Preliminary Study on the Effect of Silencing Nucleostemin Com- bined with Rapamycin on Autophagy and Apoptosis of HL-60 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of knocking down nucleostemin ( NS) combined with rapamycin (RAPA) on autophagy and apoptosis in HL-60 cells , and to explore its role in HL-60 cells .@*METHODS@#The expression of NS protein was detected using Western blot , after transfection of HL-60 cells was achieved by the recombinant lentviral vector NS -RNAi-GV248 . Flow cytometry was used to detect changes in cells apoptosis after NS silencing/ rapamycin for 24 , 48 hours , and the expressions of NS , LC3 , p62 , BCL-2 and Bax proteins in cells were detected by Western blot.@*RESULTS@#The expression of NS in HL-60 cells was successfully down-regulated by recombinant lentiviral vector. After treatment with rapamycin for 24 and 48 h , the apoptosis rate of cells in each group increased (P < 0.05) , and the apoptosis was more obvious at 48 hours . Compared with the NS silencing group or rapamycin group , after treated with NS down-regulation combined with rapamycin for 48 hours , the apoptosis of HL-60 cells was significantly increased ( P < 0.05 ) , LC3 -II/LC3 -I ratio was significantly increased ( P < 0.05 ) , p62 protein expression was significantly decreased (P < 0.05) , and BCL-2/Bax ratio was significantly decreased ( P < 0.05) .@*CONCLUSION@#NS down-regulation combined with rapamycin can enhance the apoptosis and autophagy of HL-60 cells , and the induction of apoptosis of HL-60 cells may be related to the expression of BCL-2 and Bax proteins .

Millimeter-wave Exposure Promotes the Differentiation of Bone Marrow Stromal Cells into Cells with a Neural Phenotype / 华中科技大学学报（医学）（英德文版）

t of the cells treated with BME alone (P<0.05). It was concluded that MMW exposure enhanced the induc-ing effect of BME on the differentiation of BMSCs into cells with a neural phenotype.

Expression of Nucleostemin gene in tumor cells derived from human embryomic bone marrow mesenchymal stem cells / 肿瘤研究与临床

Objective To study the tumorigenesis mechanism in bone marrow mesenchyme stem cells (MSC). Methods The bone marrow MSC could be induced into turnout (F6 cells) in vitro. The difference between gene expression of F6 cells and MSC was distinguished by fluorescent differential display (FDD). Verification of the result was detected by Real time RT-PCR and Western blotting, and immunocytochemistry. Results FDD analysis confirmed that Nucleostemin (NS) was positively up-regulated in F6 cells compared with MSC. Similar results were obtained by PCR and Western blotting. The NS gene expression levels in MSC, F6, 176-4, F6-6 and F6-7 were significantly different(F =160, P <0.05). The NS gene expression level in F6 (0.0372±0.0019) was 18 folds higher than those of MSC(0.0021±0.0002,P <0.05). Expression levels in F6-4, F6-6 and F6-7 tissue were 0.0504±0.0083, 0.0995±0.0026 and 0.0614±0.0036, and were significantly higher than that in MSC(P <0.05). The expression of NS increased significantly with the accreting volume of turnour, and high-level protein expression of NS was confirmed by Western blotting and immunocytochemistry. Conclusion The expression level of NS might be one of the factors playing important roles during turnour genesis, especially in MSC mutation.

Effect of specific short hairpin RNA of nucleostemin on differentiation antigens and biological properties of HL-60 cells / 第三军医大学学报

Objective To study the role of the specific short hairpin RNA(NS-shRNA) of nucleostemin in inhibiting the expression of nucleostemin(NS) gene and evaluate the effect of NS-shRNA on differentiation antigen and biological properties of HL-60 cells,so as to explore the relationship between the biological functions of NS and acute leukemia,the potential perspective of NS gene therapy with RNA interference(RNAi).Methods HL60 cells were directly transfected NS-shRNA by using special transfection reagent.After 48 h,the inhibition rate of NS-shRNA to NS gene was detected by RTPCR,the proliferation ability of HL-60 cells was detected by MTT,the differentiation antigens were assayed by flow cytometry(FCM),the morphologic peculiarities such as cell shape,growth condition were observed,and the volume and granularity of HL-60 cells were analyzed by blood cell analyzer.Results The expression of NS gene were significantly inhibited by both two NS-shRNA,the inhibition rate were 37.82% and 71.88%,respectively.The significant inhibition effect of NS-shRNA to the proliferation of HL-60 cells existed in a time-dependent and concentration-dependent manner,and the best time was 48 h,the best concentration was 10 nmol/L.The change of differentiation antigen included increase of CD11b,CD33,CD14,CD64,HLA-DR and decrease of CD38,indicating continuous maturation of HL-60 cells to granulocytes and redifferentiation trend to monocytes.The aggregation of cells declined and the cell fragments increased.Myeloperoxidase(MPO) and ?-naphthol acetate esterase(?-NAE) activity increased after NS-shRNA-2 transfection.Furthermore,some HL-60 cells changed from round to fusiform shape even to pseudopod.The cells of small volume and karyorrhexis increased. Conclusion Through blocking the expression of NS gene,NS-shRNA can inhibit the cell proliferation and induce the differentiation of HL-60 cells,weaken malignant degree of HL-60 cells and trigger cell apoptosis.NS-shRNA is of clinical potential in gene therapy for acute leukemia.

Nucleostemin specific RNAi influences cell proliferation in HeLa cells in vitro and in vivo / 中国病理生理杂志

AIM: To examine Nucleostemin (NS) expression in tumor cells, and observe the effect of NS specific RNA interference on the cell proliferation in Hela cells. METHODS: Total RNA was extracted from 6 kinds of cultured tumor lines, the NS expression level was measured by RT-PCR and Northern blot. An NS-specific siRNA expression vector was constructed to transfect HeLa cell (NS-siRNA-HeLa), and the proliferation of the cell was observed. RESULTS: NS was highly expressed in 6 kinds of tumor cells. NS expression level in the NS-siRNA-HeLa cells was remarkably reduced, and the percentage of G_0/G_1 cells increased. The neoplasm forming ability in nude mice by the NS-siRNA-HeLa cells was decreased. CONCLUSION: NS is highly expressed among tumor cells. NS-specific siRNA inhibits the entry of the cell cycle into the S phase, and remarkably reduces the proliferation ability of HeLa cells in vitro and in vivo.

Nucleostemin gene expression in human breast tumor tissues and its relation with clinical pathology / 中国病理生理杂志

AIM: To study the expression of nucleostemin (NS) gene in human breast tumor tissues and the relations of NS gene expression level with histological grades, histological types and TNM stages of the tumor.METHODS: Total RNA was isolated from human breast tumor tissue. The methods of electrophoresis and RT-PCR were used in measuring NS gene expression level, and the relations of NS gene expression level with histological grades, histological types and TNM stages of the tumor were analyzed. RESULTS: The results indicated that there was no NS gene expression detected in normal breast tissues, and NS gene expression in malignant breast tumor tissues (P0.05). CONCLUSION: It is suggested that there is no relation of NS gene expression level with histological types of the breast cancer, but there is a marked correlation of NS gene expression level with the histological grades and TNM stages.

Construction of specific short hairpin RNA (shRNA) for Nucleostemin by T7 RNA polymerase promoter in vitro / 中国药理学通报

Aim In order to further explore the feasibility of gene therapy with RNA interference(RNAi) for acute leukemia,we synthesized short hairpin RNA(shRNA)for nucleostemin(NS)in vitro.Methods The designed DNA template consists of the sequence complementary to target mRNA,which was screened out consensus motif of three variants of NS gene,using 5′-UCUCUUGAA-3′ as loop sequence,The sequence of 5′-GGATCCTAATACGACTCACTATA-3′ acts as T7 promoter.Two shRNA were produced by T7 RNA polymerase and named as shRNA-NS-1 and shRNA-NS-2,respectively.The purified shRNA was quantified by gel electrophoresis.The interference effect of shRNA-NS transfected into HL-60 cell was examined by resultant cell morphology and inhibition rate of NS-mRNA expression.Results The concentrations of two shRNA-NS without degradation and diffusion were 5.24 ?mol?L~(-1)and 3.35 ?mol?L~(-1),respectively.There were significant decline in density and aggregation of cells and significant differences in size between cells after interfering HL-60 cell for 48 h.Furthermore,some of HL-60 cells treated by shRNA-NS-2 were changed from round to fusiform shape even with pseudopod.Compared with control group,the inhibition rates of shRNA-NS-1 and shRNA-NS-2 to NS-mRNA expression were 37.82% and 71.88%,respectively.Conclusion The two shRNA for NS gene were successfully constructed,which suppress NS gene expression significantly.shRNA-NS-2 also can chang HL-60 cell′s morphology and might be a useful tool to explore NS gene function and possible therapy for acute leukemia.

The effect on proliferation and apoptosis by Nucleostemin gene specific RNA interference in gastric-carcinoma cells / 西安交通大学学报(医学版)

Objective To investigate the effect on proliferation and apoptosis by RNA interference to inhibit Nucleostemin(NS) gene expression in gastric carcinoma SGC-7901 cells.Methods The NS-siRNA expression vector was transfected into gastric-carcinoma cells with LipofectamineTM2000 reagent.Then we detected the cell proliferation inhibition ratio by MTT assay,the levels of NS gene expression in all groups by RT-PCR,cell cycle by flow cytometry,cell apoptosis ratio by Annexin-V-FITC/PI kit.Results Compared with that in the control group,cell proliferation in S group decreased;at 24,48 and 72 h the cell proliferation inhibition ratio was 53.21%,71.54% and 87.47%,respectively,the level of NS gene expression reduced in S group.G0/G1 phase cell was 58.34%,S phase cell was 20.68%,and the cell apoptosis was 26.85%.Conclusion RNA interference could substantially inhibit NS gene expression in gastric carcinoma SGC-7901 cells,decrease cell proliferation,arrest cell cycle and increase apoptosis ratio.