Study On Effect And Mechanism Of ADAMTS9Gene In Multiple Myeloma

Objective: Multiple myeloma (MM) is a plasma cell malignancy withhigh incidence in the blood tumor diseases. At present, the pathogenesis ofMM remains unclear, clinical treatment is very difficult. Numerousstudies show that epigenetic regulation imbalance closely related with theoccurrence of MM. ADAMTS9(a disintegrin-like and metallopeptidasewith thrombospondin type1motif,9) is one member of ADAMTS family,research has shown that ADAMTS9is involved in the occurrence anddevelopment of tumor. This study aims to observe the ADAMTS9geneexpression and promoter methylation status in multiple myeloma, toexplore the role of ADAMTS9in the pathogenesis of MM.Methods:1. We examined the expression levels of ADAMTS9mRNA inRPMI-8226、KM3cells,15MM and healthy adults bone marrow (BM)samples with reverse transcription-polymerase chain reaction (RT-PCR).2. we used methylation-specific PCR (MSP) to analyze themethylation status of ADAMTS9in RPMI-8226、KM3cells and56MMBM samples, and15normal adult BM samples were included as controls. We collected the clinical information of MM patients, to searched forpotential correlations between the ADAMTS9methylation status and theseclinicopathological characteristics.3. Cultured the KM3cells in vitro, cells were treated with the DNAmethyltransferase inhibitor5-aza-2’-deoxycytidine (Aza), combined withthe histone deacetylase inhibitor trichostatin A (TSA). RT-PCR and MSPwere used to detect whether the mRNA expression and methylation statusof ADAMTS9have changed before and after the drug intervention.4. The ADAMTS9-expressing vector pCEP4-ADAMTS9and emptyvector pCEP4were transfected into KM3cells. The cells were thenselected with G418to obtain stablely transfected cells. Using RT-PCR toobserve the mRNA expression of ADAMTS9after transfection.5. The colony formation assay, Cell Counting Kit-8(CCK-8) and flowcytometry analysis to observe the biological behavior of KM3cells beforeand after transfection.Results:1. The RT-PCR results revealed that the ADAMTS9expression wasdetected in the RPMI-8226cells, In contrast, KM3showed no ADAMTS9expression. ADAMTS9was detected0%(0/15) of the MM samples, whileexpression was readily detected100%(15/15) in the normal adult BMsamples.2. The MSP results showed that methylation was not observed in RPMI-8226cells, while ADAMTS9was methylated in KM3. TheADAMTS9gene promoter methylation rates of MM and normal bonemarrow samples were66%（37/56）、0%（0/15）.3. No significant correlations were observed between the MM patientswith methylated ADAMTS9and these clinicopathological characteristicssuch as age, gender, MM subtype and so on (P＞0.05）.4. ADAMTS9expression was substantially induced following drugtreatment in KM3cells, along with a decrease in methylated alleles.However, no difference was observed in the RPMI-8226cells.5. The ADAMTS9can be transfected into KM3cells successfully.Ectopic expression of ADAMTS9led to a sharp reduction in cell colonyforming ability in the KM3-ADAMTS9cells、the proportion of cells in theS phase decreased significantly in the ADAMTS9-transfected KM3cells、the proliferation trend was significantly decreased in ADAMTS9-expressing KM3cells（P <0.05）.Conclusion:1. The ADAMTS9was silenced in KM3cells and MM patients BMsamples, while expression was readily detected in RPMI-8226cells andnormal adult BM samples.2. The aberrant promoter methylation of ADAMTS9is directlyinvolved in transcriptional silencing in MM.3. ADAMTS9suppresses MM cell clonogenicity and cell proliferation,suggesting ADAMTS9may be a TSG in MM.