Project description:One of most common events in carcinogenesis is loss of DNA methylation (hypomethylation) from sequence 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 fully methylated CpG sequences in order to keep prevent incorrect transcriptional start site (TSSs) that cause genome instability. To study character 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 of LINE-1’s host gene within cell. 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. Since LINE-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, DICER1 gene can investigate possibility role that LINE-1 RNA regulate LINE-1 host gene. Within yeast cell, overexpress retrotransposon transcript was control by endo-siRNA pathway, which require DICER1 like protein for produce endo-siRNA from precursor molecule that include retrotransposon transcript. 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. According to CU-DREAM analysis for HNSCC cell panel, DICER1 involved pathway may induce hypermethylation on gene body region which relate to TSS and intragenic LINE-1 promoter 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.

Project description:One of most common events in carcinogenesis is loss of DNA methylation (hypomethylation) from sequence 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 fully methylated CpG sequences in order to keep prevent incorrect transcriptional start site (TSSs) that cause genome instability. To study character 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 of LINE-1M-bM-^@M-^Ys host gene within cell. 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-1M-bM-^@M-^Ys host genes expression, which confirm by 5M-bM-^@M-^Y- aza-2-deoxycytidine induced DNA hypomethylationin HNSCC cell that cause EPHA3 become greater down- regulated. Since LINE-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 hostM-bM-^@M-^Ys 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, DICER1 gene can investigate possibility role that LINE-1 RNA regulate LINE-1 host gene. Within yeast cell, overexpress retrotransposon transcript was control by endo-siRNA pathway, which require DICER1 like protein for produce endo-siRNA from precursor molecule that include retrotransposon transcript. 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. According to CU-DREAM analysis for HNSCC cell panel, DICER1 involved pathway may induce hypermethylation on gene body region which relate to TSS and intragenic LINE-1 promoter 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. Oligonucleotides specific target on LINE-1 was inserted into Psilencer 3.1 vector hygro H1 promoter. (Ambion, Austin, Texas, USA) and transfection was mediated by FuGENE M-BM-. HD (Roche), sequence shown in appendix. Oligosynthesis siRNA specific to DICER1 purchased from santa cruz biotechnology for transient tranfection, inc was dilute with RNASE free dH2O to reach 10M-NM-<M. In a 12 well plate, seed 6 x 105 cells per well in 1 ml antibiotic-free normal growth medium supplemented with 10% FBS. In next day, perform transfection those siRNA at 100 nM with Lipofectamine 2000 reagent (Invitrogen Cat: 11668-027) according to the manufacturer's instructions. Cell was collect at 48h after transfection step by Trizol reagent (Life technologies, Inc.) in order to collect total RNA from each sample for hybridize on beadchip according to the protocol.

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Homo sapiens

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