The Relationship Between DNA Methylation And RPRM Expression By Silencing DNMTs In Gastric Cancer

DNA methylation is one of the important epigenetic modifications,and DNA abnormal methylation leads to disease and cancer occurrence.Therefore,it is an inevitable trend to study cancer genomics and epigenetics together in order to understand the mechanism regarding cancer development.The RPRM gene has been reported to be methylated in different types of malignant tumors,including gastric cancer;in addition,RPRM promoter methylation is frequently detected in plasma samples of gastric cancer.So it could be a potential biomarker for early detection of gastric cancer.Besides,RPRM's expression might be regulated by its promoter's methylation.However,the mechanism regarding RPRM functions have not been well understood and the DNA methylation has been proposed as a potential mechanism regulating RPRM expression.In the present study,we modified the bisulfite conversion methods based on the previously published papers,and established a comprehensive method in detecting RPRM promoter's methylation.RPRM mRNA transcription level was also detected by using RT-PCR and qRT-PCR techniques.The results indicated that the RPRM expression was down-regulated if the gene was hypermethylated;otherwise,the RPRM expression remained in a normal level.Thus,the results show that RPRM promoter methylation has a significant correlation in gastric cancer occurrence,and RPRM expression is regulated by DNA methylation.After confirming the regulation of expression RPRM by DNA methylation,we studied the effects of DNMT on the RPRM expression by using DNMT inhibitors and corresponding RNA interference.AGS and SGC-7901 cell lines were treated with zebularine,a DNMT inhibitor,to assess its effect on the RPRM expression.The results showed that under the influence of zebularine,the RPRM methylation degree in these two cell lines was lowered,and RPRM expression was restored consequently.Since zebularine was unable to inhibit all types of DNA methyltransferases,we used RNA interference to silence DNMT1,DNMT3A and DNMT3B genes and decided which of the DNA methyltransferase was associated with the expression of RPRM.The result indicated that the inhibition of each of above DAN methyltransferases could upregulate RPRMexpression in AGS or SGC-7901 cell lines.And DNMT3B showed more profound role in the regulation of RPRM expression.