FESH And Microarray Study On Apoptosis Of K562 Cells Induced By BCR-ABL Gene Silence

Leukemia is a serious disease to human health. 20% of Leukemia is Chronic myeloid leukemia (CML). CML is a hematopoietic stem cell disorder in which 90% of patients display a reciprocal translocation involving chromosomes 9 and 22, resulting in what has been designated the Philadelphia (Ph) chromosome. The BCR-ABL fusion gene is the molecular manifestation of the Ph~1 chromosome, which arises from a head-to-tail fusion of the breakpoint cluster region (bcr) at band q11 of chromosome 22 with the proto-oncogene (c-ABL) at band q34 of chromosome 9.This leads in turn to the expression in virtually all Ph~1 CML patients of the chimeric fusion protein P210 BCR-ABL, a constitutively active tyrosine kinase. This kinase activity is assumed to be sufficient and necessary to initiate CML.P210~BCR-ABL is the characteristic protein of CML. It can stimulate diverse downstream signal transduction pathways and provide affected cells with anti-apoptotic and proliferative advantages. So BCR-ABL fusion gene and its mRNA and fusion protein are the ideal targets. The study of inhibition of BCR-ABL fusiongene at different levels now is attracting more and more attention. Finding effective mehods of inhibition of BCR-ABL fusion gene and P210 ~BCR-ABL kinase activity and signal transduction pathways is very important to the mechanism study and clinic therapy of CML.The identification and characterization of human genes differentially expressed in tumor cells will hopefully shed light on uncovering the mechanisms of tumor development and provide useful genetic markers for screening, diagnosis, prognosis, therapeutic monitoring and development of therapeutic vaccines. There are many techniques that aim at producing an inventory of differential transcripts between two populations of mRNAs. Subtractive hybridization technology is a common method to clone differentially expressed genes and construct subtraction library. Microarray is a new technology to analyze the gene expression profile in a sensitive, specific and efficient manner. RNAi is a newly developed technology. It can specifically and efficiently interfere with the expression of genes. The combination of these technologies will be a powerful method to screen and identify differentially expressed genes between the corresponding tissues or cells.In this study, a novel method, called Filter Enhanced Subtractive Hybridization (FESH), was developed to construct a subtraction cDNA library of K562 cell lines. The inhibitory effect of BCR-ABL gene expression induced by RNAi technology on apoptosis of K562 cell lines was studied. The changes of gene expression were screened with the K562 subtraction cDNA microarray using the cDNA fragments of subtraction library as probes.The researches address the following 3 aspects:1. The subtraction cDNA library of K562 cell lines was constructed with FESH and restriction display (RD) technology. The total RNA was isolated from the K562 cells (tester) and normal peripheral blood lymphocytes (driver) respectively.Double-strand cDNA molecules were synthesized by reverse transcription and digested with restriction enzyme. The fragments were ranged from 200-2000 bp. The driver cDNA fragments were immobilized on nylon filter column in excess. The tester cDNAs were ligated with the double stranded DNA oligodexynucleotide linker. The ligation products were purified with PCR Purification kit. The tester cDNAs were resuspended in SSC, heat denatured, added onto the driver nylon filter column. After several round of subtraction, differentially enriched subtracted cDNAs were amplified with selective universal primers. The selective primers were designed by copying the linker sequence and adding one nucleotide at the 3' end. As this nucleotide can be either A or T or C or G, when each PCR uses a pair of such selective primers, by primer combinations, the original one group of subtracted cDNA fragments could be rationally divided into ten subgroups. The results of each step were verified correct before each following step. Subtraction efficiency was validated by GAPDH, a housekeeping gene, according to PCR cycles needed in subtracted sample and unsubtracted sample, with which the gene could be observed on agarose gel. The subtracted ten subgroups PCR products were ligated with pMD18 T vector. These ligated cDNA fragments were transformed into Escherichia coli DH5 a . So the subtraction cDNA library containing cDNA fragments only expressed in K562 cell lines was built up successfully. The library clones were selected by blue - white screening. The inserts in plasmid were amplified by PCR using vector-specific primers. The sequences were further analyzed by DNA sequencing. The sequence data were pumped online to the NCBI GenBank for BLAST analysis.Results: By formaldehyde agarose gel electrophoresis, the integrity and size of total RNA were analyzed and clear bands of 18s and 28s were obvious. Theamplified products of tester cDNAs with adaptor ranged from 200-2000 bp on 2.0 % agarose gel showed that ligation efficiency was high. After PCR amplification, the housekeeping gene GAPDH appeared in unsubtraction samples and did not appear in subtraction samples after 35 cycles. This result indicated that GAPDH expressed in both parts have been greatly decreased through the subtraction method. It implied that other genes expressed in both tissues have been reduced and the specially expressed genes in the test sample were selected. The inserts were amplified from 50 randomly picked colonies. The clones contained inserts ranging from 200~800 bp. Comparison of the sequences of clones to the GenBank database revealed some known genes and new ESTs, but no housekeeping gene.Conclusion: The FESH represents a faster, more economical and easier method for constructing subtraction cDNA library. Construction of subtraction cDNA library of K562 cell lines was necessary, which laid a foundation for screening and cloning new and specific genes of K562 cell lines. Differentially expressed genes of the subtraction cDNA library were closely associated with BCR/ABL-induced abnormalities in signal transduction, gene transactivation, cell cycle, apoptosis, adhesion, DNA repair, differentiation, metabolism and malignant progression. MUC3 and TPTl may be used in diagnosis and immunotherapy. The structure and function of differentially expressed genes could be further studied with bioinformatics.2. Small interfering RNAs (siRNA) were designed to target the breakpoint of BCR-ABL oncogene and control siRNAs were prepared by inverting a 2-nt fragment in the middle of the sequence and performing a BLAST search of the resulting sequence against GenBank. Transfections were performed by usinglipofectamine? 2000. The expression change of BCR-ABL examined by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and the effect of the siRNAs on the cell cycle and cell poliferation was analyzed by flow cytometry and MTT method respectively.Results: BCR-ABL siRNAs knocked down the BCR-ABL expression in K562 cellls obviously, inhibited the cell proliferation significantly and promoted cell apoptosis.Conclusion: We have demonstrated that siRNA is a highly specific tool for targeted gene knockdown. It can inhibit BCR-ABL expression and induce apoptosis. Our data establish siRNA-mediated gene silencing as a reliable and valuable approach for large-scale screening of gene function and drug target identification and validation.3. A set of cDNA clones including 250 FESH inserts were amplified with PCR using primers from sequences flanking the cloning site. PCR fragments were visualized on 1% agarose gel. The cDNA microarray was produced by spotting each PCR product 3 times on glass slide. The control group cDNAs was labeled with Cy3 dyes and siRNA group was labeled with Cy5 dyes using RD technology. The gene expression profile of the K562 cells after BCR-ABL gene knocked down was studied with cDNA microarray. RT-PCR analysis was used to confirm the results of microarray hybridization.Results: The probes length is comparatively homogeneous and suitable for microarray fabrication. Combination of subtraction hybridization and restriction display technology could provide a fast, easy and specific method for probe-preparation of microarray. Two sets of adaptors used in probe-preparing and sample-labling solved the problem that the same sequence in probe andsample may led to cross hybridization. 50 genes were found differently expressed,with 14 up-regulated and 36 down-regulated, in K562 cells. RAS> C-MYC^ cyclin D2-, BCL-XL found down-regulated were involved in the signal transduction pathways related to BCR-ABL. With the inhibition of BCR-ABL fusion gene, expression of these genes decreased, the RAS/MAPK^ JAK STAI\ PI-3K and C-MYC signal transduction pathway were inhibited, BCL-Xl protein decreased, Caspase was activated, the K562 cells were induced apoptosis.Conclusion: Enriched cancer-specific libraries constructed by FESH and RD can serve as ideal target sources for microarray screening. This study provided a fundamental tool for identifying important candidate genes involved in the BCR-ABL-mediated signal transduction pathways of K562 cells. Coupling of FESH with microarray analysis creates a convenient, high throughput means to profile gene expression patterns.In summary, with combination of FESH and microarray and RNAi technologies, the genes involved in the BCR-ABL-mediated signal transduction pathways of K562 cells were studied. This study provides theoretic basis for the diagnosis, therapy of CML. The combination of these technologies will be a powerful method to screen and identify differentially expressed genes between the corresponding tissues or cells.