Study On Effect And Mechanism Of SFRP2, PLCD1Gene In Chronic Myeloid Leukemia

Objectives:1To examined the expression and methylation status of SFRP2genein chronic myeloid leukemia(CML), and try to find the role in the promotermethylation of SFRP2during the tumorigenesis of CML.2To explore methylation status and possible mechanism of PLCD1gene in CML, to evaluate the role of tumorigenesis and as a TSG(tumorsuppressor gene) in CML.Methods:1We investigated the expression of SFRP2and PLCD1genes inCML cell line K562and bone marrow cells by using semi-quantitativePCR(RT-PCR), respectively. K562was treated with pharmacologicaldemethylation5’-aza-2’deoxycytidine (Aza) and trichostatin A (TSA).2The methylation status of these two genes (SFRP2gene andPLCD1gene, respectively) promoters were detected in K562and CMLbone marrow samples by methylation-specific PCR(MSP).3Building a PLCD1expression vector pcDNA3.1PLCD1, with liposomes2000vector transfection of human CML cell line RPMI-K562cells, G418screening the clone which PLCD1gene expression. RT-PCRand Western blot identificated PLCD1mRNA and protein expression.4Study the effect of PLCD1gene expression K562cells onapoptosis and cell biological characteristics of the expression of PLCD1positive cloning: colony formation assays and flow cytometric analysis.Results:1SFRP2was silenced in K562and7/9CML bone marrow whilereadily detected in normal bone marrow samples(0/8). The frequencies ofSFRP2gene promoter methylation were100%,66%(25/38) and0(0/13) inK562, CML bone marrow and normal bone marrow samples, respectively.SFPR2in mRNA expression was dramatically induced afterpharmacological demethylation treatment, this reactivation was associatedwith an increase of unmethylation alleles. There were no significantcorrelations between the methylation status of PLCD1promoter and theclinical features, such as clinical stages, ages, sex, hemoglobin level, whiteblood cells(WBC), platelet counts(p＞0.05).2we investigated the PLCD1expression in CML Cell LineK562(0/1) and15%(2/13) of bone marrow mononuclear cells with CML byusing RT-PCR. The CpG island (CGI) methylation status of PLCD1promoter was detected in K562(0/1) and56%(23/41) of CML patients byMethylation-specific PCR(MSP), but none of normal adult bone marrow mononuclear cells. Furthermore, the DNA demethylation agent can restorethe expression of PLCD1in K562. Clinical statistical analysis showed thatthe PLCD1gene methylation status has no obvious statistical differencewith patients’ clinical stages, ages, sex, hemoglobin level, white bloodcells(WBC),platelet counts(p＞0.05).3The expression vector encoding full-length PLCD1or vector alonewere transfected into K562cells, in which PLCD1was fully silenced bymethylation. stable overexpression of PLCD1as shown by RT-PCR andWestern blot, was successfully obtained. Ectopic expression of PLCD1dramatically reduced the colony formation efficiencies of K562cells downto40-50%of vector controls,(p＜0.05), we investigated the effect ofPLCD1on cell cycle distribution by flow cytometry. The percentage ofcells in G1phase was increased in PLCD1-transfected cells compared withvector K562cells(p＜0.05).Conclusion:1The promoter of SFRP2gene are hypermethylated in CML, themethylation of SFRP2might be one of the molecular mechanisms of CML,it might serve as a new biomarker for CML.2The promoter of PLCD1gene are hypermethylated in CML, themethylation of PLCD1might be one of the molecular mechanisms of CML,PLCD1is a novel functional tumor suppressor gene and it is involved inangiogenesis and proliferation correlated with CML.