[Show abstract][Hide abstract] ABSTRACT: Secreted protein acidic and rich in cysteine (SPARC) plays key roles in erythropoiesis; haploinsufficiency of SPARC is implicated in the progression of the 5q- syndrome. However, the role of SPARC in other subtypes of myelodysplastic syndrome (MDS) is not fully understood, particularly in the del(5q) type with a complex karyotype, which has a high risk to transform into acute myeloid leukemia (AML). In the present study, we investigated the role of SPARC in the proliferation and apoptosis of SKM-1 cells, an acute myeloid leukemia cell line transformed from an MDS cell line. SKM-1 cells were infected with SPARC-RNAi-LV or NC-GFP-LV lentivirus. Apoptosis and cell cycle profiling were assessed by flow cytometry, and cell proliferation was evaluated by MTS assay. The mRNA and protein expression levels of SPARC, p53, caspase-3, caspase-9 and Fas were detected by RT-PCR, real-time PCR and western blot assay. The SPARC shRNA constructed by us led to a significant reduction in SPARC expression in SKM-1 cells. SPARC knockdown inhibited the proliferation of SKM-1 cells by inducing cell cycle arrest at the G1/G0 phase and apoptosis. SPARC knockdown elevated the expression of p53, caspase-9, caspase-3 and Fas at both the mRNA and protein levels. SPARC silencing inhibited the growth of AML transformed from MDS by activating p53-induced apoptosis and cell cycle arrest. These data indicate that SPARC acts as an oncogene in transformed MDS/AML and is a potential therapeutic target in MDS/AML.
International Journal of Molecular Medicine 01/2014; 33(4). · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ribosomal protein S14 (RPS14) plays a key role in erythropoiesis and causes p53 activation in 5q- syndrome. However, the oncogenic potential of RPS14 is not understood in leukemia and high-risk myelodysplastic syndrome (MDS). Here, we investigated the changes of proliferation and apoptosis of SKM-1, an acute myeloid leukemia (MDS/AML) cell line transformed from MDS, and explored the role of RPS14 in them.
SKM-1 cells were transfected with recombined lentiviral vector shRPS14. Reverse-transcribed polymerase chain reaction and western blot assay were carried to detect the expression of RPS14 and p53. Cell proliferation was determined by MTT assay. Cell cycle and apoptosis were detected through flow cytometry.
When compared with negative control, the proliferation rate of SKM-1 cells transfected with RPS14 hairpin siRNA dropped by 30%. Transfected SKM-1 cells presented with activation of p53. Transfection also arrested cells in G0/G1 phase and induced apoptosis, indicating that RPS14 is involved in the pathophysiology of MDS/AML.
These findings indicate that partial silencing of RPS14 inhibits the proliferation of MDS/AML cells, and RPS14 may negatively regulate p53 activation in MDS/AML cells.
[Show abstract][Hide abstract] ABSTRACT: Minimal residual disease (MRD) occurrence with some chemotherapy drugs that promote tumor cell escape is also a key factor in blood malignancy relapse. We observed that cytarabine promotes multiple myeloma (MM) cell escape and that the number of cells in the lower chamber increased with increasing clinical disease stage in vitro model which was constructed by a Boyden chamber, matrigel glue and serum from MM patients in different disease stages. The mechanism of cytarabine promotes MM cell escape is closely associated with the up-regulation of CXCR4. SDF-1α can up-regulate the expression of MMP9 and RHoC proteins in MM cells with up-regulated CXCR4, and further promote the cell escape. Fucoidan, a sulfated polysaccharide in the cell wall matrix of brown algae, has attracted much attention for its multiple biological activities, and we further explored the effects and possible underlying mechanisms of fucoidan on MM cell escape from cytarabine cytotoxicity. The results show that fucoidan may decrease MM cell escape from cytarabine cytotoxicity, and that fucoidan can down-regulate CXCR4, MMP9 and RHoC expression. This research provides new direction for investigating MRD occurrence and prevention.
[Show abstract][Hide abstract] ABSTRACT: To construct a lentiviral vector expressing small-hairpin RNA(shRNA) targeting SPARC gene and investigate its silenced effect on SPARC in human myelodysplastic syndromes(MDS) cell line SKM-1.
The targeting sequence of SPARC gene which can be effectively silenced in RNA interference was confirmed in our previous study. The designed and synthesized single-stranded primers were annealed to double-stranded oligo sequences and subcloned into linear pGCSIL-GFP lentiviral plasmid digested by enzyme Age I and EcoR I to produce GC-shSPARC lentiviraL vector. After being identified by PCR and sequencing, plasmids GC-shSPARC with pHelper 1.0 and pHelper 2.0 were cotransfected into 293T cells to package lentiviral particles. The recombinant lentiviral vector was transfected into human SKM-1 cells, transfection efficiency was evaluated with expression of green fluorescent protein(GFP) determined by fluorescent microscope. Expression of SPARC in SKM-1 cells was detected using RT-PCR and Western blotting.
A recombinant lentiviral vector, GC-shSPARC, expressing shRNAs targeting SPARC gene was constructed and confirmed by DNA sequencing. The recombinant lentivirus was harvested from 293T cells with a viral titer of 1×10(9); TU/mL. GFP was observed in the 70% of SKM-1 cells after transfection. Expression of SPARC mRNA and protein was significantly reduced in the GC-shSPARC transfected group than that in the control group (P<0.05).
The lentivirus RNAi vector targeting SPARC has been successfully constructed, and can effectively inhibit the expression of SPARC in SKM-1 cell line, which shed light on the foundation for researching the inhibition of SPARC siRNA target against human MDS cells proliferation, induction apoptosis and gene therapy.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 05/2012; 28(5):466-9.