| Background:Leukemia is a hematopoietic systems tumor that certain hematopoietic cells are hindered by specific differentiation stage and characterized by malignant clone amplification. Leukemia is classed as acute leukemia and chronic leukemia depending on the degree of leukemia cell maturity. It can be also classed as lymphocytic leukemia and non-lymphocytic leukemia (or myeloid leukemia). Acute lymphoblastic leukemia (ALL), the most common malignant tumor in childhood, is a cancer of the white blood cells, characterized by the overproduction and accumulation of cancerous lymphocytic cells. ALL carried with exceeding 80%-85% of individuals now cured. Nevertheless, around 20%-30% of pediatric patients will ultimately relapse and succumb to their disease. Acute myeloid leukemia (AML), most common acute leukemia affecting adults, is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormalwhite blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. To explore the pathogenesis of different types of leukemia is of great significance for their better treatment.Leukemia stem cell (LSC) is a kind of cancer stem cells and has the ability to differentiate into hematopoietic cells. LSC, is generally accepted to be responsible for treatment failure and relapse, plays a key role in survival, proliferation, metastasis and recurrence of leukemia. Bone marrow mesenchymal stem cell (BM-MSC), an important components of bone marrow microenvironment (BME), is a particularly key hematopoietic regulator due to their capacity to differentiate into specialised stromal cells and produce cytokines, chemokines, adhesion molecules, and extracelluar matrix molecules necessary for that process. Previous studies have shown that the interactions of LSCs and BM-MSCs contributed to the development and progression of leukemia. However, the role and potential mechanisms of LSCs in regulating BM-MSCs immunophenotypes and hematopoietic function are not fully understood.BM-MSCs are important non-hematopoietic progenitors with the capacity to differentiate toward multiple mature cell types, and it has been shown that MSCs protect tumor cells against the effects of chemotherapy. Studies have demonstrated that AML derived MSCs have abnormal morphology, and the abnormal MSCs in the BME are involved in promoting the pathophysiological process of AML. Peripheral CD4+ T lymphocytes mediate vigorous suppression via contact-dependent and contact-independent mechanisms in AML. The effect of CD4+ T lymphocyte on BM-MSCs proliferation remains unclear, and its underlying mechanism is needed to be further studied.Objective:1. To evaluate the effect and the mechanism of Nalm-6 derived LSCs on the immunophenotypes, hematopoietic factor expressions and differential gene expressions of BM-MSCs after co-incubating.2. To investigate the effects and the mechanisms of CD4+ T lymphocytes on BM-MSCs proliferation.Methods:1. The role and mechanism of B-ALL cell line Nalm-6 derived LSCs in the biological characteristics and differential gene expressions of BM-MSCs1.1 BM-MSCs cultures were established from fetal bone marrow sample and with explant cultures. Light microscope and scanning electron microscope were used to observe the cellular morphology of BM-MSCs. Flow cytometry was used to detect the BM-MSCs immunophenotypes including CD29, CD31, CD34, CD44, CD45, CD73, CD90 and CD 105.1.2 CD34+ cells from Nalm-6 cells were isolated using immunomagnetic bead-positive selection and defined this subpopulation as LSCs. Co-incubated LSCs with BM-MSCs for 24h to 72h, flow cytometry was used to detect the immunophenotypes of BM-MSCsLSC. Real-time PCR was used to measure the hematopoietic factors expression of BM-MSCs, including SDF-1, IL-6, IL-10, G-CSF, IL-3, IL-7, SCF, IL-11, IL-1α, IL-1β and LIF.1.3 Co-incubated LSCs with BM-MSCs for 24h to 72h, the differentially expressed genes were identified with Illumina Genome Analyzer/Hiseq 2000. According to the RNA-Seq results, real-time PCR was performed to further confirm the differentially expressed genes.1.4 Constructed the pcDNA3.1-LUM vector and synthesized the siRNA-LUM (siRNA437 and siRNA467). Transfected pcDNA3.1-LUM vector or siRNA-LUM into BM-MSCs with Lipofectamine2000 reagents. Western blot was used to detect the lumican expression.1.5 BM-MSCs transfected with pcDNA3.1-LUM vector or siRNA-LUM, and then co-incubated with Nalm-6 cells. Flow cytometry was used to detect the cell cycle, Annexin V/PI staining assay was used to detect cell apoptosis, CCK8 assay was performed to determine the sensitivity of Nalm-6 cells for VP-16.2 The role and mechanism of AML patients derived CD4+ T lymphocyte in the BM-MSCs proliferation2.1 Collected the BM-MSCs from 20 AML patients and 20 healthy subjects. Light microscope was used to observe the cellular morphology. Flow cytometry was used to detect the BM-MSCs immunophenotypes. MTT assay was used to measure the cell proliferation.2.2 CD4+T lymphocytes from AML patients were separated and purified with magnetic beads and analyzed by flow cytometry. CD4+T lymphocyte from AML patients and BM-MSCs from healthy donors were co-cultured in different ratio (0:1,5:1,10:1 and 20:1). MTT assay was used to measure the cell proliferation.2.3 Collected the peripheral CD4+ T lymphocyte from AML patients and healthy subjects. The miR-10a expression was detected by Real-time PCR.2.4 Isolated and cultured the BM-MSCs from healthy subjects, and then transfected with miR-10a mimic and miR-10a inhibitor, MTT assay was used to detect cell proliferation. AML derived CD4+ T lymphocytes were transfected with miR-10a antagomir, real-time PCR confirmed the transfection efficiency. Co-incubated AML derived CD4+T lymphocyte with BM-MSCs, MTT assay was used to detect cell proliferation.2.5 Bioinformatics software was used to predict the binding of miR-10a and BCL6 3’-UTR. Luciferase reporter gene assay was used to determine their combination in BM-MSCs. BM-MSCs were transfected with miR-10a mimic and miR-10a inhibitor, the BCL6 mRNA and protein were quantified with real-time PCR and western blot, respectively.2.6 Collected the BM-MSCs from 20 AML patients and 20 healthy subjects. MiR-10a and BCL6 mRNA expression were detected by real-time PCR. The single factor correlation analysis was performed to analyze the correlation between serum miR-lOa expression and BM-MSCs BCL6 level.Results:1 The role and mechanism of B-ALL cell line Nalm-6 derived LSCs in the biological characteristics and differential gene expressions of BM-MSCs.1.1 BM-MSCs were collected and cultured, adherent cells had the typical characteristics and morphology of MSCs from 14 days following establishment of explant cultures. Three or four passage MSCs exhibited a fibroblast-like, bipolar spindle-shaped appearance with a whirlpool-like morphology under a light microscope. Two morphologically distinguishable cell types within the fibroblastoid cell population were identified by scanning electron microscopy. Flow cytometric analysis showed that BM-MSCs were positive for the following adhesion molecules:CD29, CD44, CD73, CD90 and CD105, and were negative for CD31, CD34 and CD45.1.2 The expression levels of cell surface molecules on BM-MSCs were evaluated by flow cytometry after LSCs simulation for 24 to 72h. The results show that no significant differences in the percentage expression of surface markers (CD29, CD31, CD34, CD45, CD73, CD90 and CD105) were observed, except for CD44, which appeared significantly up-regulated. Real-time PCR showed that SDF-1 and IL-6 levels were decreased, and other hematopoietic factors, including IL-10, G-CSF, IL-3, IL-7, SCF, IL-11, IL-la, IL-1β and LIF showed increased in BM-MSCs after the treatment of LSC, especially IL-10 and G-CSF.1.3 Illumina Genome Analyzer/Hiseq 2000 was performed to identify differentially expressed genes between the BM-MSCs with or without the treatment of LSCs. We observed the significant up-regulation of two genes (SLC7A5 and TRIB3) and significant down-regulation of five genes (WSB1, NKTR, LUM, OGT, and LENG8) in BM-MSCs after LSCs simulation for 24 to 72h. Repeated quantitative real-time PCR analysis were performed to confirm the expression profiles obtained by RNA-Seq. TRIB3 was up-regulated and LUM, and LENG8 were down-regulated in the BM-MSCs with the treatment of LSCs. The different expression abundance of LUM was more remarkable.1.4 Transfected the pcDNA3.1-LUM plasmid and siRNA-LUM (siRNA437 or siRNA467) into BM-MSCs. The results revealed that lumican expression in pcDNA3.1-LUM group was more than 2 fold higher than expression in mock and pcDNA3.1-transfected cells. In siRNA-LUM group, lumican mRNA were effectively down-regulated siRNA437-transfection group compared to siRNA467-transfection group and control siRNA group. So we used siRNA437 only to inhibit the expression of LUM in subsequent experiments.1.5 F low cytometry analysis results demonstrated that the fraction of GO/Gl phase cells increased little and the proportion of G2/M phase cells decreased in Nalm-6 cells when co-culture BM-MSCs. Down-regulation of LUM in BM-MSCs resulted in Nalm-6 cells a markedly increase in the percentage of cell population in G0/G1 phase and a decrease in S and G2/M phase, total apoptotic cells were significantly decreased, and cell survival rate (SR) to VP-16 was significantly increased. Remarkably, increased LUM expression had little effect on the distribution of cell cycles. Meanwhile, overexpression of LUM in BM-MSCs further reduced the Nalm-6 cell SR compared with BM-MSCs group, but this effect was not significant.2 The role and mechanism of AML patients derived CD4+ T lymphocyte in the BM-MSCs proliferation2.1 Isolated the BM-MSCs from AML patients and healthy subjects, we found that AML BM-MSCs appeared to be more sparse and heterogeneous in morphology under light microscopy compared with healthy BM-MSCs. Flow cytometry showed that the positive rates (positive makers including CD73, CD90 and CD105) were over 98%, and the negative rates (negative makers including CD45, CD14, CD19, CD34 and HLA-DR) were below 2% in two groups. BM-MSC from AML grew slower than that from healthy people.2.2 CD4+ T lymphocytes from AML patients and BM-MSCs from healthy donors were co-cultured in different ratio (0:1,5:1,10:1 and 20:1). After removing the suspended T cells, MTT assay showed that CD4+ T lymphocytes from AML reduced the BM-MSCs proliferation, the reduction was in a concentration-dependent manner of CD4+ T cells.2.3 MiR-10a was highly expressed in CD4+ T lymphocytes from AML patients.2.4 MiR-10a mimic strongly inhibited the growth of BM-MSCs and miR-10a inhibitor highly enhanced their growth. CD4+ T lymphocytes from AML patients highly inhibited the growth of MSCs. Furthermore, the inhibited extend of CD4+T lymphocytes transfected with miR-lOa control were stronger than that with miR-lOa antagomir.2.5 Bioinformatics prediction showed that miR-lOa could bind the 3’-UTR of BCL6, and luciferase report gene assay confirmed that BCL6 was a target gene of miR-10a. miR-lOa mimic reduced the mRNA and protein levels of BCL6 in BM-MSCs, and miR-10a inhibitor up-regulated their expression.2.6 Comparing to the miR-lOa level in serum from the healthy donors, the miR-lOa level was greatly higher in AML patients. The mRNA level of BCL6 in BM-MSCs from AML patients was highly lower than that from healthy donors. Correlation analysis revealed that serum miR-10a level was negatively correlated with BCL6 mRNA of BM-MSCs in AML patients (R2=0.5777, P<0.05).Conclusion:1. LSCs up-regulated CD44 expression of BM-MSCs, and the interaction of LSCs and MSCs in the BME contributed to the complex alterations of hematopoietic factors.2. After LSCs simulation for 24 to 72h, significant upregulation of 2 genes and downregulation of 5 genes in BM-MSCs were observed. The different expression abundance of LUM was more remarkable than other genes and was confirmed again by repeated real-time PCR. Down-regulation of LUM in BM-MSCs decreased apoptosis in Nalm-6 cells and decreased the sensitivity of Nalm-6 cells to VP-16.3. AML patient derived CD4+T lymphocytes inhibited BM-MSCs proliferation through the secretion of miR-10a. |
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