Journal: Cellular & Molecular Biology Letters

Article Title: The low-complexity domains of the KMT2D protein regulate histone monomethylation transcription to facilitate pancreatic cancer progression

doi: 10.1186/s11658-021-00292-7

Figure Lengend Snippet: KMT2D LCDs modulate the stability of WDR5 protein and influence the formation of the KMT2D-enzyme complex. a Representative images of cell immunofluorescence detecting the WDR5 expression in modified-PANC-1 cells. The WDR5 level had markedly reduced in the LCD-depleted cells, but normal expression was recovered in OE1 and OE2 cells. b Western blotting analyses of the KMT2D-enzyme complex, including WDR5, ASH2L, and RBBP5, in HEK293T and PANC-1 cells. Knockout of KMT2D LCDs significantly attenuated WDR5 expression, but the expressions of ASH2L and RBBP5 were not affected. In OE1 and OE2 cells, WDR5 expression is restored. ASH2L, 69 KDa; RBBP5, 59 KDa; WDR5, 36 KDa; GAPDH, 37 KDa. c , d RT-PCR analyses of WDR5 gene expression. After LCD deletion in HEK293T and PANC-1 cells, the gene expression of WDR5 was similar to that in OE1 cells, OE2 cells, and control group (vehicle; error bars denote standard deviation). e , f Western blotting analyses of WDR5 protein expression with CHX or MG132 treatment. The WDR5 protein level in KMT2D LCD knockout cells gradually decreased with the time after CHX treatment compared with the wild-type cells, indicating that the stability of WDR5 protein is closely related KMT2D LCDs. g Co-immunoprecipitation analyses of the interactions between KMT2D and other components. The interactions between KMT2D and WDR5 were significantly reduced in KMT2D LCD-depleted PANC-1 cells. Similar results were found for the interactions of KMT2D with ASH2L and RBBP5. In OE1 and OE2 cells, these interactions were restored. All values are represented as means ± SD. Data are representative of three independent experiments

Article Snippet: Subsequently, the cells were stained overnight with primary fluorescent antibodies to KMT2D (Atlas Antibodies, HPA035977), H3K4me1, WDR5, EpCAM and CDH2.

Techniques: Immunofluorescence, Expressing, Modification, Western Blot, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control, Standard Deviation, Immunoprecipitation

Journal: Cellular & Molecular Biology Letters

Article Title: The low-complexity domains of the KMT2D protein regulate histone monomethylation transcription to facilitate pancreatic cancer progression

doi: 10.1186/s11658-021-00292-7

Figure Lengend Snippet: The LLPS microenvironment formed by the KMT2D protein influences its functions and modulates the stability of WDR5. a Representative images of cell immunofluorescence detecting the KMT2D expression in wild-type PANC-1 cells following treatment with 1,6-hexanediol (HD). KMT2D protein expression was markedly reduced and scattered after HD treatment. KMT2D, 593 KDa; GAPDH, 37 KDa. b , c Western blotting analyses of the KMT2D-enzyme complex in PANC-1 cells treated with HD. The expression of WDR5 decreased following HD treatment in a concentration- and time-dependent manner, while the expressions of ASH2L and RBBP5 were not affected. ASH2L, 69 KDa; RBBP5, 59 KDa; WDR5, 36 KDa; H3K4me1, 17 KDa; GAPDH, 37 KDa. d RT-PCR assays of metastasis-associated markers in PANC-1 cells treated with HD. The transcription levels of CDH1 and EpCAM significantly increased, while those of the CDH2 and vimentin decreased (error bars denote standard deviation). e RT-PCR assays of WDR5 in PANC-1 cells treated with HD. After HD treatment, the gene expression of WDR5 did not change (error bars denote standard deviation). f Representative images of cell immunofluorescence determining the WDR5 expression in PANC-1 cells treated with HD. The protein expression of WDR5 significantly decreased. g Western blotting analyses of the WDR5 expression after treatment with HD and CHX. Following HD treatment for 6 h at a concentration of 10 mg/mL, the stability of the WDR5 protein was sensitive to CHX. h Representative images of cell immunofluorescence determining the H3K4me1 expression in PANC-1 cells treated with HD. The level of H3K4me1 significantly decreased in cells treated with HD. i Co-immunoprecipitation analyses of the interactions between KMT2D and other components in PANC-1 cells treated with HD for 6 h at a concentration of 10 mg/mL. The interactions between KMT2D and WDR5, ASH2L and RBBP5 were markedly weaker following HD treatment. All values are represented as means ± SD. Data are representative of three independent experiments

Article Snippet: Subsequently, the cells were stained overnight with primary fluorescent antibodies to KMT2D (Atlas Antibodies, HPA035977), H3K4me1, WDR5, EpCAM and CDH2.

Techniques: Immunofluorescence, Expressing, Western Blot, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Gene Expression, Immunoprecipitation

Journal: Cellular & Molecular Biology Letters

Article Title: The low-complexity domains of the KMT2D protein regulate histone monomethylation transcription to facilitate pancreatic cancer progression

doi: 10.1186/s11658-021-00292-7

Figure Lengend Snippet: a Nude mice bearing established PANC1 xenograft tumors, including the vehicle group and sgRNA A1 × B2 group. After 2 weeks, tumor formation was monitored for a further 3 weeks. Interestingly, we could not detect tumor formation for PANC1 cells modified with LCD1 knockout, while the vehicle group could form tumors (red circle). b The proposed mechanistic model of for KMT2D LCDs in gene transcription. The knockout of KMT2D LCDs results in a loss of ability to form a stable LLPS microenvironment. The stability of WDR5 protein significantly decreased, and the protein–protein interactions between the components involved in the KMT2D–enzyme complex were attenuated. Some transcription factors, such as LIFR and KLF4, were markedly downregulated, attributed to the decrease in the H3K4me1 level. The left panel indicates the mechanism for full-length KMT2D, while the right panel indicates the mechanism for knocked out KMT2D LCDs

Article Snippet: Subsequently, the cells were stained overnight with primary fluorescent antibodies to KMT2D (Atlas Antibodies, HPA035977), H3K4me1, WDR5, EpCAM and CDH2.

Techniques: Modification, Knock-Out, Protein-Protein interactions

Journal: Journal of Hepatocellular Carcinoma

Article Title: LncRNA-CCDC144NL-AS1 Promotes the Development of Hepatocellular Carcinoma by Inducing WDR5 Expression via Sponging miR-940

doi: 10.2147/JHC.S306484

Figure Lengend Snippet: Sequence of Primers and siRNAs

Article Snippet: GeneGo analysis revealed that a large number of DNA fragments of MMPs and CDKs were enriched by anti-WDR5 antibody in cells overexpressing CCDC144NL-AS1compared to normal cells ( ).

Techniques: Sequencing

Journal: Journal of Hepatocellular Carcinoma

Article Title: LncRNA-CCDC144NL-AS1 Promotes the Development of Hepatocellular Carcinoma by Inducing WDR5 Expression via Sponging miR-940

doi: 10.2147/JHC.S306484

Figure Lengend Snippet: WDR5 is involved in CCDC144NL-AS1 regulating cell invasion and proliferation. ( A ) Luciferase assays, ( B ) RNA pull-down assay, and ( C ) Western blotting analysis confirmed the interaction between WDR5 mRNA and miR-940. ( D and E ) Overexpression of WDR5 in MHCC97H significantly blocked the effects of siCCDC144NL-AS1 on cell invasion and Ki67 expression, and knockdown of WDR5 also reversed the effects of pLV-CCDC144NL-AS1. ( F ) Expression of WDR5 was significantly upregulated in human HCC tissue compared to that in the matched normal tissue. ( G ) Expression of WDR5 in HCC tissue was negatively correlated with the 5-year prognosis of patients. The scale bar is 50 um. Data were expressed as mean ± SEM. * P <0.05, ** P <0.001.

Article Snippet: GeneGo analysis revealed that a large number of DNA fragments of MMPs and CDKs were enriched by anti-WDR5 antibody in cells overexpressing CCDC144NL-AS1compared to normal cells ( ).

Techniques: Luciferase, Pull Down Assay, Western Blot, Over Expression, Expressing, Knockdown

Journal: Journal of Hepatocellular Carcinoma

Article Title: LncRNA-CCDC144NL-AS1 Promotes the Development of Hepatocellular Carcinoma by Inducing WDR5 Expression via Sponging miR-940

doi: 10.2147/JHC.S306484

Figure Lengend Snippet: The Relationships Between WDR5 Expression in HCC Tissue and Patients’ Clinical Characteristics

Article Snippet: GeneGo analysis revealed that a large number of DNA fragments of MMPs and CDKs were enriched by anti-WDR5 antibody in cells overexpressing CCDC144NL-AS1compared to normal cells ( ).

Techniques: Expressing

Journal: Journal of Hepatocellular Carcinoma

Article Title: LncRNA-CCDC144NL-AS1 Promotes the Development of Hepatocellular Carcinoma by Inducing WDR5 Expression via Sponging miR-940

doi: 10.2147/JHC.S306484

Figure Lengend Snippet: MMPs and CDKs are the targets of WDR5. ( A ) Results of ChIP-seq and bioinformatics analysis on L02 with or without CCDC144NL-AS1 overexpression showed the involvement of cancer metastasis (MMP2 and MMP9) and the cell cycle (CDK1, CDK2, and CDK4) pathways in CCDC144NL-AS1-overexpressed cells. ( B ) Results of Western blotting showed that the expression of MMPs and CDKs are regulated by CCDC144NL-AS1/miR-940/WDR axis.

Article Snippet: GeneGo analysis revealed that a large number of DNA fragments of MMPs and CDKs were enriched by anti-WDR5 antibody in cells overexpressing CCDC144NL-AS1compared to normal cells ( ).

Techniques: ChIP-sequencing, Over Expression, Western Blot, Expressing

Journal: Journal of Hepatocellular Carcinoma

Article Title: LncRNA-CCDC144NL-AS1 Promotes the Development of Hepatocellular Carcinoma by Inducing WDR5 Expression via Sponging miR-940

doi: 10.2147/JHC.S306484

Figure Lengend Snippet: CCDC144NL-AS1 regulates cell cycle by miR-940/WDR5 pathway. ( A ) Analysis of flow cytometry showed that CCDC144NL-AS1 significantly upregulated the proportion of cells in S/G2/M through miR-940/WDR5 pathway. ( B ) Apoptosis-related proteins such as caspase-3, Bcl-2, and Bax were also involved in the pathway regulated by CCDC144NL-AS1/miR-940/WDR5. * P <0.05, ** P <0.001.

Article Snippet: GeneGo analysis revealed that a large number of DNA fragments of MMPs and CDKs were enriched by anti-WDR5 antibody in cells overexpressing CCDC144NL-AS1compared to normal cells ( ).

Techniques: Flow Cytometry

Journal: Journal of Hepatocellular Carcinoma

Article Title: LncRNA-CCDC144NL-AS1 Promotes the Development of Hepatocellular Carcinoma by Inducing WDR5 Expression via Sponging miR-940

doi: 10.2147/JHC.S306484

Figure Lengend Snippet: CCDC144NL-AS1 promotes WDR5 to be recruited to the promoter region of MMPs and CDKs. ChIP assay showed that ( A ) the recruitment of WDR5 and ( B ) the H3K4me3 level in the promoter region of MMP2, MMP9, CDK1, CDK2, and CDK4 was regulated by CCDC144NL-AS1/miR-940 axis. Data were expressed as mean ± SEM. * P <0.05, ** P <0.001.

Article Snippet: GeneGo analysis revealed that a large number of DNA fragments of MMPs and CDKs were enriched by anti-WDR5 antibody in cells overexpressing CCDC144NL-AS1compared to normal cells ( ).

Techniques:

Journal: Journal of Hepatocellular Carcinoma

Article Title: LncRNA-CCDC144NL-AS1 Promotes the Development of Hepatocellular Carcinoma by Inducing WDR5 Expression via Sponging miR-940

doi: 10.2147/JHC.S306484

Figure Lengend Snippet: Expression pattern of CCDC144NL-AS1/miR-940/WDR5, MMPs, and CDKs in mice HCC model. ( A ) RT-qPCR showed that the expression of CCDC144NL-AS1 increased markedly in mouse HCC tissue compared to that in the normal liver tissue, while the expression of miR-940 was significantly decreased. ( B ) IHC assay showed that the expression of WDR5, CDK1, CDK2, CDK4, MMP2, and MMP9 were all increased significantly in mouse HCC tissue compared to those in normal tissue. The scale bar is 50 um. Data were expressed as mean ± SEM. ** P <0.001.

Article Snippet: GeneGo analysis revealed that a large number of DNA fragments of MMPs and CDKs were enriched by anti-WDR5 antibody in cells overexpressing CCDC144NL-AS1compared to normal cells ( ).

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of Hepatocellular Carcinoma

Article Title: LncRNA-CCDC144NL-AS1 Promotes the Development of Hepatocellular Carcinoma by Inducing WDR5 Expression via Sponging miR-940

doi: 10.2147/JHC.S306484

Figure Lengend Snippet: Targeting CCDC144NL-AS1/WDR5 or overexpressing miR-940 significantly suppresses tumor growth and improves the prognosis of HCC in mice model. ( A ) Mouse burdening xenograft HCC. ( B ) Targeting CCDC144NL-AS1 or WDR5 suppressed tumor growth significantly, and similar results were observed in miR-940-overexpressed group. ( C ) Targeting CCDC144NL-AS1/WDR5 or overexpressing miR-940 could all improve the prognosis of HCC mice significantly.

Article Snippet: GeneGo analysis revealed that a large number of DNA fragments of MMPs and CDKs were enriched by anti-WDR5 antibody in cells overexpressing CCDC144NL-AS1compared to normal cells ( ).

Techniques:

Journal: Scientific Reports

Article Title: Upregulated WDR5 promotes proliferation, self-renewal and chemoresistance in bladder cancer via mediating H3K4 trimethylation

doi: 10.1038/srep08293

Figure Lengend Snippet: (A and C) The images of animals and tumors are shown in this study. (B and D) The volume of tumor growth was measured every 3 days. The results are presented as the means ± SD of values (n = 5). Statistical significance was calculated using the Student's t-tests. *p < 0.05, **p < 0.01. (E) The expression of WDR5 and Ki67 in the tumor was examined by IHC. (F) The correlation between WDR5 protein level and Ki67 protein level was measured in the animal tumor tissues. The H-score values were subjected to Pearson correlation analysis (n = 20). (G) The expression of WDR5 and Nanog was detected in the xenograft tumors by Western blotting.

Article Snippet: The anti-WDR5 and anti-Ki67 antibodies (1:1000, Zhongshan Bio-Tech Co. Ltd, Beijing, China) were used to detect the expression of WDR5 and Ki67 in mice tumors.

Techniques: Expressing, Western Blot