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.     

Identification of UCHL1 as a potential biomarker for human gastric adenocarcinoma by subcellular proteomics

AIM: To search the biomarker for illustrating the pathogenesis of human gastric adenocarcinoma by differential proteome analysis of the cytosol of human gastric adenocarcinoma cell line SGC7901 and the corresponding immortalized normal gastric gland cell line GES. METHODS: The differential expression of cytosol protein between SGC7901 and GES cells was identified with subcellular protein separation and two-dimensional SDS-PAGE combined with MALDI-TOF-MS. The differential proteins were confirmed by Western blotting and semi-quantity RT-PCR. RESULTS: Ten repeatable differential proteins were identified. Compared with the expression level in normal gastric cell line GES, ubiquitin carboxyl-terminal hydrolase L1(UCHL1) was obviously down-regulated one both at protein and mRNA levels in gastric adenocarcinoma cell lines of SGC7901, AGS, BGC823 and MKN45. The expression of UCHL1 was also far lower in gastric cancer tissues than that in the corresponding normal tissues. CONCLUSION: Our finding suggests that UCHL1 has potential value as a biomarker for the diagnosis of gastric adenocarcinoma.     

Inhibitory effects of nm23-H1 gene on proliferation and invasion of A549 cell line

AIM:To study the inhibitory effects of nm23-H1 gene on proliferation and invasion of human lung adenocarcinoma A549 cell line. METHODS:Recombinant eukaryotic expression vector pcDNA3.1-nm23-H1 containing full length of human nm23-H1 cDNA was constructed and transfected into a human lung adenocarcinoma A549 cell line by lipofectamine. Cell strain that expressed nm23-H1 stably was screened out by G418 and named pcDNA-nm23-A549. Expression of nm23-H1 was identified by RT-PCR and immunohistochemistry. Growth curves were drawn to detect the inhibitory effects on cell proliferation. Cell cycle of pcDNA-nm23-A549 was examined by flow cytometry. Atomic force microscopy was used to observe the filopodia on the surface of the cells. RESULTS:Introduction of nm23-H1 obviously inhibited the proliferation of A549. Expression of nm23-H1 did not induce apotosis in A549 cells but increased the percentage of phase G₁ cells and decreased phase S cells. Meanwhile, phase G₁ to phase S transition was restrained. Filopodia in the cell surface was much fewer and its structure changed in cells transformed. CONCLUSION:nm23-H1 is capable of inhibiting A549 proliferation and decreasing its metastatic ability, probably by interfering with cell cycle and cell surface structure.     

Expression of Wip1 in non small cell lung cancer tissue and cells

AIM: To detect the expression level of wip1 in lung cancer tissue and three lung cancer cell lines, and to explore the relations between the expression level of Wip1 in lung cancer and various clinical and pathological features. METHODS: Real-time PCR was employed to detect the expression of Wip1 mRNA in 44 specimens of non small cell lung cancer tissues and normal tissues. The relations between the expression of Wip1 mRNA and various clinical and pathological features were analyzed. Real-time PCR was also employed to detect the expression of Wip1 mRNA in A549, NCI-1299, NCI-H460 and HBE for relative quantitative analysis.RESULTS: In the 44 specimens, the expressions of Wip1 mRNA in both cancer tissues and normal lung tissues were positive. Wip1 gene was over-expressed in 17 specimens in 44 non small cell lung cancer specimens. The rate was 38.6%. The relative level of Wip1 mRNA in NSCLC tissues was significantly higher than that in normal lung tissues (ratio=2.1644±1.3940, P<0.01). The expression of Wip1 mRNA was also correlated with pathological grading (P<0.05). The relative level of Wip1 mRNA in three kinds of lung cancer cells was significantly higher than that in HBE cells. The difference was statistically significant (P<0.05).CONCLUSION: The Wip1 mRNA is over-expressed in non small cell lung cancer, indicating that Wip1 is related to the tumorigenesis and may become the new target of non small cell lung cancer gene therapy. The expression of Wip1 mRNA is related to tumor cell differentiation and may use for the molecular biological reference index to estimate the malignant degree of cancer.     

Effect of IL-8 on mitochondrial fusion and fission gene expression in human lung adenocarcinoma cell line A549 during cell migration

AIM: To investigate the changes of migration of lung adenocarcinoma cells promoted by IL-8 and the inner and outer mitochondrial membrane dynamic changes during this process.METHODS: Human lung adenocarcinoma cell line A549 was divided into control group and IL-8 group. Cell migration was analyzed by scratch detection and Transwell assay. The secretion of endogenous IL-8 was detected by ELISA. The protein levels of mitochondrial cytochrome C (Cyt C) and mitochondrial outer membrane protein Tom20 was detected by Western blot. The mRNA expression of mitochondrial fusion genes Mfn1, Mfn2 and OPA1 and fission genes Fis1, Drp1 and MTP18 was detected by RT-PCR. The morphological changes of mitochondria were observed by MitoTracker Red CMXRos dye staining and confocal microscopy.RESULTS: The migratory rate of A549 cells and endogenous secretion of IL-8 in A549 cells were higher than those in SPC-A-1 cells. The migratory rate of A549 cells was improved by IL-8 in a time-dependent manner. Compared with control group, the Tom20 protein expression was increased (P<0.05), and the Cyt C protein expression was decreased (P<0.05). The expression of mitochondrial outer membrane fusion genes Mfn1 and Mfn2 was increased (P<0.05), and the expression of mitochondrial inner membrane fusion gene OPA1 was decreased (P<0.05). The expression of fission genes Drp1 and MTP18 were decreased (P<0.05), while the expression of Fis1 was no change (P>0.05). Under confocal microscope, the punctate aggregates in the mitochondria of the A549 cells treated with IL-8 were observed.CONCLUSION: The migratory rate of A549 cells is increased by IL-8, which is related to the changes of mitochondrial fusion genes and the fission genes.     

Role of HGF/c-Met signaling pathway in crizotinib-induced apoptosis of different lung carcinoma cell lines

AIM: To investigate the role of HGF/c-Met signaling pathway in crizotinib-induced apoptosis of different lung carcinoma cell lines and to analyze its potential regulatory mechanisms. METHODS: EML4-ALK positive cell line H2228, c-Met proliferation cell line H1993 and control cell line A549 were treated with crizotinib at different doses for different time periods. The viability of the cell lines was measured by MTT assay. The apoptosis was analyzed by flow cytometry with PI staining. The protein levels of MET and phosphorylated MET(p-MET) of HGF/c-Met signaling pathway as well as its down-stream key proteins AKT, ERK, p-AKT and p-ERK in the cell lines before and after crizotinib treatment were examined by Western blot. RESULTS: The growth of H1993, H2228 and A549 cell lines was inhibited after crizoti-nib treatment for 72 h in a dose-dependent manner. Apoptotic rates of H1993 cells and H2228 cells were increased with the crizotinib concentration and exposure time. Down-regulation of p-MET, p-AKT and p-ERK at protein levels in H1993 cells and H2228 cells after exposure to crizotinib for 72 h was confirmed by Western blot. No obvious change of the related-proteins of HGF/c-Met signaling pathway was found in A549 cell line. CONCLUSION: HGF/c-Met signaling pathway may contribute to crizotinib-induced apoptosis of H1993 cells and H2228 cells, which provides the experimental basis for MET-targeting treatment of lung cancer.     

Salinomycin inhibited proliferation and induced apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP

AIM: To investigate the effect of salinomycin on the proliferation and apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP. METHODS: The inhibitory effect of salinomycin on the growth of A549/DDP cells was tested by MTT method in vitro . The apoptosis and mitochondrial membrane potential (ΔΨ_m) of A549/DDP cells were assayed by flow cytometry. The activity of caspase-3, 8 and 9 was determined by the method of colorimetry. The levels of cytochrome C, Bcl- 2, Bax, β-catenin, and phosphorylated low-density lipoprotein receptor-related protein 6(p-LRP6) were measured by Western blotting. RESULTS: Salinomycin inhibited the growth of A549/DDP cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L decreased ΔΨ_m level, and increased reactive oxygen species (ROS), cytochrome C and cytosolic Ca²⁺ release in the cells. Salinomycin also increased the acti-vity of caspase-3, 8, and 9 in the cells, reduced the ratio of Bcl-2/Bax, and decreased the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin depresses the cell growth by inhibiting Wnt signaling, and induces the apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP via mitochondria-dependent and Bcl-2/Bax pathways.     

Construction of HLCDG1 gene siRNA expression vector and its regulation on cell cycle and proliferation in A549 cells

AIM:HLCDG1 is a novel gene cloned recently, and its expression inhibits significantly the growth of A549 cells and tumorigenesis of A549 cells transplanted in nude mice. In this study, our aim was to construct HLCDG1 gene short/small interference double-strand RNA (siRNAs) expression vector and to observe its influence on cell cycle and proliferation of A549 cells. METHODS:Using RNA interference (RNAi) techniques, a DNA vector-driven siRNAs expression vector was constructed, and a lung carcinoma cell line stably expressing siRNAs was also selected. Sequentially, using flow cytometry analysis and MTT assay, the changes of cell cycle and cell proliferation in this cell line were observed. RESULTS:Four site-match and one site-mismatch plasmids were constructed, which were named pHL-si-1, pHL-si-2, pHL-si-3, pHL-si-4 and pHL-si-c. These plasmids were co-transfected with a pcDNA3.1(+)/HLCDG1 plasmid into A549 cells, respectively. Among five co-transfected A549 cell lines, a A549 cell line co-transfected by the pcDNA3.1(+)/HLCDG1 and pHL-si-1 plasmids, namely A549-HLCDG1-si-1, showed nearly complete inhibition of HLCDG1 expression. MTT assay and flow cytometry analysis indicated that A549-HLCDG1-si-1 cells, namely the HLCDG1 gene-silencing cells, got a faster growth compared with other HLCDG1 expression cell lines, and that HLCDG1 gene-silencing induced A549-HLCDG1-si-1 cells into S phase and G2+M phase significantly. CONCLUSION:These results suggest that the HLCDG1 gene is proved to have a markedly inhibitory effect on growth in A549 lung carcinoma cells. This study might provide some understanding of the biological function and molecular mechanism of HLCDG1 gene.     

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.     

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.     

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.     

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.     

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.     

Effects of Buzhong Yiqi decoction-medicated serum on drug resistance of human lung adenocarcinoma cell line A549/DDP to cisplatin

AIM:To investigate the effects of Buzhong Yiqi decoction-medicated serum on the drug resistance of human lung adenocarcinoma cell line A549/DDP to cisplatin. METHODS:Medicated serum containing Buzhong Yiqi decoction was prepared. The optimal dose of the medicated serum was selected by MTT assay. The A549 cells and A549/DDP cells were treated with the optimal medicated serum and cisplatin at different concentrations. The IC 50, resistance index and reversal index were determined. The cells were divided into control serum group, optimal medicated serum group, LY294002 group, LY294002 + optimal medicated serum group (combination group) and negative group. The expression of PI3K and Akt at mRNA and protein levels was detected by the methods of immunocytochemistry, Western blotting and real-time PCR. RESULTS:Treatment with 10% middle dose of medicated serum for 48 h was the optimal dose and time for medicated serum. The sensitivity of A549/DDP cells to cisplatin was obviously enhanced when the cells were exposed to the optimal medicated serum with the reversal index of 2.46. The expression of PI3K and Akt at mRNA and protein levels in optimal medicated serum group and combination group was significantly decreased. CONCLUSION:Buzhong Yiqi decoction decreases the resistance index by reducing the expression of PI3K in A549/DDP cells, thus increasing the sensitivity of A549/DDP cells to cisplatin.     

Expression of GFP and Luc reporter genes driven by human myocardial myosin light chain gene promoter in human cardiomyocytes

AIM:To construct the lentiviral vectors with green fluorescent protein(GFP) and luciferase(Luc) reporter genes driven by human myosine light chain 2v gene promoter(pMLC2v) and to investigate their expression in human cardiomyocyte(HCM) cell line and human lung cancer cell line A549. METHODS:Human pMLC2v-specific lentiviral vectors with GFP(pMLC2v-GFP) or Luc(pMLC2v-Luc) were constructed and transfected into HCM and A549 cell lines. The expression characteristics of the reporter genes were observed by confocal fluorescent microscopy and bioluminescence detection. Common(nonspecific) promoter-driven GFP(GFP_C) or red fluorescent protein(RFP_C) lentiviral vectors were used as controls. RESULTS:Both cell lines expressed GFP and RFP 3 days after transfected with the nonspecific vectors. HCM specifically expressed GFP and Luc 3 weeks after transfected with the pMLC2v-GFP or pMLC2v-Luc vectors. However, A549 cells didn't show the similar expression pattern. CONCLUSION:The pMLC2v-GFP and pMLC2v-Luc lentiviral vectors are specific for newly proliferative cardiomyocytes, indicating that they can be used as reliable tools for tracking the differentiation of stem cells into cardiomyocytes in vivo.     

Let-7a-3p inhibits activity of cancer stem cells in human lung cancer A549 cells by targeting IGF1R

AIM:To study the effect of let-7a-3p on the activity of cancer stem cells in human lung cancer A549 cells and its molecular biological mechanism. METHODS:The exepression levels of let-7a-3p in lung cancer cell lines A549, NCI-H1299, SPC-A1, H1650 and HCC-827, and human normal bronchial epithilial cell line BEAS-2B were compared by RT-qPCR. The lung cancer A549 cells were transfected with let-7a-3p mimic and negative control mimic, as let-7a-3p group and negative control group, respectively, and non-transfected control group was also set up. The content of let-7a-3p in each group was detected by RT-qPCR. Tumor sphere formation assay was used to detect the tumor sphere formation ability in 3 groups of the cancer stem cells. The proportion of cancer stem cells was detected by flow cytometry. The protein levels of NANOG, OCT4 and insulin-like growth factor 1 receptor (IGF1R) were determined by Western blot. The target gene of let-7a-3p was predicted by the bioinformatic method. The relationship between let-7a-3p and IGF1R was analyzed by double luciferase assay. Western blot was used to detect whether IGF1R over-expression antagonized the inhibitory effect of let-7a-3p on the activity of cancer stem cells. A subcutaneous transplantation tumor model was also established and the effect of let-7a-3p in vivo was observed. RESULTS:The expression level of let-7a-3p in the lung cancer cell lines was significantly lower than that in the normal bronchial epithelial cell line (P<0.01). The expression level of let-7a-3p in the A549 cells of let-7a-3p group was significantly up-regulated compared with non-transfected group (P<0.01). The number of tumor spheres in let-7a-3p group was significantly lower than that in non-transfected group. The percentage of CD133⁺ cells in let-7a-3p group was significantly lower than that in non-transfected group (P<0.01). The protein expression of NANOG and OCT4 in let-7a-3p group was significantly lower than that in non-transfected group (P<0.01). Bioinformatic prediction showed that let-7a-3p complementarily matched the 3'-UTR of IGF1R, and IGF1R might be the target gene of let-7a-3p. Luciferase assay confirmed that IGF1R was the direct downstream target gene of let-7a-3p. The protein expression of IGF1R in let-7a-3p group was significantly decreased (P<0.01). Subcutaneously transplantated tumor in let-7a-3p group was significantly smaller than that in non-transfected group. CONCLUSION:Let-7a-3p may affect the expression of lung cancer stem cell-related proteins and inhibit the potential of lung cancer stem cells by down-regulating its downstream target gene IGF1R.     

Antisense nucleic acid of K-ras inhibits growth of lung adenocarcinoma cancer cell A549

AIM: To explore the mechanism of inhibiting lung adenocarcinoma cancer mediated by antisense nucleic acid of K-ras.METHODS: The expression of K-ras was detected in A549 cells and 6 lung adenocarinoma samples. The antisense expression vector of K-ras was successfully constucted (named antisense- K-ras-pcDNA3.1). After transfection, the growth curve and Apoptosis were determined by MTT assay and Annexin V staining, respectively. Cyclin A, cyclin D1, cyclin E, CDK2, CDK4, P53, Rb and caspase-3 were detected by Western blotting. RESULTS: K-ras was highly expressed in 4 samples of lung adenocarcinoma and A549 cells. In A549 cells transfected with antisense nucleic acid of K-ras, the cell growth was significantly inhibited and the apoptosis was visible.The expression levels of cyclin A, cyclin D1, cyclin E, CDK2 and CDK4 were significantly decreased, and the expression levels of P53, Rb and caspase-3 were significantly increased. CONCLUSION: The growth inhibition in A549 cells mediated by antisense nucleic acid of K-ras is related to the decreases in the expression of cyclin A, cyclin D1, cyclin E, CDK2 and CKD4 , and the increases in the expression of P53, Rb and caspase-3.     

Effect of rapamycin on proliferation,invasion, adhesion,apoptosis and autophagy of human lung adenocarcinoma A549 and resistant A549/DDP cells treated with cis-diamminedichloroplatinum

AIM: To study the effect of rapamycin (Rap) on the proliferation, invasion, adhesion, apoptosis and autophagy of human adenocarcinoma A549 and resistant A549/DDP cells treated with cis-diamminedichloroplatinum (DDP). METHODS: Human adenocarcinoma A549 and resistant A549/DDP cell lines were cultured. The inhibitory effects of Rap alone or combined with DDP on A549 and resistant A549/DDP cells were detected by MTT assay. The in vitroinvasion abilities of the 2 cell lines treated with Rap alone or combined with DDP were detected by Transwell methods. The in vitroadhesion abilities of the 2 cell lines treated with Rap alone or combined with DDP were detected by adhesion experiments. The apoptosis of A549 and resistant A549/DDP cells induced by Rap alone or combined with DDP was analyzed by flow cytometry. The cell autophagy marker proteins beclin-1 and LC3 in A549 and resistant A549/DDP cells treated with Rap alone or combined with DDP were detected by Western blotting.RESULTS: Compared with Rap or DDP alone group, the combination of Rap and DDP significantly inhibited the proliferation, invasion and adhesion of A549 and resistant A549/DDP cells in vitro, and promoted the cell apoptosis and autophagy marker proteins beclin-1 and LC3 expression (all P＜0.05).CONCLUSION: Rap enhances the effect of DDP through promoting the cell autophagy, thereby inhibiting the proliferation, invasion and adhesion of A549 and resistant A549/DDP cells and inducing the cell apoptosis with a synergistic effect.