4P-1014 Serum PON1 activities are reduced with ageing: effects on the antioatherogenic properties of HDL
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OBJECTIVE:To investigate the effect and the molecular mechanism of Gab2 on migration and invasion of human lung adenocarcinoma cell line A549.METHODS:The plasmid pGCsilencerU6/GFP/Neo-RNAi-Gab2#1,pGCsilencerU6/GFP/Neo-RNAi-Gab2#2 and the empty vector were transfected into A549 cells respectively to establish Gab2-deficient A549 cells(siGab2/A549#1,siGab2/A549#2 cells) and the SCR/A549 cells(as the control cells).RT-PCR and Western blot analyse were used to test the mRNA and protein expression levels of Gab2.The abilities of migration and invasion were determined by chemotaxis assay and invasion assay.The IGF-1 induced expression of MMP-2,MMP-9 and the activation of pAkt,and pmTOR in Gab2-deficient A549 cells and SCR/A549 cells were detected by Western blot.The Gab2 mRNA expression of A549 cell,Scr/A549 cell,siGab2#1/A549 cell and siGab2#2/A549 cell were 1.340±0.009,1.201±0.074,0.315±0.008 and 0.289±0.007 respectively.The protein expression of A549 cell,Scr/A549 cell,siGab2#1/A549 cell and siGab2#2/A549 cell were 0.205±0.003,0.241±0.004,0.128±0.002 and 0.066±0.001,respectively.RESULTS:The Gab2 mRNA expression level of Gab2 silencing siGab2#2/A549 cells was obviously lower than that of A549 cells(t=168.032,P0.001);Gab2 protein expression in siGab2#2/A549 cells was also significantly down-regulated than that of A549 cells(t=64.352,P0.001).The abilities of migration and invasion of Gab2 silencing A549 cells were obviously decreased as compared with those of the SCR/A549 cells(t=5.101,P=0.029;t=10.812,P=0.002).In addition,our research showed that IGF-1-induced expression of MMP-2,MMP-9(t=-2.051,P=0.054;t=-2.652,P=0.064) and the phosphorylation of pAkt,pmTOR were obviously suppressed in Gab2 silencing A549 cells as compared with those in the SCR/A549 cells.CONCLUSIONS:This study identifies that Gab2 is a critical factor in A549 cell invasion.It seems that Gab2 promotes invasive potential via activating the Akt/mTOR pathway.
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Long-term culture of the human lung adenocarcinoma cell line A549 promotes the differentiation of these cells toward an alveolar type II cell phenotype. Here, we evaluated the susceptibility of long-term cultured A549 cells to human influenza viruses. A549 cells were cultured continuously for 25 days (D25-A549) or 1 day (D1-A549) in Ham's F12K medium. Six human influenza A viruses grew much faster in D25-A549 cells than in D1-A549 cells; however, two influenza B viruses replicated poorly in both cell types. Two avian influenza viruses replicated efficiently in both cell types, with similar titres. Expression levels of human virus receptors were higher in D25-A549 cells than in D1-A549 cells. D25-A549 cells thus more efficiently support the replication of human influenza A viruses compared with D1-A549 cells. Our data suggest that long-term cultured A549 cells will be useful for influenza A virus research.
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Renal interstitial fibrosis(RIF) represents the final common pathology pathway of most forms of kidney disease.Epithelial-mesenchymal transition(EMT) of tubuloepithelial cells plays an important role in RIF.Although a number of factors may initiate EMT in the kidney,the most potent factor is transforming growth factor-β(TGF-β).That the molecular pathways for TGF-β induced EMT have critical relation with the mechanism of RIF.Such represent a potential therapeutic approach,offering a mechanism to slow or even redress established renal interstitial fibrosis.
Myofibroblast
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To explore the effects of oxidative damage induced by mainstream smoke (MS) on A549 and A549-R cells of hOGG1 deficient cell.A549 cells and A549-R cells with down-regulated hOGG1 gene were treated at the concentrations of MS for 2h. The cellular sensitivities and DNA damages were measured by MTT assay and comet assay, respectively. The contents of ROS and 8-OHdG in both cells were examined by fluorescence method and HPLC-ECD.The cell viabilities decreased with increases of concentration of MS. The IC50 of hOGG1 deficient A549-R cell was more lower than that in A549 cell (P < 0.05). With dose increases of MS, the contents of ROS increased in both cell lines. When MS was more than or equal to 1.25 No. of cigarette/L, the contents of ROS in A549-R cells were much higher than those in A549 cells. The comet assay indicated that DNA damages of A549-R cells were more higher than those in A549 cells, and comet rate, tail length and OTM in A549-R cells were more higher in comparison with A549 cells (P < 0.05) at the levers of all concentrations. Furthermore, the levels of 8-OHdG in A549-R cells were higher than those of A549 cells at the doses of 2.5 and 5 No. of cigarette/L (P < 0.05).Mainstream smoke could induce oxidative damage in A549 and A549-R cells and hOGG1 deficiency cell could increase sensitivity to MS.
Comet Assay
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Abstract Bladder cancer (BC) is one of the most frequent urological malignancies, and its molecular mechanism still remains unclear. Recent studies have revealed that MicroRNA (miRNAs) acted as oncogenes or tumor suppressors in a variety of cancers. MiRNA‐96 has been reported to play a significant role in the development and progression of many cancers. In the current study, we found that transforming growth factor (TGF)‐β1 played a significant role in the progression that miR‐96 conducted. And TGF‐β1 could also regulate the expression of FOXQ1, which is the target gene of miR‐96. Furthermore, miR‐96 induced epithelial‐mesenchymal transition in BC cells, which is driven by TGF‐β1. In conclusion, our data revealed that miR‐96 regulates the progression and epithelial‐mesenchymal transition, which is driven by TGF‐β1 in BC cells; it may provide a new thought for the therapy of BC.
Tumor progression
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Objective: To investigate whether cancer-associated- fibroblasts (CAF), the key component of tumor microenvironment, regulate the chemoresistant capacity of lung cancer cell line A549 through SDF-1 secretion. Methods: Primary cell isolation techniques was used to isolate cancer-associated-fibroblasts from lung cancer patients. MTT assay was applied to determine the proliferation and chemoresistance of A549 cells. Quantative PCR was used to detect the mRNA changes of Bcl-xL. Western blotting was used to detect the protein expression of Bcl-xL. ELISA was applied to detect the SDF-1 secretion from normal fibroblasts (NF) and CAF. Results: CAF promoted the proliferation of A549 cells, while NF had no significant effect on them. After 72 hrs incubation, the absorbance value of A549+ CAF medium group was 0.814±0.006, significantly different from the 0.753±0.006 of the A549+ NF medium group (P<0.05). The Q-PCR assay indicated that mRNA expressions of Bcl-xL in the A549 group, A549+ NF medium group and A549+ CAF medium group were 1.00±0.11, 1.10±0.09 and 3.50±0.30, respectively, showing a significant difference between the A549+ NF medium group and A549+ CAF medium group (P<0.05). The Western blot showed that protein expressions of Bcl-xL in the A549 group, A549+ NF medium group and A549+ CAF medium group were 1.00±0.08, 1.10±0.12 and 3.10±0.25, respectively, with a significant difference between the A549+ NF medium group and A549+ CAF medium group (P<0.05). The ELISA results showed that the SDF-1 concentrations in the A549+ NF medium group and A549+ CAF medium group were 3.23±0.02 and 9.53±0.10, respectively, significantly different from each other (P<0.05). The MTT assay indicated that the absorbance values of OD of A549 group, A549+ AMD3100 group, A549+ NF medium group, A549+ NF medium+ AMD3100 group, A549+ CAF medium and A549+ CA Fmedium+ AMD3100 group were 0.43±0.03, 0.25±0.02, 0.48±0.03, 0.31±0.03, 0.72±0.06 and 0.45±0.03, respectively. The data of A549+ NF medium group was significantly different from that of A549+ CAF medium group (P<0.05). Conclusions: Cancer-associated-fibroblasts enhance the drug resistance of A549 cells through SDF-1 secretion, upregulating the expression level of Bcl-xL through interaction with CXCR4. Our study not only illustrates that tumor microenvironment is able to enhance drug resistance of tumor, but also provides experimental evidence for the cancer-associated-fibroblasts as a potential therapeutic target for the treatment of lung cancer.目的： 探讨肿瘤相关成纤维细胞(CAF)是否通过分泌基质细胞衍生因子1(SDF-1)调节肺癌A549细胞的化疗耐药性。 方法： 采用原代细胞分离方法获得肺癌患者的CAF，采用四甲基偶氮唑蓝(MTT)法检测A549细胞的增殖能力和对化疗药物的耐药性，荧光定量PCR检测Bcl-xL mRNA水平，Western blot检测Bcl-xL蛋白水平，酶联免疫吸附试验(ELISA)检测CAF分泌SDF-1的情况。 结果： CAF可以促进肺癌A549细胞的增殖，而正常纤维细胞对A549细胞增殖的影响不明显。培养72 h后，A549＋CAF上清组和A549＋NF上清组的吸光度(A)值分别为0.814±0.006和0.753±0.006，差异有统计学意义(P<0.05)。荧光定量PCR检测结果显示，A549组、A549＋NF上清组和A549＋CAF上清组的Bcl-xL mRNA表达水平分别为1.00±0.11、1.10±0.09和3.50±0.30，A549＋CAF上清组与A549＋NF上清组比较，差异有统计学意义(P<0.05)。Western blot检测显示，A549组、A549＋NF上清组和A549＋CAF上清组的Bcl-xL蛋白表达水平分别为1.00±0.08、1.10±0.12和3.10±0.25，A549＋CAF上清组与A549＋NF上清组比较，差异有统计学意义(P<0.05)。ELISA检测显示，A549＋NF上清组和A549＋CAF上清组中SDF-1含量分别为3.23±0.02和9.53±0.10，差异有统计学意义(P<0.05)。MTT检测显示，A549组、A549＋AMD3100组、A549＋NF上清组、A549＋NF上清＋AMD3100组、A549＋CAF上清组和A549＋CAF上清＋AMD3100组的A值分别为0.43±0.03、0.25±0.02、0.48±0.03、0.31±0.03、0.72±0.06和0.45±0.03，A549＋CAF上清和A549＋NF上清组比较，差异有统计学意义(P<0.05)。 结论： CAF通过分泌SDF-1激活肺癌A549细胞中的趋化因子受体4(CXCR4)，提高Bcl-xL的表达，增强肺癌A549细胞对顺铂的化疗耐药性，这为CAF作为肺癌潜在的治疗靶点提供了实验依据。.
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Transforming growth factor β (TGF-β) plays an essential role in the process of pulmonary fibrosis.TGF-β regulates pulmonary fibrosis by stimulating and activating the high expression of collagen mRNA of fihroblasts,to promote the synthesis of extra cellular matrix and improve the stability of the collagen molecule after transcription.The latest research suggests that TGF-β induced epithelial-mesenchymal transition (EMT) may be the key mechanisms of pulmonary fibrosis.This article reviews the present condition and prospects of the association between TGF-β induced EMT and pulmonary fibrosis.
Key words:
pulmonary fibrosis; Transforming growth factor-β; Epithelial-mesenchymal transition; Smad
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Objective To investigate different gene expressions of EGFR and HER-3 in human lung adenocarcinoma cells A549 and pemetrexed disodium(PEM)-resistant cells A549/PEM.Methods A549/PEM cell line was established with large dose of PEM.Growth inhibitions of A549 and A549/PEM cells were determined by CCK-8 assay.Cell cycle analysis was performed by flow cytometry(FCM).RT-PCR was used to analyse mRNA expressions of EGFR and HER-3 in A549 and A549/PEM cells.Results CCK-8 assay showed that the IC50 of PEM for A549 cells was 3.43×10-6mol/L.PEM-resistant cell line A549/PEM was established after being exposed to PEM repeatedly for 4 months,and the resistance index(RI) was 5.15.Flow cytometry analysis showed that proportions in G1/G0 phase of A549 and A549/PEM cells were(56.25±0.10)% and(65.12±0.40)%(P0.05).The proportions in S phase were(40.87±0.30) % and(31.74±0.40)%(P0.05).The mRNA expressions of EGFR in A549 and A549/PEM were 0.55±0.08 and 0.64±0.07,and mRNA expressions of HER-3 in A549 and A549/PEM were 1.29±0.03 and 1.56±0.12.The differences were statistically significant(P0.05).Conclusion Gene expressions of EGFR and HER-3 increase in A549/PEM cells,compared with that in A549 cells.It suggestes that changes of the two genes expressions appear to be associated with resistance to PEM.
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