Screening And Identification Of The Specific Markers Of Human Lung Adenocarcinoma Stem Cells With CDNA Microarrays

Backgroud and objectiveStatistic data of WHO revealed the fact that cancer patients in the incidence of lung cancer ranked first in male and second in female .The average of 5-year-survival rate ranged from 9 to 13 percent, even that in patients with surgery in primary stage is approximately 18 percent. The proportion of lung adenocarcinoma in lung cancer has risen rapidly, which became the main pathological type and its morbidity is about 30%-40% of all lung cancer. Nowadays therapies for it are not satisfied because of its relapse and the metabasis in primary stage. It has been considered that the tumor stem cells being capable of self-renewal and multipotential differentiation are the origin of the tumors, which is the reason of cancers'relapse and metabasis as well as insensitivity to the chemotherapy and radiotherapy. Eramo et al. identified the lung cancer stem cells in 2007, which facilitated the further research of lung carcinoma. How the normal lung stem cells transform to cancer stem cells? What are the specific markers of the lung adenocarcinoma stem cells? The answers of these questions are important to the pathogenesis , early diagnosis and treatment of lung adenocarcinoma. Therefore, we arranged the following experiments: Isolation and identification of the lung adenocarcinoma stem cells; Screening the differentially expressed genes profiles between lung adenocarcinoma stem cells and normal lung stem cells with cDNA microarrays ; Then analysis and identification of the specific markers of lung adenocarcinoma stem cells.MethodPart I Isolation and identification of human lung adenocarcinoma stem cells1. Distribution of the stem cells of normal lung and lung adenocarcinoma : Sections were double labelled with CD133-PE and CD326-FITC,and were observed with laser confocal microscopy.2. Isolation of stem cells: Stem cells of lung adenocarcinoma were isolated from the single cell suspension labelled by CD133-PE with the magnetic cell sorting system(MACS).3. Culture of the lung adenocarcinoma stem cells: Cells were incubated in serum-free medium, which were supplied with fresh medium every 3 days.4. Evaluation of cell proliferation of lung adenocarcinoma stem cells: Stem cells were incubated in 96 wells plate, cells proliferation were tested with WST-8 method on day 0, 4, 6, 8.5. Dertermination of tumor formation: CD133+ and CD133- cells were transplanted in 4-weeks-old NOD/SCID mice in defferent concentrations, tumor formation were observed in 8 weeks.Part II Screening of differentially expressed gene profiles of stem cells between human lung adenocarcinoma and normal lung with cDNA microarrays1. Sorting stem cells with flow cytometry: The two types of cell suspension were double labelled with CD133-PE and CD326-FITC, and observed with fluorescence microscopy.2. Screening of expression profiles with the cDNA microarrays : Total RNA of sorted stem cells were isolated, reverse transcribed, labelled with Cy3, and hybridized with chips, fluorescent scanned, and finally expressed genes analysed.3. Analysis of differential expression profile: Gene profiles of the two types of cells were analysed with GO and KEGG pathway, especially the proto-oncogenes and genes associated with wnt signal pathway.4. Real-time qRT-PCR to verify the result of the microarrays: Upregulated expression genes Rab25 and TLE2 , downregulated genes FGR and MACF1 obtained from microarrays screening were selected for real-time qRT-PCR, the data between microarrays and qRT-PCR were compared with t test.Part III Analysis and identification of the specific markers of human lung adenocarcinoma stem cells1. Analysis of the candidate markers Rab25 , TLE2 , LRP5 and PYGO2 with bioinformation: The length of mRNA and protein , chromosomal assignment , molecular weight , classification in GO , pathways involved, hydrophobic regions and homologization sequences,etc. were inquested in NCBI GENBANK , SWISSPROT/TrEMBL , GO , KEGG pathway , Blastp and Protscale databases, data analysed.2. Detection of Rab25 expression in tissue sections of lung adenocarcinoma and normal lung with laser confocal microscopy: After the bioinformation analysis of candidate markers, Rab25 was determined as the specific marker(Rab25 belongs to small GTPase superfamily,RAS family). And then the Rab25 expression in normal lung and adenocarcinoma tissue sections were verified with CD133 and Rab25 monoclonal antibodies labelled.3. Effection of Rab25 monoclonal antibody in the proliferation experiment of lung adenocarcinoma stem cells: The Rab25 monoclonal antibody in different concentrations were added to cultured cells, and the cells'reproductive activity were examined.ResultPart I Isolation and identification of human lung adenocarcinoma stem cell1. Distribution of the stem cells in normal lung and lung adenocarcinoma :A small quantity stem cells in tissue sections were found, which was located in the bronchioal-veolar duct junction (BADJ) in normal lung, and in the BADJ as well as alveolar wall in adenocarcinoma of lung.2. Isolation of stem cells: The purity of isolated stem cell was about 60-70%, and the cells appearing typical morphology of stem cells.3. Culture of the stem cells of lung adenocarcinoma: On day 6, small cells conglobation observed, on day 20, sphere of stem cells was obvious.4. Evaluation of cell proliferation of lung adenocarcinoma stem cells: Stem cells proliferated obviously.5. Determination of tumor formation: Only the group of 1×104 CD133+ cells formed tumor in a mouse and the other groups didn't.Part II Screening of differentially expressed gene profiles between human normal lung stem cells and lung adenocarcinoma stem cells with cDNA microarrays1. Sorting stem cells with flow cytometry: The ratio of CD133+ and CD326+ double positive cells(stem cells) in normal lung was 0.31%, and obtained 1×105 stem cells from 5×107 cells sorted; meanwhile, there were 0.65% stem cells in adenocarcinoma of lung ,and obtained 4×105 stem cells from 7×107 cells sorted.2. Screening of expression profiles with the cDNA microarrays :Isolated total RNA were tested with formaldehyde denaturing gel electrophoresis which showed no degradation ,as well as high purity examined with UV spectrophotometer. After hybridization, the specificity activity of the cRNA labeled with Cy3 was 21.8 and 26.9 pmolCy3/μgcRNA in adenocarcinoma and normal lung respectively, and there were 5798 genes differencially expressed found in two types of stem cells (cut-off value:2.0).3. Analysis of differential expression profile: Gene hierarchy cluster showed different expression profiles between two types of stem cells. GO Analysis of differencially expressed genes revealed that in biological process the highly enriched categories included those related to biological regulation(41%) , cellular component organization biogenesis , intracellular signaling cascade , cell differentiation and cell cycle. In molecular function the highly enriched categories included those related to binding(72%) , transferase activity , kinase activity and enzyme regulator activity,meanwhile, in cellular component the highly enriched categories included those ralated to membrane (13%) , non-membrane-bounded organelle , extracellular region , cell fraction and macromolecular complex assembly. KEGG pathway analysis showed that wnt signal pathway was of significant difference between two types of cells (p<0.01), and 13 differentially expressed genes involved in the pathway. In addition, 11 differentially expressed genes of proto-oncogenes were detected(cut off value : 2.0).4. Real-time qRT-PCR to verify the results of the microarrays: t test showed the two methods had no significant differences(p>0.05).Part III Screening and identification of the specific markers of human lung adenocarcinoma stem cells1. Candidate markers Rab25 , TLE2 , LRP5 and PYGO2 were analysed with bioinformation:After inquested the bioinformation databases ,data of the length of mRNA and protein , chromosomal assignment , molecular weight , classification in GO , pathways involved , hydrophobic regions and homologization sequences, etc. were obtained.2. Detection of Rab25 expression in sections of lung adenocarcinoma and normal lung tissue with laser confocal microscopy: In the sections of adenocarcinoma, we found stem cells membrane-stained yellow(CD133+ and Rab25+) as well as cytoplasma-stained green(Rab25+) in some area ,which means stem cell marker- CD133-positive cells expressed Rab25 on membrane and in cytoplasma. But in normal lung sections, in those cells, only membrane-stained red(CD133+ only), cytoplasma-stained green were rarely observed, which indicate that almost no Rab25 were expressed in normal stem cells.3. Effection of Rab25 monoclonal antibody in the proliferation experiment of lung adenocarcinoma stem cell: The antibody can inhibit the cells'proliferation and its effect was dose-dependent.Conclusion1. A small quantity of stem cells were found in both normal lung and lung adenocarcinoma tissue sections , which were located in the BADJ in normal lung and the BADJ and alveolar wall in adenocarcinoma of lung.2. Lung adenocarcinoma stem cells proliferated obviously and posseses the ability of tumor formation.3. There were 0.31% stem cells in normal lung and 0.65% in lung adenocarcinoma.4. Diversity expression profiles were observed between normal lung stem cells and lung adenocarcinoma stem cells with GO analysis, which is composed of three main categories. In biological process category, highly enriched expressed genes included those related to biological regulation(41%) , cellular component organization biogenesis , intracellular signaling cascade , cell differentiation and cell cycle. In molecular function category, the highly enriched included those related to binding(72%) , transferase activity , kinase activity and enzyme regulator activity. And in cellular component category, the highly enriched included those ralated to membrane (13%) , non-membrane-bounded organelle , extracellular region , cell fraction and macromolecular complex assembly. Analysis of KEGG pathway uncovered that wnt signal pathway was of significant difference between two types of stem cells, and 13 differentially expressed genes were involved in the pathway. In addition, 11 differentially expressed genes were detected in proto-oncogenes.5. Distinct expression of Rab25 were observed in lung adenocarcinoma stem cells but not in normal lung stem cells.6. Rab25 monoclonal antibody inhibited the proliferation of stem cells of lung adenocarcinoma and the effect was dose-depended.