Methylation Analysis In Hydatidiform Mole

Hydatidiform mole, a common gestational trophoblastic cells disease, were characterized by trophoblastic cells hyperplasia, placental villi matrix edema and lack of blood vessel. It can be divided into partial hydatidiform mole(PHM) and complete hydatidiform mole(CHM) according to histopathology and genetic origins. Over-invasion of trophoblastic cells in hydatidiform mole was the same biological behavior with tumor[1]. In cancer research, epigenetic study had shown that DNA methylation was changed in the process of cancer development. In view of the same features of "no control" between hydatidiform mole and tumor, the DNA methylation were studied in the hydatidiform mole to open up new paths for the mechanism of hydatidiform mole.MethodsThe expression of Dnmts(DNA methyltransferases) in complete hydatidiform mole(CHM) and normal early chorionic villi were detected by Real Time RT-PCR, Western blot and immunohistochemistry.Differential methylation pattern of genes in CHM and normal early chorionic villi were examined by MeDIP-chip (Methylated DNA Immunoprecipitation-chip).Differential methylation genes and functional characters were analyzed by bioinformatics.Part of differential methylation pattern of genes were confirmed by MSP(Methylation Specially PCR).Result1. The Dnmts mRNA expression results showed that Dnmt1(p=0.032) and Dnmt3b(p=0.009) in CHM were significantly higher than that in normal chorionic villi, but Dnmt3a(p=0.018) level was lower(p<0.05).2. The Dnmts protein level were detcted by Western blot. The results showed that Dnmt1(p=0.047) and Dnmt3b(p=0.006) in CHM were significantly higher than that in normal chorionic villi, but Dnmt3a (p=0.009) was lower (p<0.05).3. Immunohistochemistry results showed that Dnmt3a(p=0.027) expression was lower in CHM than that in normal chorionic villi, but Dnmt1(p=0.042) and Dnmt3b(p=0.016) were significantly higher(p<0.05). Dnmt1, Dnmt3a, Dnmt3b were mainly expressed in trophoblast cell cytoplasm in CHM. Dnmt1,Dnmt3b were mainly expressed in the part of the cytoplasm of trophblastic cells in normal early chorionic villi. Dnmt3a was mainly expressed in nucleus and cytoplasm of trophblastic cells in normal early chorionic villi.4. MeDIP-chip results showed 122 genes in CHM were methylated, of which 112 genes can be mapped to genome. Functional classification of these genes showed that (1) TRPC4, KCNT2, KCNQ1, KCNE1L, GABRE, KCNJ8, CLCNKA and KCNH7 were connected with ionic channel, (2) KCNT2, KCNH7, KCNQ1 and KCNJ8 were involved in potassium transport, (3)UROD and GAD1 were related with Carboxy-lyase, (4)CLCNKA, KCNH7, KCNJ8 and KCNQ1 were concerned with Voltage-gated channe.5. Part of differential methylation pattern of genes were confirmed by MSP and the results showed ELN was mainly unmethylation in normal chorionic villi, but mixed methylation in CHM. Other genes as E-cadherin,RASSF5,FGF12,SHROOM2,TIMP3 were mixed methylation both in CHM and normal chorionic villi.6. The methylation level (M/U value) of RASSF5, FGF12, SHROOM2, TIMP3, E-cadherin in CHM and normal early chorionic villi were analyzed, it showed that the methylation level of E-cadherin (p = 0.043)in CHM was higher than that in normal early chorionic villi(p <0.05).Conclusion1. Compared CHM with normal chorionic villi, Dnmt1, Dnmt3b expression were increased, Dnmt3a was decreased and Dnmt3a mainly expressed in trophoblast cell cytoplasm, nucleus hardly any. It suggested that over-expression of Dnmt1 and Dnmt3b, lower-expression of Dnmt3a and lost expression of Dnmt3a in trophoblast cell nucleus in CHM might be an important cause for CHM genesis.2. Compared CHM with normal chorionic villi, 122 genes were methylated in CHM, ELN and E-cadherin methylation level were higher in CHM than that in normal chorionic villi by MSP. It suggested that methylation of the genes might be correlated with genesis of CHM. Hypermethylation of ELN and E-cadherin may be uesd as the cure target of CHM.