Journal: Journal of Translational Medicine

Article Title: DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3

doi: 10.1186/s12967-024-05960-8

Figure Lengend Snippet: Association analysis of RORA expression and ESCC diagnosis and prognosis. ( A ) Data of RNA-seq in ESCC tumor specimens and matched healthy esophageal samples were retrieved from UCSC XENA. R package ggplot2 showing the under-expression of RORA in ESCC tumor specimens versus normal tissues. ( B ) IHC assay was implemented to measure the expression of RORA in five pairs of ESCC tumor tissues and adjacent normal tissues. ( C ) ROC curve and AUC value analyzed by pROC package in R demonstrating the diagnostic potential for RORA. ( D and E ) Survival package in R indicating the correlation of RORA expression with overall survival of ESCC. *** P < 0.001

Article Snippet: For analysis of protein expression, we used primary antibodies from Proteintech (Wuhan, China) including rabbit anti-RORA polyclonal antibody (pA) (1:1,500, #10616-1-AP), rabbit anti-HIF1A polyclonal antibody (pA) (1:8,000, #20960-1-AP), rabbit anti-Snail pA (1:800, #13099-1-AP), rabbit anti-N-cadherin pA (anti-N-cad, 1:5,000, #22018-1-AP), rabbit anti-E-cadherin pA (anti-E-cad, 1:30,000, #20874-1-AP), rabbit anti-SLC2A3 pA (1:4000, #20403-1-AP), and mouse anti-β-actin monoclonal antibody (mAb) (1:10,000, #81115-1-RR).

Techniques: Expressing, Biomarker Discovery, RNA Sequencing, Diagnostic Assay

Journal: Journal of Translational Medicine

Article Title: DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3

doi: 10.1186/s12967-024-05960-8

Figure Lengend Snippet: Inhibitory activity of RORA in ESCC cell viability and motility under hypoxia. ( A and B ) RT-qPCR and western blotting demonstrating that RORA expression increased in cells exposed to hypoxia. ( C ) The regulatory effect of HIF1A on RORA protein expression under hypoxia or normoxia was analyzed by western blotting. ( D ) The transfection efficiencies of shRORA and RORA ectopic expression plasmid in ESCC cells under hypoxia or normoxia were assessed by western blotting assay. ( E ) Under hypoxic conditions, CCK-8 assay revealing that RORA knockdown (KD) promoted cell viability, and RORA overexpression (OE) decreased cell viability. * P < 0.05, *** P < 0.001

Article Snippet: For analysis of protein expression, we used primary antibodies from Proteintech (Wuhan, China) including rabbit anti-RORA polyclonal antibody (pA) (1:1,500, #10616-1-AP), rabbit anti-HIF1A polyclonal antibody (pA) (1:8,000, #20960-1-AP), rabbit anti-Snail pA (1:800, #13099-1-AP), rabbit anti-N-cadherin pA (anti-N-cad, 1:5,000, #22018-1-AP), rabbit anti-E-cadherin pA (anti-E-cad, 1:30,000, #20874-1-AP), rabbit anti-SLC2A3 pA (1:4000, #20403-1-AP), and mouse anti-β-actin monoclonal antibody (mAb) (1:10,000, #81115-1-RR).

Techniques: Activity Assay, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Plasmid Preparation, CCK-8 Assay, Knockdown, Over Expression

Journal: Journal of Translational Medicine

Article Title: DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3

doi: 10.1186/s12967-024-05960-8

Figure Lengend Snippet: Suppressive function of RORA in ESCC cell migration and invasion under hypoxia. ( A ) Under hypoxic conditions, wound-healing assay showing that RORA KD enhanced cell motility, and RORA OE hindered migration. ( B ) Under hypoxic conditions, transwell assay demonstrating that cell invasiveness was facilitated by RORA KD and repressed by RORA OE. ( C and D ) IF microscopy revealing that RORA KD increased the levels of MMP2 and MMP9, which the two factors were downregulated following RORA OE. ( E ) Western blotting demonstrating the expression alteration of E-cad, N-cad and Snail following RORA KD and RORA OE. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For analysis of protein expression, we used primary antibodies from Proteintech (Wuhan, China) including rabbit anti-RORA polyclonal antibody (pA) (1:1,500, #10616-1-AP), rabbit anti-HIF1A polyclonal antibody (pA) (1:8,000, #20960-1-AP), rabbit anti-Snail pA (1:800, #13099-1-AP), rabbit anti-N-cadherin pA (anti-N-cad, 1:5,000, #22018-1-AP), rabbit anti-E-cadherin pA (anti-E-cad, 1:30,000, #20874-1-AP), rabbit anti-SLC2A3 pA (1:4000, #20403-1-AP), and mouse anti-β-actin monoclonal antibody (mAb) (1:10,000, #81115-1-RR).

Techniques: Migration, Wound Healing Assay, Transwell Assay, Microscopy, Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3

doi: 10.1186/s12967-024-05960-8

Figure Lengend Snippet: Suppression of RORA in KYSE150 xenograft growth. ( A and B ) Images of mice and xenografts showing the inhibitory role of RORA in KYSE150 xenograft growth. ( C and D ) Reduced tumor volume and weight after RORA OE indicating the repression of RORA in KYSE150 xenograft growth. ( E ) RT-qPCR indicating the upregulation of RORA in lenti-RORA-transduced xenograft tumors. ( F ) Western blotting was employed to detect the expression of HIF1A, a biomarker of hypoxia, in the two groups of xenograft tumors. ( G ) IHC revealing that RORA OE reduced the ratio of the Ki-67-positive cells in KYSE150 xenograft tumors. ( H ) IF microscopy indicating that RORA OE repressed the growth of xenografts as measured by the reduced ratio of the PCNA-positive cells. ( I and J ) IF microscopy showing that RORA OE downregulated the levels of MMP2 and MMP9 in KYSE150 xenografts. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: no statistically significant difference

Article Snippet: For analysis of protein expression, we used primary antibodies from Proteintech (Wuhan, China) including rabbit anti-RORA polyclonal antibody (pA) (1:1,500, #10616-1-AP), rabbit anti-HIF1A polyclonal antibody (pA) (1:8,000, #20960-1-AP), rabbit anti-Snail pA (1:800, #13099-1-AP), rabbit anti-N-cadherin pA (anti-N-cad, 1:5,000, #22018-1-AP), rabbit anti-E-cadherin pA (anti-E-cad, 1:30,000, #20874-1-AP), rabbit anti-SLC2A3 pA (1:4000, #20403-1-AP), and mouse anti-β-actin monoclonal antibody (mAb) (1:10,000, #81115-1-RR).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Biomarker Discovery, Microscopy

Journal: Journal of Translational Medicine

Article Title: DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3

doi: 10.1186/s12967-024-05960-8

Figure Lengend Snippet: DNMT1 induces methylation of RORA in hypoxia-induced ESCC cells. ( A ) GSEA analysis revealing the association between RORA and DNA methylation. ( B ) TCGA samples from Ualcan database showing the augmentation of the promoter methylation level of RORA in esophagus cancer (ESCA). ( C and D ) TCGA samples from Ualcan database revealing the correlation of RORA’s promoter methylation level and tumor lymphatic metastasis (N) and pathological grade. ( E ) Schematic showing the four biotin-labelled RORA promoter probes and five fragments divided by the four probes. ( F ) Venn diagram revealing the 69 specific bound proteins in the F1 fragment and 97 specific bound proteins in the F2 fragment. ( G ) Venn diagram revealing the 20 specific bound proteins in the F3 fragment. ( H ) Venn diagram revealing the 119 specific bound proteins in the F1 fragment and 101 specific bound proteins in the F2 fragment. ( I ) RNAseq data of TCGA-ESCA item was downloaded from TCGA database ( https://portal.gdc.cancer.gov ), and DNMT1 mRNA expression in 174 cases of tumor samples and 11 cases of normal samples was analyzed. ( J ) IHC assay was employed to measure the expression of DNMT1 in five pairs of ESCC tumor tissues and adjacent normal tissues. ( K ) RT-qPCR demonstrating that in hypoxia-induced ESCC cells, decitabine (DCA) elevated RORA mRNA level, and DNMT1 upregulation decreased the mRNA level of RORA. ( L ) Western blotting demonstrating that in hypoxia-induced ESCC cells, decitabine (DCA) elevated RORA protein expression, and DNMT1 upregulation inhibited the protein level of RORA. ( M ) ChIP qPCR confirming the binding of DNMT1 and the RORA promoter in hypoxia-induced KYSE150 ESCC cells. ( N and O ) DNA pyrosequencing indicating the enhancement of DNMT1 in the promoter methylation level of RORA in hypoxia-induced KYSE150 ESCC cells. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For analysis of protein expression, we used primary antibodies from Proteintech (Wuhan, China) including rabbit anti-RORA polyclonal antibody (pA) (1:1,500, #10616-1-AP), rabbit anti-HIF1A polyclonal antibody (pA) (1:8,000, #20960-1-AP), rabbit anti-Snail pA (1:800, #13099-1-AP), rabbit anti-N-cadherin pA (anti-N-cad, 1:5,000, #22018-1-AP), rabbit anti-E-cadherin pA (anti-E-cad, 1:30,000, #20874-1-AP), rabbit anti-SLC2A3 pA (1:4000, #20403-1-AP), and mouse anti-β-actin monoclonal antibody (mAb) (1:10,000, #81115-1-RR).

Techniques: Methylation, DNA Methylation Assay, Expressing, Quantitative RT-PCR, Western Blot, ChIP-qPCR, Binding Assay

Journal: Journal of Translational Medicine

Article Title: DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3

doi: 10.1186/s12967-024-05960-8

Figure Lengend Snippet: The downstream targets of RORA are screened by RNA-seq. ( A ) GO function annotation of the significantly down-regulated genes in RORA + hypoxia group versus vec + hypoxia group was performed. ( B ) The differentially expressed genes were shown by the volcano plot, and blue for down-regulated genes, red for up-regulated genes. ( C ) The mRNA expression levels of SLC2A3 and SLC2A14 in vec + hypoxia and RORA + hypoxia groups were determined by RT-qPCR. ( D ) Western blot assay was employed to detect the protein level of SLC2A3 in vec + hypoxia and RORA + hypoxia groups. ( E ) The binding motif of RORA and the binding sequence between RORA and SLC2A3 promoter region were predicted by JASPAR database. ( F ) Three ChIP-qPCR primers of SLC2A3 promoter were designed based on the three predicted binding sites, and ChIP-PCR was employed to verify the binding sites between RORA and SLC2A3 promoter. ( G ) Dual-luciferase reporter assay was conducted to verify the binding sites between RORA and SLC2A3 promoter. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: non significant

Article Snippet: For analysis of protein expression, we used primary antibodies from Proteintech (Wuhan, China) including rabbit anti-RORA polyclonal antibody (pA) (1:1,500, #10616-1-AP), rabbit anti-HIF1A polyclonal antibody (pA) (1:8,000, #20960-1-AP), rabbit anti-Snail pA (1:800, #13099-1-AP), rabbit anti-N-cadherin pA (anti-N-cad, 1:5,000, #22018-1-AP), rabbit anti-E-cadherin pA (anti-E-cad, 1:30,000, #20874-1-AP), rabbit anti-SLC2A3 pA (1:4000, #20403-1-AP), and mouse anti-β-actin monoclonal antibody (mAb) (1:10,000, #81115-1-RR).

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Western Blot, Binding Assay, Sequencing, ChIP-qPCR, Luciferase, Reporter Assay

Journal: Journal of Translational Medicine

Article Title: DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3

doi: 10.1186/s12967-024-05960-8

Figure Lengend Snippet: The anti-tumor activity of RORA on migration and invasion of hypoxia-induced ESCC cells is largely based on its target SLC2A3. ( A ) RT-qPCR was employed to assess the overexpression efficiency of SLC2A3 plasmid. ( B-F ) ESCC cells were transfected with vector, RORA, or RORA + SLC2A3 and treated with hypoxia. ( E ) Cell migration ability was evaluated by wound-healing assay. ( C ) Transwell assay was employed to assess cell invasion ability. ( D and E ) The abundance of cell motility-related indicators (MMP2 and MMP9) was determined by IF assay. ( F ) Western blotting was conducted to detect the expression of EMT-associated markers (E-cad, N-cad, and Snail) in hypoxia-induced ESCC cells. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For analysis of protein expression, we used primary antibodies from Proteintech (Wuhan, China) including rabbit anti-RORA polyclonal antibody (pA) (1:1,500, #10616-1-AP), rabbit anti-HIF1A polyclonal antibody (pA) (1:8,000, #20960-1-AP), rabbit anti-Snail pA (1:800, #13099-1-AP), rabbit anti-N-cadherin pA (anti-N-cad, 1:5,000, #22018-1-AP), rabbit anti-E-cadherin pA (anti-E-cad, 1:30,000, #20874-1-AP), rabbit anti-SLC2A3 pA (1:4000, #20403-1-AP), and mouse anti-β-actin monoclonal antibody (mAb) (1:10,000, #81115-1-RR).

Techniques: Activity Assay, Migration, Quantitative RT-PCR, Over Expression, Plasmid Preparation, Transfection, Wound Healing Assay, Transwell Assay, Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3

doi: 10.1186/s12967-024-05960-8

Figure Lengend Snippet: RORA restrains the glycolysis of hypoxia-exposed ESCC cells largely by targeting SLC2A3. ( A-C ) ESCC cells were transfected with vector, RORA, or RORA + SLC2A3 and treated with hypoxia. Cell glycolysis was analyzed by measuring the uptake of glucose and the generation of lactic acid and ATP. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For analysis of protein expression, we used primary antibodies from Proteintech (Wuhan, China) including rabbit anti-RORA polyclonal antibody (pA) (1:1,500, #10616-1-AP), rabbit anti-HIF1A polyclonal antibody (pA) (1:8,000, #20960-1-AP), rabbit anti-Snail pA (1:800, #13099-1-AP), rabbit anti-N-cadherin pA (anti-N-cad, 1:5,000, #22018-1-AP), rabbit anti-E-cadherin pA (anti-E-cad, 1:30,000, #20874-1-AP), rabbit anti-SLC2A3 pA (1:4000, #20403-1-AP), and mouse anti-β-actin monoclonal antibody (mAb) (1:10,000, #81115-1-RR).

Techniques: Transfection, Plasmid Preparation