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Objective To explore the effect of Yes-associated protein 1 (YAP1) and potential mechanism on erlotinib ( ER) resistance in lung adenocarcinoma.Methods In PC-9 cells and acquired ER resistant PC-9 ( PC-9/ER) cells, the expression changes in YAP1 gene were measured by quantitative real-time PCR( RT-qPCR) and Western blot.Indirect immunofluorescence was adopted to observe the location of YAP1.PC-9/ER cells were treated with the verteporfin ( VP, YAP1 inhibitor) for 24 h and 48 h, respectively.Expression changes in mRNA and proteins of YAP1, AKT and p-AKT were detected in the presence or absence of VP.The effect of VP was analyzed by drug resistance index using Cell Counting Kit 8(CCK-8) assay.Results The resistance index of PC-9/ER cells was (99.80 ±25.81).Compared with PC-9 cells, the expression levels of YAP1 mRNA and protein were increased in PC-9/ER.The inhibitory efficiency of VP was (50.96 ±5.86)%, and the levels of AKT and p-AKT proteins were down-regulated by the inhibition of YAP1 simultaneously.The half maximal inhibitory concentration (IC50) of PC-9/ER decreased from (11.10 ±2.72) to (1.47 ± 0.32)μmol/L (P =0.024).Resistance index was reduced to one eighth of the original.Conclusion These results indicate that the YAP1 mediates ER resistance in lung adenocarcinoma.Suppression of YAP1 can reduce the resistance through PI3K/AKT signaling pathway.Therefore, YAP1 may be a potential target for lung cancer gene therapy.
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Objective To investigate the value of detecting plasma microRNA‐125a‐3p(miRNA‐125a‐3p) ,IGF‐2 on monitoring invasion and metastasis in NSCLC ,and to study the correlation between miR‐125a‐3p and IGF‐2 .Methods miR‐125a‐3p transcripts of 20 controls ,73 NSCLC were performed in plasma by quantitative reverse transcription‐polymerase chain reaction(qRT‐PCR) and PCR data was analyzed by the 2‐ΔΔCT method .The expression of IGF‐2 in plasma was detected by ELISA .Results The expression of miR‐125a‐3p in stage Ⅲ /Ⅳ was lower than stage Ⅰ/Ⅱ and the controls(P=0 .001 ,P=0 .005) .There was no statistical differ‐ence between the stage Ⅰ /Ⅱ patients and the controls(P=0 .776) .The expression of miR‐125a‐3p was related with lymph node metastas ,lower expression in positive lymph node metastasis (P=0 .003) .The expression of IGF‐2 in stage Ⅰ /Ⅱ 、stage Ⅲ /Ⅳ was higher than the controls(P=0 .036 ,P=0 .011) .There was no statistical difference between the stageⅠ/Ⅱ and stage Ⅲ/Ⅳ (P=0 .451) . The expression of IGF‐2 was related with lymph node metastas ,higher expression in positive lymph node metastasis (P=0 .037) .The re‐sults showed a negative correlation between miR‐125a‐3p expression and IGF‐2 in plasma(r= -0 .280 ,P=0 .007) .Conclusion Low ex‐pression of miR‐125a‐3p and high expression of IGF‐2 in plasma may play a role in invasion and metastasis of NSCLC .miR‐125a‐3p may play a negative regulatory role on IGF‐2 .
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Chemotherapy treatment plays an important role in the comprehensive treatment of malignant disease,but the chemotherapy related knowledge and the selection of an appropriate regimen for certain patient are hard to master.The establishment of computer-assisted cancer chemotherapy program and management system with a follow-up database of cancer patients can help the oncologist to master the designing skill of chemotherapy regimen and cancer-related knowledge quickly,improve the teaching qnality and the efficiency of treating malignant diseases.
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Objective To survey membrane inhibitor of reactive lysis(MIRL) expression in non-small cell lung careinoma(NSCLC) and to analyze the relationship between MIRL expression and clinical staging, adjuvant chemotherapy and disease-free survial. Methods The expression of MIRL in 8 adjacent tissues and 36 NSCLC sam-pies were determined by immunohistochemistry. Furthermore, the relationship between MIRL expression and clinical stage ,adjuvant chemotherapy and disease-free survival was assayed by follow-up. Results Among 36 samples of non-small-cell lung cancer,there were 10(27.8%) samples expressing MIRE. Out of 18 samples of squamous carcinoma, 4(22.2%) expressed MIRL,while 6(37.5%) expressed it in 16 samples of adenocarcinoma,there was no statistical significance between them(P>0.05). There were no expression in 2 samples of large cell carcinoma. There was no correlation between MIRL expression and disease-free survival(P>0.05). MIRL positive expression rate in patients with preoperational adjuvant chemotherapy was significantly lower than that of those without preoperational adjuvant chemotherapy(P<0.05). Conclusions There is great percentage of MIRE expression in NSCLC. Our present study suggests that the immunological inhibition of MIRL should be blocked when monoclonal antibody is used in the treat-merit of NSCLC.
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<p><b>BACKGROUND</b>As a new member of inhibitor of apoptosis protein(IAP) family,Livin,especially Livin α,is known to be involved in occurrence and development of lung cancer.Livin is an important mechanism of chemotherapy resistance of lung cancer cell.The aim of this study is to set up Livin isoform(α & β)-specific gene silencing system in SPC-A1 cells by gene transfection and RNA interference(RNAi),and to explore the different functions and value of the isoforms in enhancing chemosensitivity of SPC-A1 cells.</p><p><b>METHODS</b>Livinα+β,Livinα and Livinβ specific siRNA were expressed stably in SPC-A1 cells,respectively.MTT was performed to study sensitivity of the cells to chemotherapy drugs.In vivo experiment was performed to test sensitivity of mouse bearing tumor to cisplatin after gene silencing of Livin.</p><p><b>RESULTS</b>After silencing of Livinα+β,Livinα and Livinβ genes,sensitivity of SPC-A1 cells to many chemotherapy drugs(including cisplatin,carboplatin,cyclophosphamide and adriblastine) was markedly increased(P < 0.05).Among them,gene silencing of Livinα+β showed the strongest enhancement effect on chemosensitivity of SPC-A1 cells(P < 0.01).Animal experiment showed that tumor inhibition rate of pSilencer-Livinα+β,pSilencer-Livinα and pSilencer-Livinβ groups was 146.1%,130.7% and 110.5%,respectively.</p><p><b>CONCLUSIONS</b>The results suggest that Livin isoform,especially Livinα+β is hopeful to be a molecular target for increasing sensitivity of lung cancer cell to chemotherapy.Gene silencing may be a new means of gene therapy for non-small cell lung cancer.</p>
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<p><b>BACKGROUND</b>Vascular endothelial growth factor-C(VEGF-C) plays a critical role in tumor-induced lymphangiogenesis and contributes to lymph node metastasis.Human antigen R(HuR) is one of the firstly identified RNA-binding proteins.It can increase the stability of a variety of growth factors and cytokines and upregulate protein expression.The aim of this study is to investigate the expression of HuR and VEGF-C protein in non-small cell lung cancer(NSCLC),and explore the relationship between the expression of HuR and VEGF-C and clinicopathological factors.</p><p><b>METHODS</b>HuR and VEGF-C protein levels were detected in 81 NSCLC tissues and 15 control benign pulmonary lesion tissues by immunohistochemistry method(SP method).</p><p><b>RESULTS</b>In NSCLC tissues,positive rate of cytoplasmic HuR,nuclear HuR and VEGF-C was 45.7%(37/81),82.7%(67/81) and 70.4%(57/81),respectively.There was a significant difference in positive expression of HuR and VEGF-C between NSCLC and benign pulmonary lesion tissues(P < 0.05).The expression of cytoplasmic HuR was closely related to pTNM stages,differentiation degree and lymph node metastasis(P < 0.05),but not correlated with sex,age and histological classification(P > 0.05).Furthermore,cytoplasmic immunoreactivity for HuR protein(P < 0.05) but not nuclear HuR expression(P > 0.05) was associated with high VEGF-C expression.</p><p><b>CONCLUSIONS</b>Cytoplasmic HuR and VEGF-C are overexpressed in NSCLC,and are related to tumor development.HuR may mediate the modulation of VEGF-C gene expression in NSCLC.</p>
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Genetic circuits are collections of basic elements that interact to produce a particular behavior. By constructing biochemical logic circuits and embedding them in cells, one can extend or modify the behavior of cells. To date, several small synthetic gene networks have been built that accomplish specific genetic regulatory functions in vivo: the autorepressor, in which a repressor regulates its own production to reduce noise in gene expression; the toggle-switch, in which two repressors inhibit each other's production to achieve a bistable system; the repressilator, in which three repressors are connected in a ring topology to produce repeated oscillation. "Rational" and "directed evolution" are currently used Genetic-circuit design tools. Someday we may be able to program cell behavior as easily as we program computers.
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Humans , Computer Simulation , Gene Expression Regulation , Genetics , Gene Regulatory Networks , Genetics , Models, GeneticABSTRACT
<p><b>BACKGROUND</b>It has been proven that the positive feedback gene circuits can increase the expression level of interested genes, and the synchronization of genetic circuits can further enhance the efficacy of gene therapy. In order to obtain an enhanced and prolonged gene expression in target cells, a radiation controlled positive feedback genetic circuit is constructed via linking the c-fos promoter with the inducible nitric oxide synthase (iNOS) cDNA, which can be synchronized by nitric oxide (NO) intercellular messenger. Ultimately, the efficacy of radiogenetic therapy for cancer will be improved.</p><p><b>METHODS</b>Using the gene recombination techniques, the vector pfos-iNOS/green fluorescent protein (GFP) was generated by cloning the radiation-responsive c-fos promoter into the plasmid vector pIRES2-EGFP to replace the primary CMV promoter, and then inserting human iNOS cDNA downstream of c-fos promoter in the vector pIRES2-EGFP. The constructed plasmids were then downloaded into A549 cells with lipofectamine. With exposure of various doses of ionizing radiation, outputs of GFP and iNOS in the treated cells were observed and analyzed.</p><p><b>RESULTS</b>The interested plasmid was successfully constructed, proved by restriction enzyme digestion analysis. The outputs of GFP and iNOS in the transfected cells were markedly increased compared with the control cells after radiation, the peak level was seen in 16 hours after radiation.</p><p><b>CONCLUSIONS</b>A positive feedback genetic circuit is successfully developed, composed by c-fos promoter and iNOS cDNA, which can be synchronized by secreting the intercellular messenger NO. This genetic circuit will be utilized in further study.</p>
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<p><b>BACKGROUND</b>Increasing evidences have shown that vascular endothelial growth factor C (VEGF-C) is involved in the tumor lymphangiogenesis via combining with its ligand, vascular endothelial growth factor receptor-3 (VEGFR-3), which is the most important factor contributing to lymph node metastasis. Futhermore, lymph node metastasis is the most familiar metastatic pathway in non-small cell lung cancer (NSCLC). Based on this knowledge, the present study aims to explore the relationship between VEGF-C and lymphangiogenesis and lymph node metastasis in NSCLC with the use of podoplanin, a novel lymphatic vessel endothelium marker to detect lymphatic vessel.</p><p><b>METHODS</b>The expression of VEGF-C and podoplanin were detected in 66 paraffin sections of NSCLC and 8 inflammatory pseudotumor tissues by immunohistochemistry (SP). Assessments of podoplanin+ lymphatic vessel density (LVD) and positive rates of VEGF-C were performed and their relationship was analysed.</p><p><b>RESULTS</b>The positive rate of VEGF-C in the inflammatory pseudotumor group (12.5%) was significantly lower than that in the NSCLC group (75.8%) (P < 0.01), the positive rate of VEGF-C in the positive lymph node group was significantly higher than that in the negative lymph node metastasis group (86.5% vs 62.1%, P < 0.05) and there was no difference between the well differentiated group and the poor differentiated group (76.3% vs 75.0%, P > 0.05). LVD in the positive VEGF-C group was significantly higher than that in the negative VEGF-C group (21.3±6.0 vs 17.7±5.1, P < 0.05), LVD in the inflammatory pseudotumor group (10.9±4.9) was distinctly lower than that in the NSCLC group (20.4±5.9) (P < 0.01) and compared with the negative lymph node metastasis group (18.5± 5.5), LVD (21.9±5.9) in the positive lymph node metastasis group increased significantly (P < 0.05).</p><p><b>CONCLUSIONS</b>Lymphangiogenesis is a significant factor for tumor lymphatic metastasis, VEGF-C may mediate lymphatic metastasis of NSCLC by the way of inducing lymphangiogenesis.</p>
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<p><b>BACKGROUND</b>It has been proved that hyaluronan was involved in adhesion and invasion behavior of varied tumor cells. Layilin is a receptor of hyaluronan found recently and has close relationship with cytoskeleton. The aim of this study is to investigate the effect of short hairpin RNA (shRNA) targeting layilin on adhesion and invasion behavior of human lung adenocarcinoma cell line A549 induced by hyaluronan in vitro.</p><p><b>METHODS</b>RNA interference plasmid that included U6 promoter and could express shRNA targeting layilin was designed, constructed, and transfected into A549 cell line. Layilin expression was examined by RT-PCR, Western blot and immunofluorescence. Adhesive and invasive ability was examined by plate adhesion model and Boyden chamber model.</p><p><b>RESULTS</b>After plasmid transfected, layilin expression in A549 cells obviously decreased (P < 0.01), and the numbers of adhesive A549 cells on plate and A549 cells permeating septum of Boyden chamber induced by hyaluronan also significantly decreased (P < 0.01).</p><p><b>CONCLUSIONS</b>The shRNA targeting layilin can efficiently inhibit the adhesive and invasive ability of A549 cells induced by hyaluronan in vitro.</p>
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<p><b>BACKGROUND</b>Gensing Rg3 is an active component from ginseng. The aim of this study is to observe the clinical anticancer effect of Rg3 in combination with chemotherapy regimen NP (vinorelbine+cisplatin) in advanced non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>Stage III-IV NSCLC patients confirmed by pathology or cytology all received vinorelbine plus cisplatin for at least two cycles, and were randomized into two groups: patients in arm A also received placebo twice a day, while patients in arm B received two tablets of Rg3 twice a day for at least two months. The endpoints of the study were the efficacy, survival and tolerance of patients.</p><p><b>RESULTS</b>From July 2000 to May 2002, 115 patients were enrolled into the trial. The patients' characteristics were well balanced in the two groups. Sex of patients: male, 79; female 36. Types of pathology: adenocarcinoma, 71; squamous cell carcinoma, 29; adenosquamous carcinoma, 8; others, 7. TNM stage: stage III, 45; stage IV, 70. Prior chemotherapy: with, 17; without, 98. Prior radiotherapy: with, 15; without, 100. Prior surgical treatment: with, 23; without, 92. Nine patients discontinued from the trial due to severe adverse effects (5) and other reasons (4), so there were 106 patients evaluable for clinical efficacy. The response rate was 14.5% (8/55) in arm A, and 33.3% (17/51) in arm B (P=0.011). The survival time in arm A was 9.7 months (mean) and 8.0 months (median), and 15.3 months (mean) and 10.0 months (median) in arm B (P=0.0088).</p><p><b>CONCLUSIONS</b>Preliminary results show improvements in response rate and survival time (median and mean) in Rg3 arm compared with placebo arm. It is worthy to confirm the results in further clinical trials.</p>
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Objective: To construct a liver targeting gene transfer vector using hepatitis B virus envelope particles. Methods: Hepatitis B viruses were obtained from the supernatant of HepG 2.2.15 cells by a PEG8000 system and were inactivated by ?-propiolactone to prepare hepatitis B virus envelope. The hepatitis B virus envelope was used to pack 5.3 kb pIRES_2-EGFP to assess their packing ability. Subsequently, the products were studied with ELISA, PCR, SDS-PAGE, and electron microscopy. Finally, the product was used to transfect HepG2 cells and the green fluorescent protein (GFP) expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometer.Results: The acquired hepatitis B virus envelope retained the surface protein HBsAg+pre S_1+pre S_2, but with no virus DNA. The prepared envelope had high packing ability for GFP and the packed GFP had a high transfection rate in HepG2 cell. Conclusion: Hepatitis B virus envelope has been successfully obtained from the supernatant of HepG 2.2.15 cells with a PEG8000 system and ?-propiolactone.
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<p><b>BACKGROUND</b>To improve the efficacy and selectivity of gene therapy for lung cancer through inducing oncostatin M (OSM) gene expression by radiation via the early growth response gene-1 (Egr-1) promoter.</p><p><b>METHODS</b>The radio-inducible OSM gene was constructed by insertion of Egr-1 promoter into upstream of the OSM gene. The expression of OSM in lung adenocarcinoma cell line A549 which was transfected with pEO and exposed to different doses of γ-ray irradiation was analyzed, and the relative survival fraction of cells and cell survival curve were observed. To examine the efficacy of this pEO gene therapy in vivo, the tumor supression effects were investigated in 40 nude mice bearing lung tumors.</p><p><b>RESULTS</b>Expression of OSM gene in A549 cells transfected with pEO plasmids was markedly upregulated in a radiation dose-dependent manner. A gene therapy experiment in vitro showed that pEO transfected A549 cells became highly sensitive to ionizing radiation. pEO transfected tumors regressed significantly after a combination therapy with irradiation in all mice (n=10), and three tumors disappeared in 3 weeks without any side effect.</p><p><b>CONCLUSIONS</b>The results indicate that tumor targeted expression of OSM gene under the control of a radio-inducible promoter represents a novel strategy for safe and effective gene therapy for lung cancer and might be widely applied in the future.</p>
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Indoleamine 2,3-dioxygenase(IDO)is the crucial rate-limiting enzyme of tryptophan outside the liver in the body and is considered as an immune defense mechanism in tumor microenvironment.IDO is expressed in a number of cells including cancer cell,dendretic cell and so on.In tumor microenvironment,the overexpression of IDO can lead to the exhaustion of tryptophan and induce the proliferation of regulatory cells,which can prevent the proliferation,diffenentiation and activity of T cells and result in the immune tolerance to tumors.
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Objective:To investigate the expression of VEGF-C in human lung carcinoma cell A549 induced by hyaluronan(HA) in vitro. Methods:A549 cells cultured in vitro were divided into three groups: control group(C): cultured with free serum medium(FSM);HA treatment group 1(HA1) and group 2(HA2): cultured with FSM containing different concentration of HA(HA1 group: 10?g/ml,HA2 group: 20?g/ml).After 48 hours,VEGF-C expression were examined by reverse transcription-polymerase chain reaction(RT-PCR)? Western blot and immunofluorescence staining. Results:Group C: both VEGF-C mRNA and protein expression were weak positive.HA1 and HA2 groups: both VEGF-C mRNA and protein expression were strong positive and increased significantly in a HA dose-depended way(P
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Objective To construct the wt-p53's eukaryotic expression vector pE6/p53/GFP that was controlled by the radiation induced promoter and research its functions.Methods Radiation response element E6 was synthesized by gene synthesis.The wt-p53 cDNA sequence was prepared from pcDNA3.1(+)/p53 plasmid by PCR.IRES2-EGFP report gene segment was prepared from double cistron expression vector IRES2-EGFP by enzyme digestion.After sequenced and identified,the recombinant plasmid was transformed into H1299(p53-/-)cell with Lipofectamine 2000,and the cell lines in stable expression was screened by G418.In the H1299(p53-/-)cell transfected with the recombinant plasmid or without,wt-p53 mRNA expression was analyzed by RT-PCR,the p53 expression by Western blot when exposed to 4 Gy 8 MV X ray for 0,3,8,12,24,36 h or when exposed to 0,1,2,4,8 Gy 8 MV X ray for 12 h.The cell cycle of H1299(p53-/-)cell transfected stably with the recombinant vector was analyzed by flow cytometry after exposed to 4 Gy 8 MV X ray.Results The recombinant pE6-p53/GFP plasmid had been constructed correctly and the expression of p53 gene in the transfected H1299 cell lines had been determined.After 4 Gy X ray radiation,the expression of wt-p53 protein had a significant rise.The transfected H1299 cell lines stopped in G1 stage after radiation and their cloning efficiency decreased notably.Conclusion We had constructed successfully the recombinant pE6/p53/GFP plasmid that was regulated by radiation induced response element E6.This provides experimental data for radiation-gene therapy of non-small lung cancer.
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Objective To explore the correlation between the expression of lung resistance-related protein (LRP) in non-small cell lung cancer (NSCLC) and that in peripheral blood lymphocytes (PBL). Methods The S-P immunohistochemistry was used to detect the expression of LRP in specimens and PBL of 49 patients with NSCLC. Para-carcinoma tissues and PBL of 10 normal people were used as controls. Results The positive expression rate of LRP was 78.2%, 46.4% and 66.8% in carcinoma tissues, para-carcinoma tissues and PBL, respectively. The expression rate of LRP in carcinoma tissues was higher than that in para-carcinoma tissues (P
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Objective To analyze the differential expressions of miR-146a,miR-206,miR-223 and let-7c-1,such as cell differentiation-related miRNAs,in CXCR4-positive and CXCR4-negative subsets of the Lewis lung cancer cell lines(LLC).Methods CXCR4-positive and CXCR4-negative subsets were isolated from LLC by immunomagnetic beads sorting,and then their total cellular RNA were extracted by Trizol,expression of 4 miRNAs were detected by real-time fluorescence quantitative PCR(TaqMan probe),and potential target genes of miRNA whose differential expression was the most significant were predicted.Immunohistochemistry was carried out to confirm differential expression of the key molecule of certain research value within CXCR4-positive and CXCR4-negative subsets growing tumor tissue,and a BLAST search was performed to identify homologies of its 3′UTR.Results Compared to CXCR4-negative subsets,the expression of 4 miRNAs were lower in CXCR4-positive subsets,and expression of miR-223 had the most significant difference(Fold change=8.26).By softwares forecasting,miR-223 had potential target sites of IGF1R,IGFBP5,Pik3cb,ELK-1 and E2F1 mRNA,such as key molecular of IGF1R signaling pathway.The expression of IGF1R of CXCR4-positive subsets growing tumor tissue was significantly higher than that of CXCR4-negative subsets.Conclusion miR-223 is lowly expressed in CXCR4 positive cells from Lewis lung carcinoma cell lines.Position 238～244 nt and 688～695 nt in target sequences of 3′UTR of IGF1R mRNA was highly homologous by screening.Close correlation is found between miR-223 and IGF1R signaling pathway.The mechanisms underlying this biologically important finding need to be further explored.
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Objective To explore the relationship between the expressions of vascular endothelial growth factor(VEGF) and matrix metalloproteinase-9(MMP-9) in prostate cancer.Methods The expression of VEGF and MMP-9 in 148 samples of pathologically verified human prostate cancer(PCa) and 10 samples of benign prostatic hyperplasia(BPH) were detected by immunohistochemical staining.Results VEGF was overexpressed in 98 of 148 tumor tissues(66.2%) and MMP-9 was overexpressed in 58 of 148 tumor tissues(39.2%).Both of them were significantly higher than those in BPH(P0.05).But the expression of VEGF was correlated with that of MMP-9.Conclusion The expressions of VEGF and MMP-9 are closely associated with the development of prostate cancer.Therefore VEGF and MMP-9 may be clinically useful as predictor of patients'survival.
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Objective To cultivate,identify and sort bronchoalveolar stem cells(BASC)derived from normal adult mouse lung.Methods After enzymatic digestion of lung tissue with dispase and collagenase in combination,the Sca-1+ cells were isolated from the obtained pulmonary cells by magnetic cell sorting.These Sca-1+ cells were cultured in dishes coated with collagen and mouse fibroblast cell line Swiss-3T3 under a serum-free culture system for BASC,which were identified by the dual-color immunofluorescent staining clara cell specific antigen(CCA)and surfactant protein C(SP-C).Finally,these pure BASC were isolated by the flow cytometry.Results One lung of normal adult mouse could yield(1.6-1.8)?107 nucleated cells in this enzyme digestion procedure.The percentage of Sca-1+ cells we sorted from lung tissue was much higher than the unsorted [(87.3?5.9)% and(9.6?1.8)%,P