Screening And Preliminary Validation Of Hypomethylated Genes Related To The Chemoresistance In Choriocarcinoma

Background and Objective:Choriocarcinoma is a highly malignant trophoblastic neoplasia, which is relatively common in women at child-bearing age. Methotrexate-based combination chemotherapy is basically the primary and the standard application which proved to be quite effective. However, a small part of choriocarcinoma patients still face the poor prognosis and the threat of death due to chemoresistance. The chemoresistance of choriocarcinoma cells may result from the alternation of genes and DNA hypomethylation is one of the main causes of chemoresistance. Therefore, the objective of our study was to screen the highly-expressed hypomethylation genes related to chemoresistance in choriocarcinoma cells in vitro, to validate screened genes both at the mRNA level and at the protein level and to explore the role of these highly-expressed hypomethylation genes in chemoresistance mechanisms of choriocarcinoma.Materials and Methods:First, Cell counting kit (CCK-8) assay was used to identify the drug sensitivity and resistance indexes(RI) of chemoresistant choriocarcinoma cell line JEG-3/MTXR compared to parent cell line JEG-3 cell line. Second, expression profiles and DNA methylation status of chemoresistant cell line JEG-3/MTXR and parent cell line JEG-3 were compared on Human Genome U133 Plus 2.0 chip and Human DNA Methylation 3x720K CpG Island Plus RefSeq Promoter Array, respectively. Candidate target genes were obtained through cross-matching select combined the result of DNA methylation chips and the result of gene expression profiles. Third, Real-time PCR and western blot were used to validate the express difference of these screened highly-expressed hypomethylation genes at the mRNA level and the protein level.Results:1. The resistance of chemoresistant cell line JEG-3/MTXR has not reversed after cultured in vitro with drug free media and refrigeration. The Rl of chemoresistant choriocarcinoma cell line JEG-3/MTXR was between 3.1-4.3.2. The ten candidate genes(ARMCX6, FSTL3, ATXN1, ASCL2, PER3, MICB, HDGFRP3, NRN1, SOHLH2, RAB40B) selected by cross-matching selection were hypomethylated and high expressed (fold change>3).At the same time, genome-wide DNA hypomethylation was observed in in the resistant JEG-3/MTXR cell lines.3. The results of real-time PCR were consistent with that of gene expression profile. The expression level of eight genes (PER3, HDGFRP3, NRN1, SOHLH2, ARMCX6, FSTL3, ATXN1, ASCL2) was increased more than 5 times in the resistant JEG-3/MTXR cell lines compared with the parental JEG-3 cell lines. The expression levels of the six proteins (PER3, HDGFRP3, SOHLH2, ARMCX6, ATXN1, ASCL2) were significantly different between these two cell lines, while the expression levels of the two protein(FSTL3 and NRN1) showed no significant difference between the two cell lines.Conclusions:Genome-wide DNA hypomethylation was observed in the resistance JEG-3/MTXR cell lines. Ten genes were found to be locally hypomethylated and high expressed in the resistance JEG-3/MTXR cell lines. Further, six genes (PER3, HDGFRP3, SOHLH2, ARMCX6, ATXN1 and ASCL2) were selected which were high expressed both at the level of mRNA and protein in the resistance JEG-3/MTXR cell lines. These 6 genes may probably involve in mediating drug resistance in choriocarcinoma since they were elevated significantly both at mRNA level and at protein level. ASCL2 might be a potential specific therapeutic target in anticancer drug resistance in the future.