Intragenic line-1 methylation controls gene expression in head and neck cancer cells

Abstract

One of most common events in carcinogenesis is loss of DNA methylation (hypomethylation) fromsequence of transposable element (TEs) including long interspersed nuclear element-1 (LINE-1). Naturally, LINE-1 and other TEs within gene body region (intragenic)will suppress by totallymethylated CpG sequences in order to keep prevent incorrect transcriptional start site (TSSs) that cause genome instability. To studycharacter of intragenic LINE-1 promoter hypomethylation or gene body methylation status, within Head and Neck squamous cell carcinoma (HNSCC) cell,LINE-1 promoter methylation status was detected with CU-L1 (unique intragenic LINE-1) and COBRALINE-1 (whole genome LINE-1) PCRs. Each intragenic LINE-1 promoter hypomethylation have unique pattern depending on cell types and impact ofLINE-1’s host gene within cell. Next, from CU-L1 PCR 16 genes, EPHA3 and PPP2R2B are only two genes that normally induce expression by hypermethylated in gene body region, intragenic LINE-1 promoter. LINE-1 promoter hypomethylation level induce global and specific LINE-1 expression and also repress LINE-1’s host genes expression, which confirm by 5’-aza-2-deoxycytidine induced DNA hypomethylationin HNSCC cell that cause EPHA3 become greater down-regulated.Then, sinceLINE-1 RNA can express via promoter hypomethylation, knockdown LINE-1 RNA can lead increasing of EPHA3 in HNSCC cell. LINE-1 RNA was concern as key factor on LINE-1 host’s gene regulation by consequence of gene body hypomethylation. RNA molecule can regulate gene by RNAi pathway and Epigenetics mechanism, within both machinery require RISC proteins. Next, knockdown RISC protein, AGO2 protein or human EIF2C2 can investigate possibility role that LINE-1 RNA regulate LINE-1 host gene. Knockdown AGO2 within HNSCC cell, EPHA3 become up-regulate that can conclude similar role between LINE-1 RNA and AGO2 on gene controlling. Immunoprecipitate by AGO2 antibody can reveal interaction of AGO2 on both LINE-1 RNA and DNA from LINE-1 promoter sequence that indicate role of LINE-1 RNA and AGO2 will mostly through Epigenetics pathway as confirm about AGO2 bind on LINE-1 promoter region. So that, by intragenic LINE-1 promoters methylation change in HNSCC cell that knockdown LINE-1 or AGO2, it may conclude that LINE-1 RNA and AGO2 regulate LINE-1’s host gene via in cis epigenetics mechanism within gene body region. Cell can control overexpress retrotransposon transcript by endo-siRNA pathway, which require DICER1 for produce endo-siRNA from precursor molecule that include retrotransposon transcript. Combine endo-siRNA pathway to epigenetics mechanism within gene body region will be determine in last step. With CU-DREAM analyse microarray results by intersection datas for check the connection between factors on gene regulation including DICER1, LINE-1 RNA, DNA hypomethylation and HNSCC carcinogenesis model. Within HNSCC cell panel, DICER1 involved pathway prefer to induce hypermethylation on gene body region while LINE-1 RNA involved pathway select to induce hypermethylation on gene without LINE-1 promoter, LINE-1 RNA have in tran function. In conclusion, intragenic LINE-1 promoter hypomethylation cause releases of LINE-1 RNA for regulate genes during HNSCC carcinogenesis via RISC protein.