| Background&ObjectivesAs the highest morbidity and mortality cancer,lung cancer has become the first killer of human health,and non-small cell lung cancer accounts for about 85%.In China,due to the number of smokers can't get well controlled,incidence and mortality of lung cancer has increased quickly.Over the past 30 years,the mortality of lung cancer in China increased by at least 465%.Lung cancer has become the fastest rising cancer and has replaced the liver cancer becoming the first cause of cancer death.The incidence of lung cancer increased 26.9%annually.If timely and effective means of control is not taken,the lung cancer patients will reach 1 million in 2015.And China will become the world's first lung cancer country.The cause of lung cancer is still not entirely clear.A large number of studies showed that long-term heavy smoking is an important risk factor for lung cancer.About 86%of lung cancer is associated with smoking,including 3%of passive smoking.Air pollution is another important factor for lung cancer.With the rapid development of global industrialization,air pollution is worsening.Heavy industry has brought a lot of 3,4-benzopyrene,nitrous oxide arsenic,radioactive substances,non-combustible aliphatic hydrocarbons and other carcinogenic substances,which are a serious threat to people's respiratory health.But as a complex polygenic disease,the pathogenesis of lung cancer is still unclear.As early symptoms of lung cancer are less,patients usually come for treatment in advanced period,with poor efficacy.Therefore,early diagnosis and early treatment are the keys to improve the survival rate of lung cancer,but there is still a lack of cost-effective lung cancer screening program worldwide.The most economical methods of lung cancer screening are X-ray and sputum cytology,but due to low sensitivity and ease pollution,the two screening methods are high rate of misdiagnosis.The rapid development of imaging techniques also provides a new tool for screening for lung cancer.Low-dose spiral CT has become one of the appropriate selections of early lung cancer screening due to its high sensitivity and security.But CT diagnosis also has shortcomings,such as high price,low rate of early detection for central lung cancer and so on.With the development of molecular biology techniques,research of tumor markers for early diagnosis of lung cancer screening has become a hot spot.Currently,carcinoembryonic antigen(CEA),carbohydrate antigen(CA19-9),cytokeratin 19 fragment(cyfra-21-1)and other serum markers have been widely used in clinical diagnosis of lung cancer,efficacy evaluation,monitoring recurrence or metastasis and prognosis judgment.But the sensitivity and specificity of those tumor markers are low,yet can be used for screening for lung cancer in high-risk populations.Treatments of lung cancer are mainly four basic instruments including surgery,radiotherapy,chemotherapy and targeted therapy.The traditional treatment surgery,radiotherapy and chemotherapy have played a certain effect,but little effects on improving long-term survival of patients.Active targeting therapy has become the focus of current research in the treatment of lung cancer because it's efficient and safe.Research on lung cancer targeted therapy drugs mainly includes the epidermal growth factor receptor(EGFR)tyrosine kinase inhibitor agents,tumor angiogenesis factor inhibitors and multi-target inhibitors.EGFR belongs to erbB(HER)family having a transmembrane protein tyrosine kinase activity.About 40%-50%of NSCLC high expresses EGFR.At present,the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib and erlotinib have achieved significant results in NSCLC treatment.Vascular endothelial growth factor(VEGF)is highly specific for vascular endothelial cell mitogen.It is now known to be the highest specificity and the most powerful regulator of angiogenesis involved in tumor angiogenesis.VEGF subtypes expression plays an important role in tumor development process.Nowadays,the drugs of anti-VEGF include monoclonal antibody bevacizumab,vandetanib,recombinant endostatin and Salle polyamines.But now most of the effectiveness of drugs is only at about 10%.The reason is that most tumors are multi-target,multi-link-control process,and now most of the drugs are for a single drug target.At the same time,tumor targets are large individual differences.Therefore,looking for specific genes for early screening of lung cancer,predicting invasion,metastasis,recurrence of and prognosing of lung cancer,providing new targets for lung cancer research and prognosis guidance,early diagnosis and targeted,individualized treatment,prolonging survival in patients with lung cancer are very important and difficult in lung cancer research.Microarray technology is an important new technology for the field of science in the 21st century.It is a highly efficient way for screening differences genes,with fast,accurate,and efficient features.It provides a strong advantage tool for the field of life science in researches of molecular mechanism of tumorigenesis,tumor type,early diagnosis,targeted therapy and prognosis assessment.The extensive application of gene chip resulted in a massive and complex data.How to interpret these massive data to reveal the limitations inherent in the data has became a bottleneck for the rapid development of gene chips.Bioinformatics solved this problem.It integrated uses the bioinformatics biology,computer science and information technology to reveal the large and complex biological data.Bioinformatics can be applied for data mining and explore the disease from molecular level basing on the biochip,sequence alignment,statistical analysis,visualization,clustering analysis,functional interpretation,pathway analysis and promoter prediction.It is an important approach to elaborate the development and progression of the disease and provides a new idea for in-depth study of the mechanism of tumor.Therefore,this study used bioinformatics to screen and analyze lung cancer gene expression data,combineding with the reported studies to discover the new NSCLC related genes SUSD2.Firstly,mRNA and protein levels were detected in clinical specimens and NSCLC cells.Secondly,the biological function was studied by up-regulated and down-regulated the expression of SUSD2 in the NSCLC cells.Finally,microarray expression profiling was used to detect the genes in SUSD2 up-regulated and down-regulated group.Then we read the literature to find the significantly differentially expressed genes and looked for the pathway that may be relevant to SUSD2.We hope that it can provide a theoretical basis for further reveal of the pathogenesis for non-small cell lung cancer,molecular diagnostics and target gene therapy research.Methods1?Two NSCLC related tissue specimens of microarray data GSE18842,GSE19188 were analyzed by the bioinformatics software Qlucore Omics Explorer(QOE).Differentially expressed genes of NSCLC and normal lung tissue were screened;Co-differentially expressed genes were got by crossing two sets of data mapping used veny.Differentially expressed genes were analyzed using bioinformatics software DAVID.A new NSCLC related gene was screened by iHOP literature mining tools.And the structure function of the gene was analyzed to predict the function.2?The expression level of SUSD2 in NSCLC cell lines was observed by detecting SUSD2 mRNA and protein expression level in a variety of NSCLC cell lines;The expression level of SUSD2 of 24 cases of NSCLC and adjacent corresponding tissue was detected by q-PCR and WB;The expression level of SUSD2 in 160 cases of NSCLC and 32 cases of normal lung tissue was detected by immunohistochemistry,and the relationship between SUSD2 expression level and clinicopathological features of NSCLC was analyzed.3.Vector that up-regulated SUSD2 and vector down-regulated SUSD2 were builded and stable cell lines H322(transfected with up-regulated SUSD2 vector)and A549(transfected with down-regulated SUSD2 vector)were screened.Transfection efficiency was detected by WB.Cells were divided into up-regulated group[SUSD2(+)vs.Vector]and down-regulated group[SUSD2(-)vs.shRNA-sramble].The proliferation ability of cells was tested by CCK-8 assay and colony formation assay,adhesion ability of cells was tested by flat adhesion assay,the invasive ability was tested by transwell invasion assay,cell cycle and apoptosis was tested by flow cytometry.4.mRNA of cells in up-regulated group and down-regulated group were detected by Agilent Whole Human Genome Oligo Microarray.Significant differences genes were selected and further bioinformatics was analyzed.In order to explore the possible ways and mechanisms of action of SUSD2,then we found the important differentially expressed genes through analyzing the protein-protein interactions by STRING and reading the literature.Results1?QOE analyzed and screened the NSCLC related genesQlucore Omics Explorer software screened 285 differentially expressed genes,of which 99 genes were upregulated,186 genes were down regulated.We used DAVID to find the functional annotation and pathway of the differentially expressed genes and found that these genes were mainly associated with the cell cycle,cell adhesion,cell replication,p53 signaling pathways and other biological pathways.The differentially expressed genes were searched by iHOP software and SUSD2 gene was discovered to be associated with NSCLC.The protein structure analysis of SUSD2 showed SUSD2 protein structure includes four parts:Somatomedin B domain;AMOP;von Willebrand factor,type D domain(vWD);Sushi/SCR/CCP domain.According to the structure,we predict that the SUSD2 may be associated with cell adhesion,migration,complement activation,the biological function of the protein hydrolysis,etc.The results laid the theoretical foundation for further functional experiments.2?SUSD2 gene expression in NSCLC cell lines and clinical samples1)q-PCR and WB were used to detect the expression level of SUSD2 in NSCLC cell lines(A549,H322,SPC-A1,and GLC-82)and human bronchial epithelial cells 16HBE.The results showed that the expression level of SUSD2 in 4 NSCLC cell lines was lower than that in 16HBE cells.And the highest expression level was in A549,lowest expression level was in H322.2)The SUSD2 expression levels of 24 cases of NSCLC and adjacent corresponding tissue was detected by q-PCR and WB.The results of q-PCR showed that,20 cases of 24 cancer tissue was SUSD2 down-expression.-?Ct of lung cancer tissue samples and corresponding adjacent tissues was tested by Paired-sample t test,the difference was significant(t=-5.062,P = 0.000).The result of WB showed that,21 cases of 24 cancer tissue was SUSD2 down-expression.Grey value of lung cancer tissue samples and corresponding adjacent tissues was tested by Paired-sample t test,the difference was significant(t=-6.733,P = 0.000)3)The SUSD2 expression level in 160 cases of NSCLC and 32 cases of normal lung tissue was detected by immunohistochemistry.The results showed that SUSD2 was down-expression in some tumor tissue samples(72/160),and was almost over-expression(32/32)in normal lung tissue.The result was significant differences(P = 0.000)by Fisher's Exact Test.Results of analysis the relationship between SUSD2 expression level and clinicopathological features showed that the expression level of SUSD2 was correlation with tumor grade(X2 = 37.982,P = 0.000),clinical stage(X2 = 4.818,P = 0.028)and lymph node metastasis(X2 14.268,P = 0.000)and no correlation with other clinicopathological features,including sex(X2=0.786,P=0.375),age(x2=0.265,P=0.607),histological type(X2=0.363,P=0.057),pT(x2=1.326,P=0.250)and distant metastasis(P=0.207).3.Biological function of SUSD2 on NSCLC cell lines1)We successfully constructed over-expression SUSD2 vector pcDNA3.1-SUSD2 and three interference vectors and screening the best interference vector shRNA-2(named psi-mHl-SUSD2),which can reduce more than 75%of SUSD2 mRNA expression levels.2)We successfully screened H322 cell lines stably transfected with positive recombinant plasmid pcDNA3.1-SUSD2 and A549 cells stably transfected with positive recombinant plasmid psi-mHl-SUSD2.3)Colony formation assay and CCK-8 assay were used to detect cell proliferation.Results of colony formation assay showed that in up-regulated group,the colonies of SUSD2(+)cells were 47.670±6.028 and the colonies of Vector cells were 99.000±16.823,which indicates that over-expression of SUSD2 significantly inhibited colony formation of H322 cell(t =-4.976,P = 0.008).In down-regulated group,the colonies of SUSD2(-)cells were 100.670±23.245 and the colonies of shRNA-scramble cells were 39.670±10.263,which indicates that down-expression of SUSD2 significantly promoted the colony formation of A549 cell(t = 4.158,P ?0.014).Results of CCK-8 assay showed that improve expression levels of SUSD2 significantly inhibited proliferation of H322 cells in 24(t =-19.964,P = 0.000),48(t=-16.745,P = 0.000)and 72(t =-14.370,P = 0.000)hours,and down-expression of SUSD2 significantly promoted the proliferation of A549 cells in 24(t = 5.296,P =0.001),48(t = 9.018,P = 0.000)and 72(t = 7.037,P = 0.000)hours.4)Flat adherent cell assay was used to test the ability of cell adherent.MTT colorimetric results showed that in up-regulated group,the OD value of SUSD2(+)cells was 0.223 ± 0.020,compared with the OD value of Vector cells 0.424 ± 0.025.Cell adhesion ability between the two groups was statistically significant(t=-14.228,P = 0.000),which reminded that over-expression SUSD2 can specifically inhibit cell adherent in H322 cells.In down-regulated group,the results showed that SUSD2(-)cells had a significantly increased ability of adhesion(t=12.572,P = 0.000).The OD value was 0.547 ± 0.011 and 0.337 ± 0.036 respectively.5)Cell invasion capacity was tested by transwell chamber assay.The results showed that in up-regulated group,the transmembrane cells of SUSD2(+)cells were 11.670±3.055,while 30.000±5.568 for Vector cells,which indicates that over-expression of SUSD2 significantly inhibit cell invasion ability of H322 cells in up-regulated group(t=-5.000,P = 0.007).In down-regulated group,the transmembrane cells of SUSD2(-)cells were 48.330±8.145,while 24.330±8.505 for Vector cells,which indicates that down-expression of SUSD2 significantly prompted cell invasion ability of A549 cells in down-regulated group(t=3.530,P = 0.024).6)Flow cytometry results showed that there was no the significant association between cell cycle and the expression levels of SUSD2(P>0.05).Apoptosis results showed that over-expression of SUSD2 significantly promote late apoptosis rate[SUSD2(+)vs.Vector:11.003±1.584 vs.6.920±1.765,t=2.982,P=0.041]and total apoptosis rate[SUSD2(+)vs.Vector:14.913±1.227 vs.8.020±0.809,t=8.126,P=0.001]of H322 cells.Down-expression of SUSD2 significantly inhibited early apoptosis rate[SUSD2(-)vs.shRNA-scramble:1.757±1.685 vs.6.810± 1.810,t=-3.537,P=0.024]?late apoptosis rate[SUSD2(-)vs.shRNA-scramble:10.340±1.492 vs.14.003±1.611,t=-2.890,P=0.045]total apoptosis rate[SUSD2(-)vs.shRNA-scramble:12.097±2.831 vs.20.810±3.414,t=-3.402,P=0.027]of A549 cells.4?The mRNA of cells in up-regulated group and down-regulated group was detected by gene expression profile chip.There were up-regulated 3392 genes and down-regulated 2883 genes in up-regulated group.In down-regulated group,1097 genes were up-regulated and 893 genes were down-regulated(fold change ?1.5).GO enrichment results showed that in the up-regulated group,differentially expressed genes involved in biological processes mainly including signal transduction,regulation of transcription,DNA-dependent,multicellular organismal development,biological processes,cell cycle and so on.Molecule function included proteins binding,nucleotide binding,metal ion binding,ATP binding and so on.In down-regulated group,biological processes of differentially expressed genes involved were signal transduction,regulation of transcription,DNA-dependent,multicellular organismal development,cell cycle,biological processes and so on.Molecule function included protein binding,metal ion binding,nucleotide binding,ATP binding,etc.;KEGG analysis showed that in up-regulated group,the difference genes mainly involved in the metabolic pathway,pathways in cancer,MAPK signaling pathways,endocytosis etc.In down-regulated group,the difference genes were mainly in the metabolic pathway,MAPK signaling pathway,pathways in cancer,focal adhesion,calcium signaling pathway.The results of STRING showed that genes in important nodes were EGFR?HSP90AA1?AKT2?NOTCH?PIK3CD?MAPK4?CXCR4 in up-regulated group and EGFR?IL6?JUN?AKT2?PHLPP1?HDACP?CDC6 in down-regulated group.Then we found the expression level of EGFR,AKT2,CXCR4 and HDAC9 genes was changed both in up-regulated group and down-regulated group,with the trend on the contrary,which prompted that the expression level of these genes may be associated with SUSD2.We also found that these genes have been confirmed playing an important role in NSCLC.ConclusionWe found the new NSCLC related gene SUSD2 by using tools for analyzing microarray array data,bioinformatics tools and literature mining tools.The mRNA and protein level of SUSD2 were tested in NSCLC cell lines and clinical samples and was found that the SUSD2 was down-regulated in NSCLC.The results of vitro experiments demonstrated that SUSD2 can inhibited the growth,proliferation,adhesion and invasion of NSCLC cells,and promoted apoptosis of NSCLC cells.Finally,we detected the differences genes by microarray.The bioinformatics analysis and literature reading results indicated that important differentially expressed genes EGFR,AKT2,CXCR4 and HDAC9 were associated with tumor development,suggesting that SUSD2 possibly plays tumor suppressor role through interacting with these genes,which laid the foundation for the study of mechanism of SUSD2 in NSCLC. |
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