Characterization, Isolation Bronchioalveolar Stem Cells And Identification Its MicroRNA Expression Profiling

Background and ObjectiveLung 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. The breakthrough on cancer stem cells provides a new avenue of research to explore the pathogenesis of tumor and improve the efficiency of treatment. Cancer stem cells are a rare population of undifferentiated tumorigenic cells responsible for initiation, maintenance and spreading of tumor. These cells display unlimited proliferation potential, ability of self-renew and capacity to generate a progeny of differentiated cells that constitute the major tumor population. 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. Some reports have described lung stem cells as cells expressing antigens typical of undifferentiated cells, such as CD34 and breast cancer resistance protein1 (BCRP1). In 2007, a rare population cells displaying the membrane antigen CD133 were identified in human lung tumor tissues, which show the ability of unlimited proliferation and self-renew, furthermore, they can differentiate into cells constituting the major tumor population. 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. We therefore aimed to set up a reliable, in-house miRNAs microarray platform for mouse lung stem cells research. Although the expression profiling of miRNAs in human and mouse organs has been detected by some groups, the expression in the lung stem cells has yet to be defined.Therefore, we first carefully looked at the existence of DPCs in the lung both from adult, neonatal mice and the patients with non-small cell lung carcinoma by immunofluorescence staining. Secondly, we propagated BASCs in vitro and isolated them from mouse lung tissues by FACS. Subsequently, miRNAs from BASCs 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 miRNA was proved by the luciferase report gene system. The present study attempts to define miRNAs profiles of BASCs, 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.Methods1. CCA and SP-C staining in human lung cancer tissues by immunofluorescence: Adjacent serial 8μm sections of lung from human adenocarcinoma, squamous carcinoma and para-cancerous tissues respectively, all were stained for anti-CCA and anti-SP-C in order to detect the CCA and SP-C double-positive cells(DPCs). Finally, the expression was observed by co-focal laser scanning microscopy in lung tissues. Each tissue was observed 4 to 6 visual fields randomly and the percentage of DPCs in every 100 lung cells was counted. All the data were analyzed by the SPSS 11.0.2. Isolation and characterization of BASCs: The Sca-1+ cells were sorted from the pulmonary single cell suspension by magnetic labeling cell sorting with anti-Sca-1 microbeads following enzymatic digestion of lung tissue with dispase and collagenase in combination. The Sca-1+ cells were seeded into tissue culture flasks pre-coated with collagen I in serum-free culture system for BASCs cell colony, which was identified by the dual-color immunofluorescent staining for anti-CCA and anti-SP-C. The cell colony of BASCs was differentiated under the 10% fetal calf serum (FCS) on day 6. Finally, the pure CD45-CD31-Sca-1+CD34+ cells (BASCs) and CD45-CD31-Sca-1-CD34-cells (controls) were both isolated by the flow cytometry.3. Identification of the miRNAs profile from BASCs: 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 LC Sciences. 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 and verified the target gene by the luciferase report gene system.Results1. Existence of DPCs in human lung adenocarcinoma tissues: The DPCs were first discovered in the human lung adenocarcinoma. However, the DPCs were not found in lung squamous cancer. Furthermore, the number of DPCs in tumor sites was much higher than that in paratumor tissues (P<0.05).2. Successfully isolated and characterized BASCs from mouse lung: One lung of normal adult mouse could yield 1.6~1.8×107 nucleated cells in the enzyme digestion procedure. The percentage of positive cells for Sca-1 markers was much higher than the unsorted mouse pulmonary cells (87.3%±5.9% vs 9.6%±1.8%, P<0.05). By day 6, the Sca-1+ cells formed into BASCs and other unknown cell colonies identified by immunostaining for anti-CCA and anti-SP-C. Under the condition with 10% FCS, the BASCs differentiated into AT 2 cells in which SP-C expression was maintained in the majority of the colony cells up to day 10, but the cell colony expressing SP-C disappeared by day 11. No CCA could be detected in the colonies during the 10-day culture period. In order to obtain the pure BASCs, we firstly measured CD45-CD31- Sca-1+ CD34+ cells which represents 0.7%~1.1% of the total number of pulmonary cells. Finally, 2×106 BASCs (CD45-CD31-Sca-1+CD34+) and 4×106 control cells (CD45-CD31-Sca-1-CD34-) were isolated from mouse pulmonary cells suspension by flow cytometry sorting.3. Identification of the miRNAs profile from BASCs: 220ng and 720ng small RNA were both extracted from BASCs and controls respectively. The miRNAs differentially expressed between BASCs and controls were performed by using mouse miRNAs array probes which included 568 mature mouse miRNAs (Chip ID miMouse 10.0 version; LC Science). Overall, 196 miRNAs of 568 arrayed miRNAs were found in normal mouse BASCs and 261 miRNAs of 568 arrayed miRNAs were found in control. The microarray identified 56 up-regulated miRNAs and 60 down-regulted miRNAs in BASCs when compared with control cells (P<0.01). Among the 116 miRNAs, miR-142-3p, miR-451, miR-106a, miR-142-5p, miR-15b, miR-20a, miR-106b, miR-25, miR-486and miR-497 were chosen from the microarray results and validated by TaqMan qRT-PCR. MiR-497 was the only up-regulated in BASCs compared to control cells, and the left 9 were down-regulated. The target gene Wee1 for miR-497 was predicted in the TargetScan,MiRbase,and miRanda, which was then verified by the luciferase report gene system( the data was not presented).ConclusionIn summary, the present findings not only validated the DPCs existing in normal lung tissues widely, but also firstly identified the DPCs in human lung adenocarcinoma. The miRNAs expression profile of mouse BASCs was first reported and miR-142-3p, miR-451, miR-106a, miR-142-5p, miR-15b, miR-20a, miR-106b, miR-25, miR-486, miR-497 might play important roles in the regulation of BASCs. The presently established platform would provide implications of the further research of miRNAs and lung stem cells in the pathogenesis of lung cancer. The present findings and consequent research based on this platform would provide new molecular targets which are in favor of the early detection, prognosis monitoring and targeted therapy for lung cancer.