| Nestin is a type VI intermediate filament protein that is known as a neural stem/progenitor cell marker.The expression of nestin has also been shown in the developing and adult organism including embryonic pancreatic cell,hepatic cells,vascular endothelial cells,skeletal muscle cells,basal cell epithelium,astrocytes after CNS damage,etc.And nestin expression was observed in cancer cells from various types of human solid tumors as well as heamtologic malignancies,such as breast cancer,lung cancer,pancreatic cancer,glioma,melanoma and leukemia etc.But the effect of nestin expression on leukemic cells biological behaviors is still unclear.Leukemia is a group of hematopoietic malignancies arising from the abnormalities of proliferation,differentiation or survival of progenitors.The treatment consisted of combination chemotherapy,radiotherapy,targeted therapy as well as hematopoietic stem cell(HSC)transplantation resulted in higher response rates of patients,but survival probability 5 years was about 20%,and did not get further improvement.Thus,it is a difficult clinical question.The aim of the project was to detect the nestin expression in patients with leukemia and test the proliferation,apoptosis and invasion in K562 cells regulated by siRNA-nestin,and evaluated its mechanism of invasion as well as migration.It is provided information to study the function of nestin expression on cell biological characteristic and its mechanism,which is benefited to elucidate the characteristics of the leukemia cell biology in order to efficiently clarify the etiology and pathogenesis of leukemia,and explore the appropriate therapy target.The blood samples were collected including from 44 patients with myeloid leukemia and 16 healthy controls,and the expression of nestin mRN A was detected by RT-PCR.The bone marrow samples were consisted of 125 acute myeloid leukemia(AML)patients and 14 iron deficiency anemia patients,and the expression of nestin protein was tested by western blot.Immunohistochemical method was used to examine the nestin expression in some samples(not all)of bone marrow smear and biopsy.Its clinical significances were discussed.Silencing in K562 cells was achieved by transient transfection with siRNA targeting nestin gene,and the mRNA and protein expression of nestin in K562 cells tranfected for 36,48 and 72 hours was determined using RT-PCR and western blot assays.The effect of nestin interference on cell proliferation was analysed by MTS.A small interference RNA targeting nestin gene was transfected into K562 cells,and the effect of nestin interference on the cycle was analyzed by flow cytometry,and apoptosis staining with Annexin V FITC/PI.Transwell cell invasion assay was used to investigate invasion abilities of the K562 cells after trasnsfection.The molecular markers,MMP2.MMP9.VEGF.HSP70,related with invasion and metastases were evaluated by western blot.Finally,the effect of scutellaria baicalensis on proliferation of KS62 cells,and the combined effects of Scutellaria baicalensis and siRNA-nestin were analysed by MTS.Nestin mRNA was expressed in most patients(36/44),but 63%(1/16)in healthy controls,and its strongly expression associated with high white blood cells(WBC)(P=0.011).However,nestin expression was not related to the level of hemoglobin,platelets,Thl/Th2 type cytokines as well as clinical complete remission(CR)(P>0.05).The positive expression of nestin protein was 12.8%(16/125)in myloid leukemia patients,as its did not express in patients with iron deficiency anemia.The overexpression of nestin protein was strongly correlated with the hyperleukocytosis myeloid leukemia(P=0.015),whereas no relationship was found with hemoglobin,platelets and complete remission(CR)of patients(P>0.05).The scatted nestin positive cells were observed by immunohistochemical method in samples of bone marrow smear and biopsy.K562 cells had the highest level of nestin expression.After transient transfection of nestin in K562 cells at 48h,there was no obviously morphological change of K562 cells under inverted microscope,but were significantly lower than in blank group and in negative control(NC)group(P<0.05).the expression levels of nestin mRNA and protein in the K562 cells transfected with siRNA were significantly lower than in the control group using RT-PCR and western blot.siRNA targeted against nestin,the proliferation of K562 cells was significantly inhibited,and the proliferation ability was decreased by(87.7±4.9)%.Comparing with control group there is a significant difference(P=0.012).The percentage of K562 cells with siRNA in G2 phase(24.12±3.81)%was higher than in the control group(4.09+2.58)%.Cell cycles were arrested at G2 phase.The apoptotic rate significantly increased to(16.83+5.99)%with respect to control cells(5.04+0.31)%(P=0.027).After silencing nestin in K562 cells,the number of cells migrating and invading across the transwell charmber was significantly lower in the nestin siRNA interference group than those in the control group(P=0.011).Compared to control group,western blot results showed that the expression of HSP-70,pro-MMP2,MMP2 and pro-MMP9 was down regulation in the siRNA-nestin group(p<0.05).But the expression of MMP9 and VEGF at protein level has no significant change(p>0.05).MTS results showed that K562 cells in the siRNA interference combined with Scutellaria baicalensis group have grown slower,and the inhibition rate significantly increased.But there is no significance difference between the combined group and control group Scutellaria baicalensis alone(p>0.05).Conclusion The expression of nestin in myeloid leukemia is considered to relateto raise high white blood cells.Transient transfection of nestin siRNA not only significantly suppressed nestin expression in K562 cells,but also inhibited the proliferation and the invasion as well as induced-apoptosis of K562 cells.Nestin may be correlated with the down-regulation of HSP70,pro-MMP2,MMP2 and pro-MMP9 protein levels.These data together provide insights into the roles of nestin during myeloid leukemia development and suggest that nestin might represent a new potential target for gene therapy. |
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