The Effects Of Lentivirus-Mediated Stable RNAi Targeting Bcr/abl Gene On Biological Characters And Curcumin Antitumor Action In K562 Cell Line

Chronic Myelogenous Leukemia(CML) originates in a pluripotent hematopoetic stem cell of the bone marrow, which is characterized by the Philadelphia(Ph) chromosome and bcr/abl oncogene. The bcr/abl fusion gene encoded Bcr/Abl protein, which possesses high activity of tyrosine kinase was known to be a resistant factor for apoptosis, cell differentiation and chemotherapy drugs resistance in chronic myelogenous leukemia (CML).RNA interference is a kind of gene-blocking technology about post-transcriptional gene silence (PTGS), which has been extensively applied to the inhibition of gene expression in the mammal cells in recent years. Compt short interference RNA (21-23nt) is obtained by the incision enzyme RNaseⅢ(Dicer), which mediate specific degradation of its homologous and complimentary mRNA, to silence the expression of target genes.In this study, K562 cells were infected by lentivirus carrying bcr/abl RNAi fragment, and further bcr/abl stable-silencing CML cellular model was constructed. As a result of long-term absence of bcr/abl, changes of biological characters, correlated signaling molecules and anti-tumor effects of curcumine were detected.Construction of lentivirus-mediated bcr/abl stable-silencing cellular model on K562 cellsIn this part, we constructed bcr/abl RNAi stable-transfecting cellular model B/A-K562, and detected the efficiency and stability of lentivirus-mediated RNA interference. Methods: (1) Plasmids pNL-EGFP-U6-brcr/abl-I was constructed, which was combined with pVSVG and pHelper to constitute vector system of three plasmids. The lentiviral vector system was transfected into 293T cell to produce EGFP- B/A-I lentivirus. (2) K562 cells were infected with both EGFP and EGFP- B/A-I lentivirus. (3) Ultimate dilution and colony forming methods were used to pick clones stably expressing EGFP and EGFP-B/A-I fragment. (4) bcr/abl mRNA level was detected by Realtime RT-PCR; (5) Cello-immunofluorescence, western blotting were used to measure the contents of P210bcr/abl.The results showed: (1) EGFP and EGFP- B/A-I lentivirus with processing infection ability were successfully produced; (2) EGFP-K562, B/A-K562 cells stably expressing EGFP and EGFP-B/A-I fragment were successfully picked; (3) After cultured in vitro for six months, bcr/abl mRNA lever was down regulated by lentivirus-mediated RNAi for 63.5 %; (4) Compared with K562 cells, P210bcr/abl content was obviously decreased by 74.5 % in B/A-K562 cells.The effect of bcr/abl gene stable-silencing on the biological characters and relevant signaling molecules in K562 cell lineSince bcr/abl plays an important role in the generation, development and turnover of human chronic myelogeous leukemia, many changes in biological characters and cell signaling molecules may be detected in human CML cell line K562 as a result of long-term absence of bcr/abl gene. This study focused on the variations of biological characters and cellular signals in bcr/abl stable-silencing cell model B/A-K562 compared with K562 and EGFP-K562 cells as normal control and vector control respectively.Methods: (1) Trypan blue exclusive staining was used to access the cell growth arrest; (2) Colony assay was used to observe the CFUs; (3) Benzidine staining was used to detect cell differentiation; (4) FCM was used to analyze cell cycle; (5) ELISA method was utilized to detect the activity of PTK; (6) AO-EB and Heochst 33342 fluorescent staining was used to observe apoptosis; (7) Chromometry was to used measure the activation of Caspase-3 and Caspase-9; (8) Western blot was used to observe the effects of RNAi on the signaling molecules which are relevant with cell growth and apoptosis.The results showed: (1) bcr/abl RNAi significantly inhibited K562 cell proliferation, doubling generation time was obviously prolonged; (2) Colony forming was arrested by RNAi; (3) Positive rate of benzdine staining was 24.83 %, low level of bcr/abl message leads to enhanced erythroid differentiation; (4) RNAi arrested cell cycle in S phase and induced apoptosis in hypodiploid peak; (5) PTK activity was descended by 41.88 % ; (6) Apoptosis was observed with 41.88 % of apoptosis rate; (7) bcr/abl RNAi triggered the release of mitochondrial Cyt-C, increases Caspase-3 and Caspase-9 activities, induces apoptosis in mitochondrial-depended pathway; (8) bcr/abl RNAi down-regulated Src, P-Src, Raf-1, P-Erk1/2, P-Stat 1, P-Stat 5, P-P38, C-myc, Bcl-2 and Hsp 90 level, and up-regulated P53, PKC and Bax level, but had no effect on the concentration of Erk1/2, P-Akt, NF-КB, Hsp 70 in K562 cells.The effect of lentivirus-mediated bcr/abl stable-silencing on the tumor formation on K562 cell xenograft in athymic mouseLentivirus-mediated RNA interference could reduce the expression of bcr/abl gene in vitro, inhibit cell growth, induce differentiation and hasten apoptosis. In this part, we mainly studied RNAi efficiency in B/A-K562 cells athymic mouse transplantation tumor, tumor formation ability and relevant signaling molecules.Methods: (1) K562, EGFP-K562, B/A-K562 cells was injected subcutaneously in order to establish athymic mouse heterogeneity transplantation tumor models; (2) Volume of the tumors were measured per 5 days and the tumor formation rates were calculated; (3) At the end of time points, the mice were killed, the tumor xenografts were removed and measured; (4) Activation of Capspase 3, Caspase-9 were detected; (5) Green fluorescence was observed in frozen section of tumor tissue by fluorescence microscope; (6) H&E staining was used to study the pathological change of tumor tissue; (7) Immunohistochemistry was used to investigate the P210bcr/abl, Bcl-2, Bax level in tumor tissue; (8) The protein was extracted from the tumor tissues, western blotting was used to analyze Src, Erk1/2, P-Stat1 and Hsp 90 protein level.The results showed: (1) Growth velocity of B/A-K562 cell xenograft was obviously lower than that of K562 group and EGFP-K562 group; (2) Tumor formation rate of B/A-K562 group was 80 % lower than that of K562 and EGFP-K562 group, which was 100%; (3) The tumor inhibition rate in B/A-K562 group was 78.4 % (compared with K562 group, P<0.01); (4) The activation of Caspase-3, Caspase-9 was elevated in B/A-K562 cell xenograft; (5) Green fluorescence was confirmed in transplantation tumor of lentivirus infected EGFP-K562 and B/A-K562 cells; (6) The results of immunohistochemistry showed decreased P210bcr/abl, Bcl-2 expression and increased Bax expression in B/A-K562 group, compared with K562 and EGFP-K562 group; (7) The results of Western blotting showed Erk1/2,Src,P-Stat 1,Hsp 90 protein level of B/A-K562 group were lower than that of K562 and EGFP-K562 group.The effect of lentivirus-mediated bcr/abl stable-silencing on anti-tumor action of curcumin and relevat mechanisms in K562 cellsbcr/abl gene and P210bcr/abl protein were important targets of Curcumin. In this part, K562, EGFP-K562 and B/A-K562 cells were treated with Curcumin in different concentration. We investigated anti-tumoral action and relevat mechanisms of curcumine when bcr/abl gene expression was blocked by lentivirus-mediated RNA interference.Methods: (1) MTT method and trypan blue exclusive staining were used to observe the cell growth arrest; (2) Colony assay was used to the cell colony forming units(CFUs); (3) Benzdine staining was utilized to investigate cell differentiation; (4) ELISA was used to measure the activity of PTK; (5) Flow cytometry (FCM) was used to analyze cell cycle; (6) AO-EB staining and DNA agarose gel electrophoresis were used to detect cell apoptosis; (7) Absorption spectrometry was used to detect activation of Caspase-3 and Caspase-9; (8) Western blotting was used to analyze the impact of Curcumine on Cell proliferation apoptosis-related signaling molecules and partner protein.The results showed: (1) Curcumin inhibited K562, EGFP-K562 and B/A-K562 cells proliferation in a time-and-dose-depended manner. IC50 values of K562, EGFP-K562 and B/A-K562 cells were 6.94, 4.93, 12.67μg/ml after 48 h of Cur exposure; (2) Cur inhibited B/A-K562 CFUs in an dose-depended manner; (3) Cur induced differentiation towards mature erythroid cells and its role in the promotion of differentiation could be weakened by bcr/abl RNAi; (4) Cur reduced PTK activity in B/A-K562 cells and bcr / abl RNAi may enhance such effect; (5) Cur induced apoptosis in hypodiploid peak in a time-and-dose-depended manner; (6) Cur effectively induced B/A-K562 cells apoptosis in a dose-depended manner; (7) Cur triggered the release of mitochondrial Cyt-C, increased Caspase-3 and Caspase-9 activities, inducesd apoptosis in mitochondrial-depended pathway in B/A-K562 cells; (8) Cur up-regulated C-fas, P53, P-P38 andBax level, and down-regulated P-Src, Raf-1, NF-КB, C-myc, PKC, Bcl-2, Hsp 70, and Hsp 90 level in B/A-K562 cells.CONCLUSIONS:1. Lentivirus-mediated RNA interference may stably silence bcr/abl gene, which brings down bcr/abl mRNA and P210bcr/abl by respectively 63.5 % and 74.5 %.2. Lentivirus-mediated stable-silencing of bcr/abl inhibits proliferation, induces differentiation towards mature erythroid cells, significantly decreases activity of PTK and hastens apoptosis in mitochondrial-depended pathway in K562 cells.3. Lentivirus-mediated stable-silencing of bcr/abl inhibits K562 cells growth and induces cell apoptosis which may be correlated with the down-regulation of P210bcr/abl, Src, P-Src, Raf-1, P-Erk1/2, P-Stat 1, P-Stat 5, P-P38, C-myc, Bcl-2, Hsp 90 and up-regulation of P 53 and Bax.4. Effect of bcr/abl RNAi retains in athymic mouse transplantation tumor,which significantly inhibits tumorous growth, induces apoptosis and necrosis in tumor tissue. Its anti-tumor effect may correlated with down-regulation of signaling molecules related with cell growth and apoptosis such as Erk1/2, Src, P-Stat 1, Hsp 90, Bcl-2 and up-regulation of Bax.5. Cur inhibits cell growth, induces differentiation, reduces PTK activity, and hastens apoptosis in mitochondrial-depended pathway in B/A-K562 cells.6. Cur decreases concentration of P210bcra/bl in B/A-K562 cells, up-regulates C-fas, P53, P-P38 and Bax, down-regulates P-Src, Raf-1, NF-КB, C-myc, PKC, Bcl-2, Hsp 70 and Hsp 90, which may contribute to growth inhibition and apoptosis in B/A-K562 cells in the background of stably bcr/abl RNAi mediated by letivirus.In conclusion, lentivirus-mediated RNAi may lead to long-term silencing of bcr/abl, inhibits cell growth, and induces apoptosis in CML cell line, so it might prove to be a promising alternative approach in CML therapy targeting bcr/abl. Curcumin synergized with lentivirus-mediated bcr/abl RNAi may further inhibits cell growth amd hastens apoptosis in K562 cells. Therefore, Curcumin may be an ideal choice to synergize with bcr/abl targeting therapy...