A Microarray Analysis Of Bortezomib-resistant Gene Expression In Multiple Myeloma

Background:Multiple myeloma(MM)is a B-cell malignant characterized by an uncontrolled proliferation of aberrant plasma cells in bone marrow.MM occurs a median age of 69 years and is one of the most common hematologic cancers Currently, many therapeutic advances like chemotherapy, stem cell transplantation and targeted drug therapy are widely used in treatment of MM. They can enhance life quality of patients and prolong survival rate. However, drug resistance cannot be prevented.As a proteasome inhibitor, bortezomib targets the ubiquitin-proteasome system to inhibit the antiapoptotic activity of nuclear factor-κB,and thus play an anti-tumor effect. Currently, bortezomib has become the first drug to MM.Chemotherapy program consists of bortezomib,dexamthasone and/or traditional chemotherapy is the standard treatment option to MM. However, primary or secondary bortezomib-resistance appears with the widespread clinical use of bortezomib. It was reported that resistant mechanisms may relate to NF-κB pathway, PSMB5 mutation and plasma cell dedifferentiation. But It has not yet been fully elucidated, especially different from the common mechanisms of multiple drug-resistant tumors. Laboratory efforts are underway to identify the mechanisms underlying resistance and develop strategies to reverse it.OBJ1ECTIVE:To search for genes related to bortezomib resistance phenotype by analyzing different gene expression patterns of cDNA microarray, and investigate the mechanism of bortezomib-resistant myeloma.METHODS:1.Gene expression profiles of KM3/BTZand KM3 cell lines was compared by the Affymetrix U 133 plus 2.0 oligonucleotide microarray.2. Specific differently-expressed genes were chosen for further verification using real-time RT-PCR.3. Par-4 was chosen for further verification using Western Blot.4.The expression level of PAWR in 7 primary diagnosed MM patients and 7 refractory recurrent MM patients were detected byreal-time RT-PCR.5. Combination of molecular annotation system MAS3.0 software and detailed analysis of documented resistance genes were used to analyze the data.RESULTS:1.It was found that the KM3/BTZ cell line has 670 significantly differently expressed genes as compared to KM3,including 32 genes was increased or decreased more than 10 folds.These 32 genes were involved in regulation of transcription and signal transduction. some members of ZNF and MS4A family and HSPB2 were hardly expressed in KM3,but obviously increased expression was discovered in KM3/BTZ.2.Except for JUN,7genes(CA12, CYP1B1、EPB41L3、HSPB2、MS4A4A、 SDPR、PAWR)were further verified using real-time RT-PCR and results were consistent with the microarray.3. Western Blot assay showed, Par4 protein level was consistent with themicroarray.4.The expression level of PAWR gene in refractory recurrent MM patients (0.034+ 0.013) was significant lower than in primary diagnosed group.5. The use of GO biological process in Molecular annotation system (MAS) 3.0 analyzes gene expression differences.Compared resistant cell lines with the primary cell lines,up-regulated genes involved in immune response, DNA-dependent transcriptional regulation, transcription, signal transduction, mitogen-activated protein kinase (MAPK), cell adhesion, and other aspects of nervous system development. However,down-regulated genes mainly involved in signal transduction, ion transport, DNA-dependent transcription regulation, transduction and cell adhesion.CONCLUSION:1.Some genes are probably related to bortezomib resistance in multiple myeloma cells:some members of ZNF and MS4A family and another 30 genes whose differential expression is more than 10 folds between KM3 and KM3/BTZ,especially of PAWR gene.2.Combination of filtering genes and detailed analysis of documented resistance genes provides a potential methodology to investigate the mechanism of drug resistance in multiple myeloma cells.