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Corelation between RISC complex, LINE-1 RNA and Global hypomethylation in head and Neck cancer cell line.

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE33493 | GEO | 2012/11/05

SECONDARY ACCESSION(S): PRJNA148743

REPOSITORIES: GEO

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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.

Project description:This is one of expressional parts of the study. These data were correlated to epigenetic marks and CG density of genes in analyzed cells. The whole study has a following summary: To elucidate possible roles of DNA methylation and chromatin marks in transcription, we performed epigenetic profiling of chromosome 19 in human bronchial epithelial cells (HBEC) and in the colorectal cancer cell line HCT116 as well as its counterpart with double knockout of DNMT1 and DNMT3B (HCT116-DKO). We found that H3K9me3 forms intragenic chromatin blocks along genes with low CpG density in the gene body. Analysis of H3K36me3 profiles indicated that this mark associates either with active genes with low CpG density and H3K9me3 in the gene body or with active genes with high CpG density and DNA hypermethylation in the gene body. In HCT116 cells with double knockout of DNMT1 and DNMT3B, transcription of genes with low CpG density in the gene body was highly elevated and associated with promoter DNA demethylation and rearrangement of H3K9me3 and H3K36me3 occupation. Our finding suggests that similar to DNA methylation, H3K9me3 may play a role in intragenic gene regulation. Further, we observed that a combination of low CpG density in gene bodies together with H3K9me3 and H3K36me3 marking is a specific epigenetic feature of zinc finger (ZNF) genes, which comprise 90% of all genes carrying both histone marks on chromosome 19. For high CpG density genes, transcription and H3K36me3 occupancy were not changed in condition of partial or intensive loss of DNA methylation in gene bodies in the HCT116-DKO cell line. siRNA experiments with SETD2 knockdown in both HBEC and HCT116-DKO cell lines failed to reduce DNA methylation in gene bodies under conditions of H3K36me3 depletion. Our study suggests that the H3K36me3 and DNA methylation marks in gene bodies are established independently from each other and points to similar functional roles of intragenic DNA methylation and intragenic H3K9me3 for CpG-rich and CpG-poor genes, respectively. Expressional changes associated with SETD2 siRNA transfection in HBEC Compare siRNA vs. control

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Corelation between RISC complex, LINE-1 RNA and Global hypomethylation in head and Neck cancer cell line.

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