MLP。序列比对及构建蛋白同源模型发现,Md MLP蛋白含有Bet_v1典型的Gly-rich loop区域结构,且为MLP家族特有的Gxxxxx G结构。经多物种MLP系统发育及共线性比较分析,Md MLP与其他蔷薇科物种MLP具有相似基因结构和蛋白质保守结构域。q RT-PCR分析表明,Md MLP在‘新疆1号’苹果14个器官组织中均有不同程度的表达,对ABA、Na Cl、PEG、低温(4℃)和高温(40℃)有一定响应,且同一亚族基因表达情况呈现相似趋势。String构建蛋白互作网络发现,Md MLP可能通过与PRSP、SNRK1/2、b HLH等应激、ABA相关转录蛋白互作,参与苹果对非生物胁迫的防御。     

Muscarinic receptor 3 agonist promotes epithelial-meschymal transition in human lung cancer A549 cells by activating PI3K/AKT signaling pathway

AIM: To investigate the possible signaling pathway that promotes epithelial-mesenchymal transition (EMT) of the lung cancer A549 cells stimulated with muscarinic receptor 3 (M3R) agonist carbachol. METHODS: The lung cancer cells A549 were treated with 400 μmol/L carbachol. The morphological changes of the cells were observed under inverted phase contrast microscope. The migration and invasion abilites were measured by Wound healing and Transwell assays. qPCR was used to detect the mRNA level of vimentin and E-cadherin. The protein levels of p-AKT, vimentin and E-cadherin were determined by Western blot. RESULTS: After treatment with carbachol, the A549 cells showed loss of the close connection and the cell morphology was transformed from irregular polygon to spindle-like cells. The results of Wound healing and Transwell assays showed that the migration and invasion abilites of the A549 cells were enhanced. Carbachol increased the vimentin expression and decreased the E-cadherin expression at mRNA and protein level (P<0.05). The phosphorylation of AKT in the A549 cells was up-regulated (P<0.05). These changes was inhibited by M3R antagonist 4-DAMP. CONCLUSION: Carbachol promotes EMT in the human lung cancer A549 cells by activating PI3K/AKT signaling pathway.     

Expression of miRNA-181a in human lung adenocarcinoma cells and its effect on cell function

AIM: To study the expression of microRNA (miRNA)-181a in different human lung adenocarcinoma cell lines, and to investigate the effect of miRNA-181a on cell function and its mechanism in human lung adenocarcinoma drug resistant cell A549/DDP. METHODS: Real-time PCR was used to detect the expression of miRNA-181a in BEAS-2B cells, A549 cells and A549/DDP cells. The A549/DDP cells were transfected with pGenesil-miRNA-181a eukaryotic expression plasmid. At the same time, the untransfection group and negative transfection group were also set up. The expression of miRNA-181a, cell viability, cell growth inhibition and apoptosis rate during cis-diamminedichloroplatinum (DDP) treatment, cell cycle, cell invasion, the protein expression of miRNA-181a target genes bcl-2 and p53 in the A549/DDP cells were determined by real-time fluorescence quantitative PCR, MTT assay, flow cytometry, Transwell method and Western blot, respectivly. RESULTS: The expression of miRNA-181a in A549 cells and A549/DDP cells was significantly lower than that in BEAS-2B cells, and the lowest expression level was observed in A549/DDP cells (P<0.05). The expression of miRNA-181a in A549/DDP cells was significantly increased after transfection with pGenesil-miRNA-181a (P<0.05). The cell viability, cell cycle and invasion ability of the A549/DDP cells were inhibited after miRNA-181a transfection (P<0.05). The cell growth inhibition rate and apoptotic rate of the A549/DDP cells were increased (P<0.05). The expression of Bcl-2 was reduced, but the expression of P53 was increased after transfection with miRNA-181a in A549/DDP cells (P<0.05). CONCLUSION: miRNA-181a may be correlated with the development of human lung adenocarcinoma. miRNA-181a can serve as a new target for treatment of lung cancer.     

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.     

CASC2/miR-18a/BTG3 signaling inhibits cell migration and invasion of non-small cell lung cancer

AIM: To explore the function and molecular mechanism of long non-coding RNA CASC2 in non-small-cell lung cancer (NSCLC) cell migration and invasion. METHODS: RT-qPCR and Western blot were used to detect the expression of CASC2, microRNA-18a (miR-18a) and BTG3 in human bronchial epithelial cell line 16-HBE, and NSCLC cell lines A549 and H1299. The interaction between CASC2 and miR-18a or miR-18a and BTG3 was predicted by bioinformatics software and verified by double-luciferase reporter assays. Transwell assays were performed to detect the migration and invasion abilities of the NSCLC cells. RT-qPCR and Western blot were used to determine the regulatory effects of CASC2 on miR-18a and BTG3 expression. RESULTS: Compared with 16-HBE cells, the expression of CASC2 and BTG3 was significantly down-regulated in the NASCL cells, while miR-18a was significantly over-expressed (P<0.05). CASC2 acted as a molecular sponge for miR-18a, and BTG3 was verified to be a target gene of miR-18a. Up-regulation of CASC2 inhibited the migration and invasion abilities of NSCLC cells, while exogenous restoration of miR-18a stimulated cell migration and invasion abilities. In addition, exogenously over-expressed miR-181a reversed the promoting effect of CASC2 on BTG3 protein expression. CONCLUSION: CASC2 promotes BTG3 expression by negatively regulating miR-18a, and then inhibits the migration and invasion abilities of NSCLC cells.     

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.     

β-estradiol promotes invasion and migration of lung cancer A549 cells via ERβ-mediated ERK1/2 membrane-initiated steroid signaling

AIM: To investigate the regulatory mechanism of β-estradiol in the invasion and migration of lung cancer A549 cells. METHODS: Breast cancer MCF-7 cells and lung cancer A549 cells were cultured in vitro. The MCF-7 cells were used as the estrogen receptor (ER) positive expression cell model. Real-time PCR and immunofluorescence were employed to measure the expression level and the localization of ER in A549 cells. The phosphorylation of ERK1/2 upon β-estradiol stimulation was quantified by Western blot. The invasion and migration abilities of A549 cells upon β-estradiol stimulation with or without ERK1/2 inhibitor PD98059 were measured by Transwell and Cell-IQ assays. RESULTS: ERβ was the dominant ER subtype in the A549 cells and primarily comprised of ERβ2 and ERβ5. Immunofluorescence revealed that ERβ expression was mainly localized in the cytoplasm. β-estradiol induced phosphorylation of ERK1/2 and promoted the invasion and migration of the cells. Inhibition of ERK1/2 signaling reversed β-estradiol-promoted invasion and migration of A549 cells. CONCLUSION: ERβ-mediated membrane-initiated steroid signaling is involved in the process of β-estradiol-promoted invasion and migration of A549 cells, through which ERK1/2 signaling plays a pivotal role.     

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.     

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.     

PAK6 signaling attenuates migratory and invasive abilities of non-small cell lung cancer cells

AIM：To investigate the roles of p21-activated kinase 6 (PAK6) and its target miRNA on the migratory and invasive abilities of non-small cell lung cancer cells. METHODS：miRNA candidates targeting PAK6 were predicted by a target prediction program. The expression of PAK6 was measured by real-time PCR and Western blotting after A549 cells were transfected with miR-23a mimics or inhibitory oligonucleotides. Luciferase reporter assay was used to determine whether PAK6 was the direct target of miR-23a. The abilities of cell migration and invasion were detected by Matrigel invasion assay and Transwell migration assay. The expression of PAK6 and matrix metalloproteinase 9 (MMP-9) was analyzed by Western blotting after A549 cells were transfected with siPAK6 or miR-23a mimics. RESULTS：miR-23a was identified by a target prediction program. Exogenetic over-expression of miR-23a resulted in a remarkable decrease in PAK6 expression (69%), whereas miR-23a inhibitory oligonucleotides induced pronounced increase in PAK6 expression (52%). The luciferase activity was significantly inhibited by 52% in wild-type PAK6 group, while there was no significant difference in the mutation group. The mRNA level of PAK6 had no change as detected by real-time PCR. Matrigel invasion assay and Transwell migration assay demonstrated there exogenetic over-expression of miR-23a markedly reduced the migration and invasion of PC-3 cells (73% and 59%, respectively). The MMP-9 expression remarkably decreased by 85％ and 76％ in the A549 cells transfected with siPAK6 and miR-23a mimics, respectively. CONCLUSION：miR-23a inhibits the migration and invasion of non-small cell lung cancer cells by repressing PAK6-MMP-9 signaling pathway.     

Sodium glycididazole combined with cisplatin efficiently promotes p21 expression in lung adenocarcinoma

AIM:To investigate the effect of sodium glycididazole (CMNa) on the sensitivity of lung adenocarcinoma to cisplatin. METHODS:The microarray data of human lung adenocarcinoma A549 cells treated with cisplatin from NCBI public database were reanalyzed for searching the genes with significantly differential expression. The A549 cells were used in the study and treated with CMNa+cisplatin. The expression levels of the verified candidate genes from database searching were detected by real-time PCR. The role of CMNa in the expression of p21 and its upstream target p53 was also determined. RESULTS:A list of candidate genes from the microarray dataanalysis was obtained, in which p21 was verified in A549 cell line treated with cisplatin by database searching and analyzing. A549 cells were treated with CMNa and cisplatin, and p21 expression was enhanced in the cells treated with CMNa+cisplatin but not CMNa alone. In addition, enhanced p21 expression in A549 cells treated with CMNa+cisplatin was mediated by promotion of the upstream p53 level. CONCLUSION:CMNa co-operates with cisplatin to improve p21 expression in human lung adenocarcinoma, which is mediated through enhancement of the p21 upstream target p53, indicating another new insight of CMNa function in radiosensitivity of human lung adenocarcinoma.     

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.