Study On Gene Expression Profiling Of K562 Cell Treated With Velcade (Bortezomib)

The proteasome is an enzyme complex that exists in all cells and plays animportant role in degrading proteins involved in the cell cycle, growth of new bloodvessels (angiogenesis), cell adhesion, cytokine production, apoptosis, and otherimportant cellular processes. Many of the processes that rely on proteasome functioncan contribute to the growth and survival of cancer cells. Velcade is a potent butreversible inhibitor of the proteasome. By disrupting normal cellular processes,proteasome inhibition promotes apoptosis. Non-clinical data has demonstrated thatcancer cells are more susceptible to the effects of proteasome inhibition than normalcells. Due to the reversibility of proteasome inhibition with Velcade, normal cells canrecover from its effects, whereas cancer cells are more likely to undergo apoptosis.Velcade is a potent, specific, and reversible proteasome inhibitor and the firstdrug of this type to enter clinical trials. Proteasomes are present in all cells andfunction to help regulate cell growth. In nonclinical studies, normal cells appear to beable to recover from intermittent proteasome inhibition, but many types of cancercells undergo apoptosis (programmed cell death) when proteasomes are inhibited, even for a short time.Our study plans to research the changes of gene expression of K562 cell treatedwith Velcade. There are many methods of screening differential expression genes,such as Differential Display(DD), Representional Difference Analysis(RDA),Suppression Subtractive Hybridization(SSH), microarray and so on. Microarray is anew technology of gene analysis, especially cDNA microarray can analyze the geneexpression widely, quickly, sensitively and effectively, so we adopt cDNA microarrayas the research tool.Use light microscope, and DNA ladder to examine the changes of K562 celltreated with velcade. Extract genome DNA and run 1.2% agarose electrophoresis.Theproliferation of some cell is inhibited, and some shrink, distort, cell nucleus condenseand cell membranes are complete. Results of DNA ladder demonstrate that velcadegroup has a DNA degradation strip, indicating K562 cell generate apoptosis.Velcadecould inhibit K562 cell proliferate effectively, interfere the process of proliferation,induce apoptosis.Apply microarray to detect the gene expression changes of K562 cell treatedwith velcade. First extract total RNA of control group and Velcade group, and assessthe RNA integrity by Agilent BioAnalyzer 2100. Second, use Low Input RNAFluorescent Linear Amplification Kit to label RNA with Cy3 and Cy5: reversetranscript RNA to synthesize cDNA, label control group with Cy3 and velcade groupwith Cy5, then synthesize fluorescently-labeled cRNA, and purify them. Then,hybridize labeled cRNA with Agilent Human 1A 60mer Oligo Microarray, after thehybridization, wash slides by wash solutions one by one. Then, scan the microarraywith Agilent scanning, collect and normalize the data with Feature Extraction. Then,use Panther to carry out biological function analysis. Last, use reverse transcriptionPCR to check the expression of Nras,Etv6.There are 640 differential expression genes in 22153 detection spots, 29 genes are up-regulated, which are with one accord tohybridization result. The gene expression changes of K562 cell treated with Velcadeshow as: apoptosis promoting genes are up-regulated while apoptosis inhibiting genesare down-regulated, proliferation promoting genes are down-regulated, The totaltendency is promoting apoptosis and inhibiting proliferation.Above all, our study used Velcade to treat K562 cell and applied DNA ladderand so on to examine the changes of K562 cell. The study indicates that velcade couldinhibit K562 cell proliferate and induce apoptosis. Then, we studied the geneexpression profiling with microarray and found there are 640 genes changed, 29genesare up-regulated while 611 genes are down-regulated. The total tendency ispromoting apoptosis and inhibiting proliferation...