Journal: JHEP Reports

Article Title: Identification and clinical implications of endogenous retrovirus elements suppressed by SETDB1 in hepatocellular carcinoma

doi: 10.1016/j.jhepr.2024.101307

Figure Lengend Snippet: Identification of the HERV elements regulated by SETDB1 through TCGA dataset analysis of HCC tissues. (A–D) Analysis of 242 patients with HCC in TCGA dataset. (A) Scatter plot showing the negative correlation between SETDB1 expression levels and median RNA expression of REs per specimen (Spearman’s rank method). (B) Box plots comparing median RE RNA expression per specimen between SETDB1 low- and high-expression groups (Mann–Whitney U test). (C, D) Box plots showing the RNA expression of SETDB1 (C) and median RNA expression of REs per specimen (D) across the MS1, MS2, and MS3 subtypes. (E) Schematic of 10 HERV elements downregulated in SETDB1 high-expression cases, with red bars indicating primer positions for qRT-PCR and qChIP-PCR. (F) Beeswarm plots of RNA expression levels for each of the 10 HERV elements per specimen in 242 HCC samples from TCGA dataset, categorized by SETDB1 expression level (Mann–Whitney U tests). (G–I) qRT-PCR analysis of HERV expression levels, and qChIP-PCR analysis of SETDB1 binding (H) and H3K9me3 levels (I) at the primer regions of four HERV elements in HuH7 and HLE cells treated with control siRNA (NC) or siSETDB1#1. Experiments were performed at least in triplicate (n ≥3). Data are presented as mean ± SE. Student’s t test, ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001. HCC, hepatocellular carcinoma; HERV, human endogenous retrovirus; NC, negative control; qChIP-PCR, quantitative chromatin immunoprecipitation-PCR; qRT-PCR, quantitative reverse transcription-PCR; RE, retroelement; TCGA, The Cancer Genome Atlas.

Article Snippet: Five human HCC cell lines (HuH7, JHH4, PLC/PRF/5, HLE, and HLF) and one murine HCC cell line (Hepa1 - 6) were obtained from the American Type Culture Collection and the Japanese Collection of Research Bioresources.

Techniques: Expressing, RNA Expression, MANN-WHITNEY, Quantitative RT-PCR, Binding Assay, Control, Negative Control, Chromatin Immunoprecipitation, Reverse Transcription

Journal: JHEP Reports

Article Title: Identification and clinical implications of endogenous retrovirus elements suppressed by SETDB1 in hepatocellular carcinoma

doi: 10.1016/j.jhepr.2024.101307

Figure Lengend Snippet: Biological effects of Setdb1 knockdown on murine HCC cells. (A) Cell proliferation assay for Setdb1 -KD Hepa1 - 6 cells (n = 8). Data are presented as mean ± SE. (B) Migration and invasion assays for Setdb1 -KD cells (n = 3), with representative images shown. (C) Photographs of xenograft tumors from Setdb1 -KD and control Hepa1 - 6 cells collected from KSN/Slc mice (top) and corresponding tumor sizes (bottom). (D) Immunohistochemistry images of Setdb1 and Ki-67 in xenograft tumors derived from scramble (shNC) and shSetdb1#2 Hepa1 - 6 cells collected from KSN/Slc mice, with relative positive cell counts. (E) Photographs of xenograft tumors from Setdb1 -KD and control cells in C57BL/6 mice (top) and the corresponding tumor sizes (bottom). (F) Immunohistochemistry images of Setdb1, CD8, and Ki-67 in xenograft tumors from shNC and shSetdb1#2 Hepa1 - 6 cells in C57BL/6 mice, with relative positive cell counts. Data are presented as mean ± SE. Student’s t test, ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001. HCC, hepatocellular carcinoma; KD, knockdown.

Article Snippet: Five human HCC cell lines (HuH7, JHH4, PLC/PRF/5, HLE, and HLF) and one murine HCC cell line (Hepa1 - 6) were obtained from the American Type Culture Collection and the Japanese Collection of Research Bioresources.

Techniques: Knockdown, Proliferation Assay, Migration, Control, Immunohistochemistry, Derivative Assay

Journal: JHEP Reports

Article Title: Identification and clinical implications of endogenous retrovirus elements suppressed by SETDB1 in hepatocellular carcinoma

doi: 10.1016/j.jhepr.2024.101307

Figure Lengend Snippet: Impact of SETDB1 on immune response and downstream target genes in HCC cells. (A) Volcano plot of differentially expressed genes in Setdb1 -knockdown (KD) vs. negative control (NC) Hepa1 - 6 cells, as analyzed using RNA-seq. Genes exhibiting a |FC| >1.5 and p <0.05 according to the DESeq2 are colored blue (log 2 FC <0) and red (log 2 FC >0). (B, C) Gene set enrichment analysis of Setdb1 -KD Hepa1 - 6 cells showing significant upregulation of the interferon α response pathway, which was induced by Setdb1 -KD ( p <0.05). (D) qRT-PCR analysis of upregulated interferon α response and interferon-stimulated genes in SETDB1 -KD murine and human HCC cells (shSetdb1#1 and siSETDB1#1), normalized to the 18S levels. (E) qRT-PCR analysis of upregulated ISGs in xenograft tumors from SETDB1-KD HuH7 cells (n = 8). (F) qRT-PCR analysis of tumor suppressor genes upregulated by Setdb1 -KD in Hepa1 - 6 cells, as identified using RNA-seq, normalized to the 18S levels. Expression changes of these genes were analyzed using human HCC cells with SETDB1 -KD. (G) qChIP-PCR analysis of H3K9me3 levels at the transcription start sites of upregulated genes in SETDB1 -KD cells (shSetdb1#1 and siSETDB1#1). ChIP was performed using anti-H3K9me3 and anti-histone H3 antibodies, with H3K9me3 enrichment normalized to that of pan H3. qChIP- and qRT-PCR experiments were performed in triplicate (n = 3). Data are presented as mean ± SE. Student’s t test, ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001. FC, fold change; HCC, hepatocellular carcinoma; qChIP-PCR, quantitative chromatin immunoprecipitation-PCR; qRT-PCR, quantitative reverse transcription-PCR.

Article Snippet: Five human HCC cell lines (HuH7, JHH4, PLC/PRF/5, HLE, and HLF) and one murine HCC cell line (Hepa1 - 6) were obtained from the American Type Culture Collection and the Japanese Collection of Research Bioresources.

Techniques: Knockdown, Negative Control, RNA Sequencing, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation, Reverse Transcription