Altered expression of MALAT1 lncRNA in chronic lymphocytic leukemia patients, correlation with cytogenetic findings

BACKGROUND: Recent studies have devoted much attention to non-protein-coding transcripts in relation to a wide range of malignancies. MALAT1, a long non-coding RNA, has been reported to be associated with cancer progression and prognosis. Thus, we here determined MALAT1 gene expression in chronic lymphocytic leukemia (CLL), a genetically heterogeneous disease, and explored its possible relationships with cytogenetic abnormalities. METHODS: MALAT1 expression level was evaluated using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) on blood mononuclear cells from 30 non-treated CLL patients and 30 matched healthy controls. Cytogenetic abnormalities were determined in patients by fluorescence in situ hybridization (FISH). RESULTS: MALAT1 expression level was up-regulated in the CLL group compared to healthy controls (P=0.008). Del13q14, followed by Del11q22, were the most prevalent cytogenetic abnormalities. We found no association between the FISH results and MALAT1 expression in patients. CONCLUSION: Altered expression of MALAT1 is associated with CLL development. Further investigations are required to assess the relationship between this long non-coding RNA and CLL patient survival and prognosis.

Effect of the receptor activator of nuclear factor [ka]B and rank ligand on in vitro differentiation of cord blood CD133[+] hematopoietic stem cells to osteoclasts

Objective: receptor activator of nuclear factor-kappa B ligand [RANKL] appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells [HSCs]. This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor [CTR] on cord blood HSC surface

Materials and Methods: in this experimental study, CD133[+] hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor [M-CSF] and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase [TRAP] staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction [RT-PCR] assay for specific genes

Results: hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells

Conclusion: presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast

Correlation between methylation and expression level of P15 and P16 genes during differentiation of cord blood stem cells into erythroid lineage mediated by erythropoietin

Several influential factors such as transcription factors and intracellular signaling components are involved in differentiation of stem cells into a specific lineage. P15 and p16 proteins are among these factors. Accumulating evidences has introduced the epigenetic as a master regulator of these factors during lineage specification. The main objective of this study is to determine the correlation between the expression level and methylation pattern of P15 and P16 genes in erythroid lineage after in vitro differentiation by erythropoietin [EPO]. The purified and expanded CD34+ cord blood stem cells were differentiated into erythroid lineage in the presence of EPO. DNA was isolated from both cord blood stem cells and differentiated cells. The Real-Time PCR performed using cDNA and the isolated DNA was used in methylation Specific PCR [MSP] reaction for methylation pattern analysis in both pre and post differentiation stages. The study demonstrated that P15 and P16 genes have partial methylation after erythroid differentiation by EPO. The Expression of P15 gene was higher after differentiation and the expression of P16 gene had a slightly decreased level in post differentiation stage. Significant increase in P15 gene expression after differentiation to erythroid lineage, suggests the remarkable efficacy of this gene in erythroid function. According to upregulation of P15 gene after differentiation despite unchanged methylation status and slight down regulation of P16 gene with slight hyper-methylation of the gene it can be suggested that although the methylation can affects the expression level of P16 gene, the P15 gene is not affected by this mechanism during erythroid differentiation mediated by EPO

Osteogenic inhibition in multiple Myeloma

Multiple myeloma [MM] is a plasma cell malignancy where plasma cells are increased in the bone marrow [BM] and usually do not enter peripheral blood, but produce harmful factors creating problems in these patients [e.g. malignant plasma cells over activate osteoclasts and inhibit osteoblasts with factors like RANKL and DKK]. These factors are a main cause of bone lesion in MM patients. Recently SOST gene which responsible to encodes the sclerostin protein was identify. This protein specifically inhibits Wnt signaling in osteoblasts [inhibition of osteoblast differentiation and proliferation] and decrease bone formation and can also cause bone lesion in MM patients. In this experimental study, human myeloma cell lines [U266 b1] were purchased from Pasteur Institute of Iran. Samples consisted of BM aspirates from the iliac crest of MM patients. BM with more than 70% plasma cell were selected for our study [6 patients] and one healthy donor. RNA extraction was done with Qiagen kit. was undertaken on mRNA of samples and cell lines. Also we purchased unrestricted somatic stem cells from Bonyakhte Company to evaluate the effect of soluble factors from myeloma cell lines on osteogenic differentiation medium. Our results showed that SOST is expressed significantly in primary myeloma cells derived from MM patients and myeloma cell lines. In other words, patients with more bone problems, express SOST in their plasma cells at a higher level. In addition, myeloma cells inhibit osteoblast differentiation in progenitor cells from umbilical cord blood stem cell [UCSC] in osteogenic inducing medium. There are many osteoblast maturation inhibitory factors such as DKK, Sfrp and Sclerostin that inhibit maturation of osteoblast in bone. Among osteoblast inhibitory agents [DKK, Sfrp, Sclerostin] sclerostin has the highest specificity and therefore will have less side effect versus non-specific inhibitory agents. Our results also show that based on SOST expression in MM, there is a potential to inhibit sclerostin with antibody or alternative methods and prevent bone lesion in MM patients with the least side effect

Zoledrinic acid induces steoblastic differentiation of mesenchymal stem cells without change in hypomethylation status of osterix promoter

Mechanism of zoledronic acid on osteoblastic differentiation of mesenchymal stem cells [MSCs] has not fully understood. With the knowledge of some drugs mechanism that alter methylation pattern of some genes, the present research sets out to evaluate osterix [OSX] promoter methylation pattern during zoledronic acid-induced osteoblastic differentiation of MSCs. In this experimental study, MSCs were isolated from human bone marrow. For osteogenic differentiation, MSCs were pulse treated with 5 micro M Zoledronic acid for 3 hours and incubated after a medium change in osteogenic differentiation medium for 3 weeks. DNA and RNA were extracted on days 0, 7, 14 and 21 of MSCs differentiating to osteoblast. After cDNA synthesis, OSX expression was evaluated by RT-PCR and quantitative Real-Time PCR. After multiplicity of infection [MOI] treatment, gene specific methylation of OSX was analyzed by methylation specific PCR [MSP]. The mRNA expression of OSX was increased in osteoblast differentiated cells induced by zoledronic acid, especially on days 14 and 21 of differentiation [p<0.05], but expression of OSX didn't change in undifferentiated MSCs. MSP revealed that, on day 0, undifferentiated MSCs are totally methylated. But, on day 7 of differentiation, MSCs treated by zoledronic acid were totally unmethylated. OSX promoter remained unmethylated, afterwards. MSP revealed that OSX had a dynamic pattern in methylation, while MSCs gradually differentiated to osteoblasts. Our finding showed that promoter region of OSX is hypomethylated independently from zoledronic acid treatment during osteoblastic differentiation. This knowledge is important to understand drug mechanisms and can be useful for developing new therapies to combat against bone diseases

[Quantitative evaluation of imatib efflux and influx transporters expression in chronic myeloid leukemia [CML] patients]

In this study quantitative expression of MDR1 and hOCT1 genes in CML patients and normal people were measured using Real-Time PCR. To study quantitative expression of these genes by real-time PCR, mastermix with syber green was used. Peripheral blood samples from 30 CML patients and 27 normal persons were harvested. Real-time PCR results were analyzed with relative quantification method. This study showed that in the patients group who were under treatment with Imatib, MDR1 gene expression was increased which was statistically significant. This increase has a direct relation with disease progress. Gene expression in AP and BP patients was also higher than CP patients. In contrast, hOCT1 expression in patients group in comparison with normal group was not statistically significant. MDR1 increase in leukemic cell membrane results in the reduction of intra-cellular drug concentration. Thus, optimal concentration of drug for inhibition of BCR-ABL tyrosine kinase is not achieved which culminated in disease progression to AP and BP phases. Moreover changes in hOCT1 gene expression as an influx transporter of Imatib could affect intracellular concentration of drug and finally determine therapy outcome. However, in this study hOCT1 gene expression was variable and was not statistically significant

[Epigenetic changes of RUNX2 and DLX5 genes in osteoblastic differentiation induced by zoledronic acid in mesenchymal stem cells]

Zoledronic acid as a main treatment for osteoporosis has an important role in differentiation of mesenchymal stem cells. However, mechanism of osteoblastic differentiation induction by zoledronic acid is not well understood until now. In this research we evaluate zoledronic acid effect on methylation status of RUNX2 and DLX5 promoters. After isolation and expansion of hMSCs from BM, they were pulse treated with 5 micro M ZA for 3h, and incubated in osteogenic differentiation medium for 3 weeks. DNA was extracted after first, second and third weeks of culture and also from undifferentiated MSCs. After SBS treatment, gene specific methylation analysis for RUNX2 and DLX5 were carried out by MSP using methylated and unmethylated primers. In the genes RUNX2 and DLX5, M and U primers of MSP amplified promoter regions of undifferentiated and osteoblastic differentiated MSCs. Therefore, methylation status in RUNX2 and DLX5 promoters present incomplete methylation. Methyltion patterns of RUNX2 and DLX5 don't change during zoledronic acid osteoblastic differentiation of MSCs. Our findings show that zoledronic acid does not induce osteoblastic differentiation via epigenetic mechanisms. Therefore, zoledronic acid can induce osteoblastic differentiation in a manner independent from DNA epigenetic changes

[Differentiation of cord blood stem cells into erythroid progenitor cells in semisolid culture media containing SCF, IL-3, IL-6 and EPO]

The study of erythropoiesis needs to develop the methods for erythroid progenitor cells [EPCs] culture using stem cell potency to differentiate into variety of hematopoietic cells lineages. In this study, we induced differentiation of cord blood stem cells into erythroid progenitor cells in a semisolid culture media. Cord blood mononuclear cells were isolated and cultured in liquid culture media consist of erythroid differentiation factors [phase I]. Non-adherent cells were cultured in semisolid media containing SCF, IL-3, IL-6 and EPO [phase II]. After one week, the appeared erythroid colonies were picked up. Characterization of differentiated cells was performed by assessment of CD235a and CD36 using flowcytometry, giemsa cytological staining and gene expression analysis of GATA-1, EKLF, -globin genes by RT-PCR. Flowcytometry analysis to detect CD235a and CD36 positive cells showed that 94.3% and 95.5% of differentiated cells have erythroid specific cell markers, respectively. Morphology of the cells in giemsa stained slides demonstrated erythroid progenitor cells, ranged from proerythroblast to orthochromatic erythroblast. The RT-PCR results, confirmed the precursor state of erythroid differentiated cells by expression of GATA-1, EKLF, -globin genes. Cord blood stem cells have high potency to differentiate into erythroid progenitor cells using described method that can be utilized in the experimental studies

miR-155 down Regulation by LNA inhibitor can reduce cell growth and proliferation in PC12 cell line

MicroRNAs [miRNAs] are a class of small non coding regulatory RNAs that have key functions in multiple cell processes. Deregulation of these tiny miRNAs are involved in various human diseases. MiR-155 is one of the multifunctional miRNA that its over-expression has been found to be associated with different kinds of cancer such as leukemia, breast and colon cancers. It is thought that deregulation and over-expression of this microRNA may be associated with PC12 cell proliferation. So, the aim of this study was to investigate the role of miR-155 expression on PC12 cell growth. For this reason, PC12 cells were cultured and transfected by 3 different concentration [25, 50 and 75 nmol] of either LNA anti-miR-155 or scramble antisense in 24-well plate. Then, total RNA was extracted from transfected cells. miRNA cDNAs were synthesized from isolated total RNA. In the second step, miR-155 expression level was analyzed using the quantitative real-time polymerase chain reaction [QRT-PCR]. MTT test was performed to evaluate cell viability. In the next step, apoptosis assay was assessed to investigate anti miR-155 effect on PC12 cells death. Obtained results were analyzed with t-test. MTT test revealed that cell viability of transfected cells with 75 nM of anti-miR- 155 to be reduced by half of the control and scramble groups [0.5 vs. 0.97 and 0.94]. Our data suggest that miR-155 over-expression is associated with PC12 cell growth. So, miR-155 down regulation by anti-miR-155 could open up new ways to restrain brain tumor growth, as anti-miR-155 causes PC12 cells to repress

high yield expansion and megakaryocytic differentiation of human umbilical cord blood CD133[+] cells

Despite of many benefits, umbilical cord blood [UCB] hematopoietic stem cell [HSC] transplantation is associated with low number of stem cells and slow engraftment; in particular of platelets. So, expanded HSCs and co-transfusion of megakaryocyte [MK] progenitor cells can shorten this period. In this study, we evaluated the cytokine conditions for maximum expansion and MK differentiation of CD133[+] HSCs. In this experimental study, The CD133[+] cells were separated from three cord blood samples by magnetic activated cell sorting [MACS] method, expanded in different cytokine combinations for a week and differentiated in thrombopoietin [TPO] for the second week. Differentiation was followed by the flow cytometry detection of CD41 and CD61 surface markers. Colony forming unit [CFU] assay and DNA analysis were done for colonogenic capacity and ploidy assay. CD133[+] cells showed maximum expansion in the stem span medium with stem cell factor [SCF] + FMS-like tyrosine kinase 3-ligand [Flt3-L] + TPO but the maximum differentiation was seen when CD133[+] cells were expanded in stem span medium with SCF + Interleukin 3 [IL-3] + TPO for the first and in TPO for the second week. Colony Forming Unit-MK [CFU-MK] was formed in three sizes of colonies in the mega-cult medium. In the DNA analysis; 25.2 +/- 6.7% of the cells had more than 2n DNA mass. Distinct differences in the MK progenitor cell count were observed when the cells were cultured in stem span medium with TPO, SCF, IL-3 and then the TPO in the second week. Such strategy could be applied for optimization of CD133[+] cells expansion followed by MK differentiation

[ role of myeloma cells in the in-vitro diffrentiation of cord blood hematopoietic stem cell to osteoclast]

Average Age of population in the industrial countries has increased. Because of aging the percent of the diseases related to the oldness such as multiple myeloma have also increased. It has both common and unique symptoms and effects. The unique effects include wide bone reabsorption. It seems necessary to understand the structure of Bone Marrow Niche and the effects of Myeloma cells on adjacent hematopoietic Stem cells with a new approach. We have studied the differentiating effect induced by the Myeloma cells through co-culturing the Myeloma cells and hematopoietic stem cells, extracted out of cord blood. In this investigation we also cocultured myeloma cells with the monoblastic cell line [U937] in order to evaluate the effect of myeloma cells on monoblastic cells differentiation. Our findings show that increased expression of myeloid and monocytoid markers in coculturing of myeloma cells and HSCs. Moreover following monoblastic and myeloid cells coculturing, we observed probably TRAP positive osteoclastic like cells. Our findings show that presence of myeloma cells in Bone Marrow play essential role in HSCs differentiation to monocytoid [osteoclastic] lineage

[Evaluation of Morpholino antisense oligos' role on BCR-ABL gene silencing in the K562 cell line]

Chronic myeloid leukemia [CML] develops when a hematopoietic stem cell acquires the BCR/ABL fusion gene. This causes these transformed hematopoietic cells to have a greater than normal proliferation rate. Scientists attempt to improve the CML treatment process by silencing the BCR/ABL oncogene. In this work, we used morpholino antisense oligos to silence the BCR/ABL oncogene. In this study, the K562 was used as a BCR/ABL fusion-gene positive cell line and the Jurkat cell line as a control. We explored the inhibiting capacity of morpholino antisense oligos in the the expression of the BCR/ABL oncogene and studied their p210 BCR/ABL suppression, inhibition of cell proliferation and stimulation of apoptosis in the K562 cells after 24 and 48 hours. Endo-Porter was used for delivery of morpholino antisense oligos into cell cytosols. Meanwhile, flow cytometric analysis was performed in order to determine the appropriate concentration of morpholino antisense oligos. Prolonged exposure of the K562 cell line to the morpholino antisense oligos targeted against the BCR-ABL gene showed proliferation inhibition as its main feature. After western blotting, we found that complete silencing of BCR/ABL was achieved, but flow cytometric analysis showed no broad apoptosis. The results indicate that the Morpholino antisense oligo is able to inhibit p210 BCR/ABL; however, it cannot induce broad apoptosis due to co-silencing of BCR

Capillary network formation by endothelial cells differentiated from human bone marrow mesenchymal stem cells

Human bone marrow derived mesenchymal stem cells [HBMSCs] have the potential to differentiate into cells such as adipocyte, osteocyte, hepatocyte and endothelial cells. In this study, the differentiation of hBMSCs into endothelial like-cells was induced in presence of vascular endothelial growth factor [VEGF] and insulin-like growth factor [IGF-1]. The differentiated endothelial cells were examined for their ability to express VEGF receptor-2 [VEGFR2] and von willebrand factor [vWF]. Then the cells were adopted to grow and develop capillary network in a semisolid gel matrix in vitro. The capillary network formation in a well of 24-well plate was found to be 85% in presence of VEGF [50ng/ml] and IGF-1 [20ng/ml] of the culture media. These data may suggest that the expression of endothelial markers in endothelial like-cells derived from hBMSCs is associated with their ability to form capillaries