Isolation,Characterization Of Pulmonary Adenocarcinoma Stem Cells And Identification Its MicroRNA Expression Profile In Conditional Knock-out Mouse

Lung cancer is the most common cause of cancer-related mortality worldwide. The overall five-year survival rate for the patients with lung cancer is still below 15% although the great progress in the early diagnosis and chemotherapy, radiotherapy, immune therapy and target therapy has been made during last fifty years.Tumorigenic cancer progenitor cells, also designated as cancer stem cells or cancer-initiating cells, has been proposed to be involved in cancer initiation and progression to metastatic disease states and resistance to conventional therapies."cancer stem cell"(CSC) display unlimited proliferation potential, ability to self-renew and capacity to generate a progeny of differentiated cells. Therefore, focusing research efforts on the CSC may drive important advances in our understanding of cancer biology and developing potential cures for these devastating diseases.Cancer stem cells have been first isolated and expanded from leukemia and subsequently been reported in several human solid tumors including melanoma, breast cancer, brain cancer, prostate cancer, retinal glioblastoma, pancreatic cancer, hepatoblastoma and colon carcinoma. The gastric cancer stem cells and the lung cancer stem cells were both identified in the animal models as well.In light of the cancer stem cells-based model, normal stem cells might be considered as proto-tumorigenic cells endowed with some properties typical of malignant cells, including the constitutive activation of survival pathways and the ability to proliferate indefinitely. Oncogenic mutations occurring in such a favorable background may turn the finely regulated growth potential of normal stem cells into the aberrant uncontrolled growth of cancer cells. The lung is an extremely complex, conditionally renewing organ composed of at least 40 differentiated cell type lineages. The candidate stem progenitor cells are the basal cells for mucosal gland development and renewal of the branched epithelium of the trachea, the Clara cells of the bronchiole, and the type-2 pneumocytes of the alveolus (AT2). The regenerative potential stem cells residing in the bronchoalveolar junction of adult lungs have been further identified and characterized in a mouse model of lung carcinogenesis as CD45~-CD31~-Sca-1~+CD34~+ cells expressing both cytoplasmic Clara cell specific antigen(CCA) and surfactant protein-C proteins(SP-C), which are specific markers for Clara cells and AT2 cells, respectively. BASCs were expanded at early stages of tumorigenesis in vivo and exhibited the first proliferative response following K-ras G12D activation in culture.They have also observed BASC expansion in association with lung tumorigenesis initiated by expression of a point mutant p53 allele .However, whether lung cancer stem cells might derive from the mutated normal lung stem cells remains to be elucidated.Previously unknown markers, such as noncoding RNA gene products, may also lead insight into the biology of lung cancer, although known genes and proteins have already yielded plenty of information. Micro RNAs (miRNAs) are a class of naturally occurring small noncoding RNA molecules, which are found in diverse organisms, involved in various biological processes, including developmental timing, apoptosis, stem cell division, disease and cancer in animals and humans. In addition, some miRNAs may function as oncogenes or tumor suppressors. More than 50% of miRNAs genes are located in cancer-associated genomic regions or in fragile sites, suggesting that miRNAs may play a more important role in the pathogenesis of a limited range of human cancers than previously thought. Increasing evidences has suggested the potential involvement of altered regulation of miRNAs in the pathogenesis of lung cancer. These findings demonstrated that miRNA splay an essential role in lung cancer pathogenesis, and the miRNAs profiles may be potentially useful for lung cancer diagnosis and prognosis. Previous studies have presented that some miRNAs are differentially expressed in stem cells, suggesting a potential role in stem cells regulation, such as self-renewal. Recent results from Drosophila and mouse have shown that miRNAs are important regulators for stem cells self-renewal, differentiation and division. MiRNAs may be involved in the mechanism that makes stem cells insensitive to environmental stimuli that would normally halt most cells at the G1/S checkpoint. The implication is that the mechanism used by stem cells to overcome this checkpoint could, possibly be usurped by tumor cells. Given that miRNAs contributing remarkably to both development of normal stem cells and cancer pathogenesis, we proposed that mutations must occur to turn BASCs to lung cancer cells, which is the one of important origin of lung adenocarcinoma.Therefore, we first carefully looked at the construction of lung carcinoma model in th mouse . Secondly, we propagated lung carcinoma stem cell in vitro and isolated them from mouse lung tissues by FACS. Subsequently, miRNAs from cancer stem cell at different time were labeled and then hybridized to microarray gene chips and ten candidate miRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). The target of miRNA was predicted by the bioinformatics. The present study attempts to isolate and identify lung carcinoma stem cell,then define miRNAs profiles of lung carcinoma stem cell in the course of the adenocarcinoma .thereby leading a new insight into both the regulation of stem cell self-renewal and the mechanisms for the turn from BASCs to lung cancer stem cells.objectiveTo amplify and purify the AdCre,construction of pulmonary adenocarcinoma mouse model by nasal infection AdCre;To isolate and characterize of tumorigenic the CD45~-CD31~-Sca-1~+CD34~+ cells by f low cytometry from the pulmonary adenocarcinoma mouse model;miRNAs from CD45~-CD31~-Sca-1~+CD34~+ cells in the course of pulmonary adenocarcinoma were labeled and then hybridized to microarray gene chips and ten candidate miRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR);The predicted target of let-7a by bioinformatics was proved by the PCR array.Methods1. Construction of the pulmonary adenocarcinoma using the Lox-stop-lox K-ras G12D mouse: After intraperitoneal injection nembuta, Lox-stop-lox K-ras G12D mouse nasally infect AdCre that were amplified and purified,then detect the mouse lung by hematoxylin and eosin stain .2. Isolation and characterization of adenocarcinoma stem celll in the mouse model: The CD45~-CD31~-Sca-1~+CD34~+cells were sorted from the pulmonary adenocarcinoma mouse model single cell suspension by magnetic labeling cell sorting with f low cytometry following enzymatic digestion of lung tissue with dispase and collagenase in combination. The CD45~-CD31~-Sca-1~+CD34~+cells were s cells were cultured in 10% FBS DMEM-F12 culture medium or serum-free medium supplemented with bFGF, EGF, and ITS. To test the hypothesis that CD45~-CD31~-Sca-1~+CD34~+cells were enriched for tumor-initiating cells, the ability of self-renewal, differentiation were evaluated in vitro. In vivo, CD45-CD31- Sca-1+CD34+cells were inoculated into nude mice at different injection dose to assess the tumorigenic distinction.3. Identification of the miRNAs profile of CD45~-CD31~-Sca-1~+CD34~+cells in the course of pulmonary adenocarcinoma: Total RNA were isolated using Trizol reagent according to the manufacturer's instructions. Small RNA were size-fractionated (<300nt) by YM-100 Microcon centrifugal filter (Millipore). RNA quality control, labeling, hybridization, scanning and data analysis were performed by Kang Cheng. The results of microarray chip were further confirmed using qRT-PCR. The targets of miRNAs were predicted using public web-based prediction tools, such as TargetScan,MiRbase,miRanda .4. The A549 cells were infected by Lenti-let-7a-2 expression vector .The target gene of let-7a were detected by tumor apoptosis PCR array. The target gene of let-7a were predicted by bioinformatics.Results1. To construct the pulmonary adenocarcinoma mouse model with Lox-stop-lox K-ras G12D mouse successfully:The lung tissue shape were not changed,but interior mucous membrane of bronchiole were thicken , heteromorphism cell were aggregated in submucous membrane after nasal infection 15 days; The tumor tissue of lung surface were emerged,cancer cell nest can be detected all over the lung tissue inside after nasal infection 30days.2. After isolation by flow cytometry, the CD45~-CD31~-Sca-1~+CD34~+cells fraction was raised . Under the serum-free medium, CD45~-CD31~-Sca-1~+CD34~+cells could grow as nonadherent, multicellular spheres. Under serum-cultured conditions, CD45~-CD31~-Sca~-服1~+CD34~+cells cells could differentiate into CD45~-CD31~-Sca~-1-CD34~-cells.In vivo, the tumor formation ability of CD45~-CD31~-Sca-1~+CD34~+cells were 25 times higher than CD45~-CD31~-Sca-1~-CD34~- cells.3. Successfully isolated and characterized CD45~-CD31~-Sca-1~-CD34~-cells from pulmonary adenocarcinoma mouse model in different period: One lung of normal adult mouse could yield 1.6~1.8×107 nucleated cells in the enzyme digestion procedure. The percentage of CD45~-CD31~-Sca-1~-CD34~-cells was 0.7—1.1%. The miRNAs differentially expressed among CD45~-CD31~-Sca-1~-CD34~-cells from pulmonary adenocarcinoma mouse model in different period were performed by using mouse miRNAs array probes (Chip ID miMouse 10.0 version; Kang Cheng). Overall, The microarray identified 145 up-regulated in turn miRNAs and 72 down-regulted in turn miRNAs in CD45~-CD31~-Sca-1~-CD34~-cells from pulmonary adenocarcinoma mouse model in different period: (P<0.01). Among the 216 miRNAs, mmu-miR-15a*,mmu-miR-203,mmu-miR-294,mmu-miR-295*,mmu-miR-19b,mmu-miR-483,mmu-miR-615-5p were chosen from the microarray results and validated by qRT-PCR. The target gene of different miRNAs was predicted in the TargetScan,MiRbase,and miRanda.4. Using TargetScan,MiRbase,and miRanda,let-7 go to chromosomes 11 122017230-122017301 [-].The 819 target genes were gotten including HMGA2,FASLG and so on. Overall, The tumor apoptosis PCR microarray identified 14 up-regulated genes and 16 down-regulted genes from A549 stable transfection Lenti-let-7a-2.According to the bioinformatics , FASLG was the targe gene in the tumor apoptosis genes.Conclusion1. To construct the pulmonary adenocarcinoma mouse model with Lox-stop-lox K-ras G12D mouse successfully, CD45~-CD31~-Sca-1~+CD34~+ is one of the markers for cancer stem cells in lung carcinoma mouse model, and CD45~-CD31~-Sca-1~+CD34~+cells exhibit characteristics of a tumor-initiating, cancer stem cell phenotype.2. lSuccessfully isolated and characterized CD45~-CD31~-Sca-1~-CD34~-cells from pulmonary adenocarcinoma mouse model in different period: The microarray identified 145 up-regulated in turn miRNAs and 72 down-regulted in turn miRNAs .Among the up-regulated genes, mmu-miR-15a*,mmu-miR-203,mmu-miR-294,mmu-miR-295* were up-regulated 2 times. Among the down-regulted genes, mmu-miR-19b,mmu-miR-483,mmu-miR-615-5p were down-regulated 0.5 times. 3. According to the bioinformatics and The tumor apoptosis PCR microarray, FASLG was the targe gene in the tumor apoptosis genes.