Study On Effect And Mechanism Of PCDH10Gene In Multiple Myloma

Multiple myeloma (multiple myeloma, MM) is a plasma cell tumor,which takes up more than10%in haematological malignancies. Beside thebone marrow transplantation，MM is difficult to cure by the existingtreatment. Studies have shown that multiple myeloma closely related withmultiple oncogene overexpression, and inactivation of tumor suppressorgenes. But at present the development of the precise molecular mechanismis not fully understood.DNA methylation in human tumors is one of the most commonphenomenon. Aberrant promoter methylation in CpG island has beenrecognized as a tumor suppressor gene silencing mechanism. In recent years,more than50gene promoter region methylation anomalies has been reported.Clearly shown, as a transcriptional regulator DNA methylation plays a keyrole in human cancers. Have found that the gene promoter hypermethylationclosely associated with MM, these susceptibility genes almost involved in allknown cell information pathways. Most of these gene is tumor suppressor,hypermethylation inactivation leads to gene expression induced by loss of function, make the individual predisposition to MM.Epigenetic mechanisms regulating PCDH10silencing occurs in avariety of human cancers. In the PCDH10promoter,5’-side462-bpfragment areas with high density of CpG Island, is DNA methylation occursregion. The PCDH10gene silencing may weaken the inhibition of cancercell migration, which contributes to the cancer development. Studies showthat PCDH10can be used as a gastric cancer suppressor gene, methylationcan be used as an independent prognostic factors, and as a tumor marker fordynamic monitoring of precancerous lesions, which has importantapplication value in the early stage of gastric cancer prevention andtreatment.Our study do some degree of interpretation the inactivation of PCDH10gene in MM.Methods and Results：1. Determinated the expression state of PCDH10in MM1.1PCDH10expression level determination: Determination theexpression level of PCDH10of MM cell lines and9random MM patientsbone marrow by RT-PCR, with3normal volunteers as controls. RT-PCRshows PCDH10in MM cell lines and MM patients’ bone marrow specimensexpress very low or silenced, while normal bone marrow PCDH10highexpressed in normal volunteers.1.2methylation assay: Determination the methylation level of PCDH10 in MM cell lines and in44MM patients bone marrow by methylationspecific PCR (MSP), with9normal volunteers as controls. And thecollection clinical information of MM patients, analysis its correlation withDNA methylation.MSP shows34/44(77.27%) patients with MM samples appearedmethylation, and in9cases of control group PCDH10were not detectedmethylation. Clinical statistical analysis showed that the PCDH10genemethylation status has no obvious statistical difference with patients’ age,gender, ISS staging, serum creatinine, serum calcium, Cr (P>0.05).1.3Explored whether PCDH10inactivation cause of DNAmethylation:A+T(5-aza-2’-deoxycytidine (Aza) and trichostatin A (TSA))application in the PCDH10gene silencing in cell lines. RT-PCR showedA+T treatment may amplified PCDH10unmethylation specific product strip,proofed that after treatment with A+T, KM3、RPMI-8226cell recoveryPCDH10’s expression.2. PCDH10tumor suppressor function in multiple myeloma:Building a PCDH10expression vector pcDNA3.PCDH10, withliposomes2000vector transfection of human multiple myeloma cell lineRPMI-8226cells, G418screening the clone which PCDH10geneexpression. Western blot identificated PCDH10protein expression. Studythe effect of PCDH10gene expression multiple myeloma cells on apoptosis and angiogenic:2.1Proliferation: Colony formation assays show that after PCDH10expression in MM cells, clone number decreased, colony formation rate isreduced; flow cytometric analysis show G1phase of the cell numberdecreased by PCDH10expression, also cells arrest in S phase;2.2Angiogenesis: Chick chorioallantoic membrane (CAM) assaysshow that if PCDH10expressed in MM, angiogenesis will generatemalformations, with vascular node number droped.Conclusion：1PCDH10inactivation in MM;2DNA methylation play a role in the silencement of PCDH10in MM;3PCDH10is involved in angiogenesis and proliferation correlated withmultiple myeloma.