Effects Of RNA Interference On Bcr/abl Fusion Gene Expression In K562 Cell Line

Chronic myeloid leukemia (CML) arises from the reciprocal translocation t(9;22) form the highly stable, constitutively active tyrosine kinase bcr/abl. This kinase activity is assumed to be sufficient and necessary to initiate CML. The fusion gene and its products served as a kind of useful molecular targets for CML therapy.Gene suppressiong by double-stranded RNAs called RNA interference (RNAi) is one of the most exciting advances in molecular genetics of recent years. Elbashir found that small interfering RNAs (siRNAs) of 21-23 nucleotedes can affect expression of homologous genes in a sequence specific manner. And it was also shown to exist in mammalian cells, including human cells, and paves the way for the development of novel RNA therapeutics. In this study, two siRNA sequences were designed to target the bcr/abl oncogene in Chronic Myeloid Leukemia cell line K562. The siRNA ihibitory effect on bcr/abl mRNA expression and growth of k562 cells were detected. Also, the changes of some relative signal transduction factors were studied. The study includes four parts as follow:Part I Application of Real-time RT-PCR to detect bcr/abl fusion gene in Chronic Myeloid LeukemiaObjective: To set up a real-time RT-PCR approach using TaqMan technology for detection and quantification of bcr/abl transcripts in CML patients and to explore the relationship between the expression level of the bcr/abl fusion transcript and the clinical status and efficiency of the therapy in CML. Method: Real-time PCR was carried out allowing the detection of both b3a2 and b2a2 transcripts. The mRNA encoding for glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as an endogenous reference. Twenty-two samples of bone marrow and peripheral blood from 14 CML patients were analyzed, and the bcr/abl mRNA levels of 2 HSCT patients after relapse were observed over time. Result: Both of the correlations of bcr/abl and GAPDH standard curves were 0.999; the sensitivity was 10^-6. The amounts of bcr/abl mRNA from 14 patients at diagnosis ranged from 2.81 to 145. The expression level of bcr/abl mRNA changed with the clinical outcome. The bcr/abl expressiong level of bone marrow and peripheral blood were almost the same.Conclusion: This real-time RT-PCR procedure is a reliable, sensitive and reproducible method of monitoring CML patients after therapy. It is useful in reflecting leukemic burden, assessing response to treatment and predicating the prognosis of the disease. Part II bcr/abl suppression by siRNAObjective: To investigate the inhibition of bcr/abl oncogene expression in K562cells using RNA interference (RNAi). Method: siRNA-S targeting bcr/abl oncogene was chemically synthesized in vitro. K562 cells stably expressing bcr/abl oncogene were transfected with the siRNAs; non-transfected cells and mismatch control siRNAs with three-point mutations (siRNAmut) transfected cells were taken as controls to investigate the speciality of RNAi. The feasibility and transfection efficiency of electroportion and liposome-based transfection were carried out. Inhibitory effect of siRNAs was demonstrated by real-time quantitative RT-PCR and Western blots. Result: The transfection efficiency of electroporation was about 70%, higher than liposome-based transfection. The siRNA-S targeting bcr/abl displayed a strong growth inhibitory effect on K562 cells, with the optimal concentration of 200 nM and the optimal time of 48 hours. The siRNA-S reduced the bcr/abl mRNA level 67% (24hours )and 72% (48hours) without exerting a significant effect on abl mRNA. The K562 cells electroporated with siRNA-S contained less bcr/abl protein than cells electroporated with siRNAmut or without any siRNA, whereas the wild-type ABL protein was not influenced by the siRNA-S, which was consistent with the results published by Scherr et al. and Wilda et al.. RNAi in mammalian cells is transient and non-heritable, and the half-life of bcr/abl protein is long. So the protein didn't decrease as significantly as the mRNA. And the siRNAmut had no effect on the mRNA and protein expression of bcr/abl or abl. Conclusion: Inhibition of CML bcr/abl oncogene expression by chemically synthesized siRNA provides the new method for the study of bcr/abl fusion gene function and downstream signal transduction pathway. It is suggested that RNAi could be used as a promising tool to purge or eliminate leukemic cells in vitro before autologous hematopoiesis stem cell transplantation.Part III Biological effects of RNAi on K562 cellsObjective: To study the biological effects of siRNA-S on K562 cells afterdown-regulating the bcr/abl fusion gene expression. Method: Cells viability and proliferation were measured by means of trypan blue exclusion and MTT assay. Apoptosis was determined by Annexin V-FITC assay and DNA Ladder. Result: SiRNA-S inhibited K562 cell proliferation with an inhibitory rate of 47% and 56%, 24 and 48 hours after transfection, respectively. The tipical DNA ladder was found.At the same time, we found that anti-bcr/abl siRNAs treatment led to an increase in apoptosis (seen in FCM) compared to controls. Wohlbold et al. found that the rate of apoptosis in K562 cells was higher than that in the controls 48 hours after transfection with dsRNA. However, in this study we showed that the percentages of dying and apoptotic cells induced by anti-bcr/abl siRNAs after 24 and 48 hours were 42.10% and 53.33%, among which 15.05% and 19.47% of the apoptotic cells were measured only, respectively. The number of dying cells (27.05% and 33.86%, 24 and 48 hours after anti-bcr/abl siRNAs treatment, respectively) rose significantly above those in the controls (0.63% and 4.55%, control and mismatch control, respectively) as the apoptotic cells increased. Therefore, siRNAs could induce cell dying as well as apoptosis. Conclusion: These results showed that siRNA could induce cell dying as well as apoptosis of CML cells and inhibited the tumor cells growth.Part IV Effect of RNAi on downstream signal pathway of bcr/ablObjective: To detect the expression levels of JAK2 and STAT5 in K562 cells after RNAi. Method: The protein levels of JAK2 and STAT5 were measured by Westorn blots with respective antibodies in the K562 cells with different siRNA. Result: The expressiong level of JAK2 and STAT5 in K562 cells tranfected with siRNA-S decreased compared with controls. But siRNAmut had no effect of the expression level of JAK2 and STAT5. Conclusion: In K562 cell line, the JAK/STAT signal pathway may be an important downstream pathway for bcr/abl fusion protein and may be a new therapeutic target for CML.Taken together, siRNA-S specifically suppressed the expression of bcr/abl oncogene, and inhibited K562 cell proliferation and resulted in the increase of cell apoptosis and dying. The results suggested that RNAi may offer a suitable techniqueto study the function of bcr/abl and may be used to develop RNAi-based targeted gene therapeutic approache against Chronic myeloid leukemia. It is also suggested that RNAi could be used as a promising tool to purge or eliminate leukemic cells in vitro before autologous hematopoiesis stem cell transplantation.