Study On Malignant Transformation And Genetic Stability Of HMSC-bm In A549 Lung Cancer Cell Microenvironment

Mesenchymal stem cells-bone marrow(MSC-bm) has the potential to self duplication, multiple differentiation, low immunogenicity, flexibly transfected by exogenous genes, inflammation and tumor-chemotaxis.It has been identified as the ideal seed cells for the cure of bone injury, tumor and angiocardiopathy.Recent studies have shown that the changes of microenvironment where stem cell lives and cell signaling pathway may induce the malignant tansformation of MSCs, even turning MSCs into tumor associated fibroblasts (TAFs). Thus, to prove the safety issue and prevent malignant tansformation in tumor microenvironment of MSCs is the crucial point to extend its clinical application.Objective:In this study, to establish the co-culture system which contains human mesenchymal stem cells-bone marrow(HMSC-bm) and lung adenocarcinoma cell line A549 through transwell technique, screen the the abnormal proliferation and differentiation of HMSC-bm, observe the change of HMSC-bm in morphology, growth and cell cycle in tumor microenvironment, detect the adhesion, migration and invasion of HMSC-bm and expression of MMP-2, MMP-9 protein, study multi-direction differentiation characteristics of MSCs in tumor microenvironment, cytoskeleton changes, chromosome heteronuclear rate and the expression of apoptosis associated protein, observe the expression of oncogene, tumor suppressor gene and key molecule in ERK1/2 related signaling pathway, analyze activities of histone deacetylase and changes of histone acetylation, to provide the scientific basis for the clinical application of MSCs.Method:Part Ⅰ:Research on the effect of A549 lung cancer microenvironment on morphology and proliferation characteristics of bone marrow mesenchymal stem cell. MSCs were incubated with A549 conditional medium.6 hole plate combined with the Transwell was applied to build co-culture system between HMSC-bm and A549 cells. A549 conditional medium incubated MSCs group, and HMSC-bm co-culture group were observation groups. Using 25%,50%,75%,100% A549 conditioned medium for co-culture incubation, proliferation activity and morphology of HMSC-bm cell were analyzed after 72h of co-culture. Co-culture groups in 3 days,7 days, respectively, were terminated co-culture and cells were collected into culture bottle for passage into P3 and P5 generation. There were also HMSC-bm and A549 control groups. Cell morphology were observed through microscopic observation. Growth curve was analyzed by MTT. Flow cytometry was used to detect cell cycle change to select appropriate conditions of abnormal proliferation and differentiation of MSCs induced by A549.Part Ⅱ:Research on the effects of A549 lung cancer microenvironment on the abilities of adhesion, migration, invasion of HMSC-bm and the expression of related molecules. Adhesion, migration and invasion of P3 and P5 HMSC-bm (CO-HMSC-bm) cells co-cultured for 7D were detected by cell matrix adhesion experiment and Transwell model method.Western blot and RT-qPCR were applied to detect MMP-2, MMP-9 protein, mRNA expression of P5 HMSC-bm co-cultured for 7d, respectively.Part Ⅲ:Research on the effect of A549 lung cancer microenvironment on cytoskeleton, clone proliferation and multilineage differentiation of HMSC-bm. Colony formation assay and MTT assay were conducted for detection of CO-HMSC-bm 7d-P5 cell proliferation. Cell cycle was detected by flow cytometry. Laser confocal microscope was used to observe the cytoskeleton. Detection of potential multi-directional differentiation were studied by observing alkaline phosphatase deposition, mineralized nodules and synapse formation after the induction of osteoblasts medium and induction of beta mercaptoethanol. Chromosome staining was applied for abnormal karyotype analysis.Part Ⅳ:Research on the effect of A549 lung cancer microenvironment on chromosome, tumor suppressor gene expression and related signaling pathway of HMSC-bm. Observed the morphological changes of CO-HMSC-bm 7d-P5 through the inverted phase contrast microscope.Cells in the logarithmic growth phase were added with colchicinet and dropped onto the icy slide and banding analysis of Giemsa staining was made. Extracting total proteins of CO-HMSC-bm 7d-P5 and expression of tumor suppressor gene P53, proto oncogene Ras and ERK signal transduction pathway of NF-κB, p-ERK protein, Capase-3 protein in each group were detected by Western blot.Part Ⅴ:Research on the effect of A549 lung cancer microenvironment on histone acetylation of HMSC-bm. Total proteins in each group was extracted and expression of histone deacetylase (HDAC) and H3K9, H4K5, H4K8 acetylation sites were detected by Western blot.Result:There were statistically significances of growth arrest between each concentration group (25%,50%,75%,100%) and the control group (P<0.05). HMSC-bm and A549 cells were co-cultured with Transwell. As the induction and passage generations increasing, the experimental group cells gradually changed significantly. Cell proliferation rate gradually increased by detection of cell growth curves and cycle, with significantly change in the CO-HMSC-bm 7d 0.05); compared with HMSC-bm 7d-P5 group, transmembrane cell number of CO-HMSC-bm 7d-P5 group decreased significantly (P< 0.05). Cell invasion assay results showed that, compared with HMSC-bm 7d-P3 group and A549 7d-P3 group, the number of CO-HMSC-bm cells of 7d-P3 group that degraded Matrigel glue invasion were increased (P< 0.05); compared with HMSC-bm 7d-P5 group, the number of CO-HMSC-bm cells in 7d-P5 group that degrade Matrigel glue film invasion obviously reduced (P<0.05). Western blot detection showed that, compared with HMSC-bm 7d-P5 group, MMP-2 and MMP-9 protein expression in CO-HMSC-bm 7d-P5 group decreased significantly (P<0.05). RT-PCR detection showed that, compared with HMSC-bm 7d-P3 and A549 7d-P3 group, MMP-2 and MMP-9 mRNA in CO-HMSC-bm 7d-P3 group were increased (P< 0.05); compared with HMSC-bm 7d-P5 group, CO-HMSC-bm 7d-P5 group was significantly decreased (P< 0.05).Differentiation induction with dosteoblast cells and neural cells showed that HMSC-bm group could be induced into osteoblastic, neural cells, and co-culture cells could not be induced into osteogenic cells or nerve cells. Microfilament was irregular in co-culture group, distributing along different directions, with aguely visible network structure, which makes the microfilament system into a grid, similar to the A549 cytoskeleton. Cell clone formation rate in coculture system was higher than that in HMSC-bm cells (P<0.01). Cell proliferation curve was S-shaped in co-culture, proliferation rate on the fourth day began faster in co-culture group than in HMSC-bm group. Detection and analysis of flow cytometry cell cycle showed G1 phase cells in co-culture group was lower than that in HMSC-bm, S (proliferating) cell ratio was significantly increased compared with HMSC-bm (P<0.05).Morphological observation showed that Co-HMSC-bm 7d-P5 cells became short and small, disorderly arrangement, large and hyperchromatic nuclei, visible pathological karyokinesis. Karyotype analysis showed that MSCs chromosome was 46, XX, which was diploid chromosome. CO-MSCs chromosome number was between 46-70, hypotriploid, triploid. Non-euploid chromosome number in some cells occurred after co-culture, obvious abnormality of chromosome appeared. Western blot showed that, compared with HMSC-bm group, the expression of caspase 3, P53 decreased, Ras, p-ERK, NF-κB expression increased in CO-HMSC-bm group (P<0.01).Acetylation level analysis by Western blot showed that HDAC4 expression in CO-HMSC-bm 7d-P5 cell was significantly higher than that in HMSC-bm group (p<0.01). High acetylation level of histone H3K9 in bone marrow mesenchymal stem decreased significantly, compared with the lung cancer cell line A549 cells, in the lung microenvironment induced by co culture conditions (p<0.01).H4K5 acetylation levels in MSCs and Co-MSCs cells were not significantly different. The high acetylation level of H4K8 in bone marrow mesenchymal stem significantly decreased in the lung microenvironment induced by co culture condition (p<0.01), closely to H4K8 in A549 cells.Conclusion1 Proliferation, cell cycle and abnormal morphology of CO-HMSC-bm in 7d-P5 group changed mostly in Transwell cella co-cultured with A549.2 Co-culture environment in A549 conditional medium can cause migration, invasion and related protein decrease of HMSC-bm,which may facilitate HMSC-bm to locate in local tumor tissue.3 Co-culture environment in A549 micro environment can induce cell pathological mitosis, cytoskeletal abnormalities such as A549 like cell morphology of CO-HMSC-bm, which make CO-HMSC-bm lose the potential differentiation into osteoblasts and neurons, increasing clone formation rate abnormally and the abnormal proliferation occurred in the cell cycle.4 Co-culture environment in A549 conditional medium can induce chromosome number into non euploid in CO-HMSC-bm, which leads to obviously abnormal chromosome. Expression of tumor suppressor gene p53 and apoptosis related protein caspase 3 decreased in induced CO-HMSC-bm group, which suggests that expression changes of Ras, p-ERK and NF-κB may be important molecular mechanism of CO-HMSC-bm malignant transformation.5 Abnormally higher level expression of histone deacetylase HDAC4 is the key cause to up-regulate the acetylation of H3K9 and H4K8 in the malignant transformation of CO-HMSC-bm.