RESUMEN
PURPOSE: Age-related cataract (ARC) is associated with the deregulation of transcription and defects in DNA repair in lens epithelial cells (LECs). DCLRE1A acted in DNA interstrand cross-links pathway to improve DNA replication and transcription. The aim of this study was to examined the further regulatory effect on DCLRE1A in the lncRNA-miRNA-mRNA network using a cell model of DCLRE1A overexpression (OE-DCLRE1A) in LECs. METHODS: The expression level of DCLRE1A in ARC tissues and SRA01/04 cells after H2O2 treatment was measured as protein and mRNA by qRT-PCR and Western Blot(WB). CCK8, and TUNEL assays detected the change in cell viability and apoptosis, respectively. Furthermore, Immunofluorescence assays detect the expression of DNA damaged and repair marker proteins after OE-DCLRE1A. The global expression profiles of lncRNAs, miRNAs, and mRNAs were determined using high-throughput sequencing. KEGG and GO enrichment analysis disclose the possible function of differentially expressed (DE) lncRNA, miRNA, and mRNA. RESULTS: The protein and mRNA of DCLRE1A were decreased in the anterior capsule of ARC and SRA01/04 cells treated by H2O2. OE-DCLRE1A improved damaged-DNA repair and enhanced cell viability against apoptosis after H2O2 treatment. Furthermore, we demonstrated the DE-molecules between the OE-DCLRE1A and control groups including 595 DE-lncRNAs, 221 DE-miRNAs, and 4718 DE-mRNAs. Next, bioinformatics analysis not only found that the DE-mRNAs are mainly involved in DNA repair-related signaling pathways after OE-DCLRE1A, but also screened two lncRNA-miRNA-mRNA networks focusing on DNA damage activated by OE-DCLRE1A, which involved 2 lncRNAs, 2 miRNAs, and 53 mRNAs. CONCLUSION: We revealed that DCLRE1A activated the lncRNA/miRNA/DNA-repair network to take part in DNA repair processes, which not only represents a new regulatory mechanism employed by DCLRE1A but also uncovers the screening lncRNA may hold potential therapeutic values in ARC formation. However, these conclusions will need to be confirmed by future studies in vitro and in vivo models.
RESUMEN
BACKGROUND: Liquid-liquid phase separation (LLPS) within the nucleus is directly linked to driving gene expression through transcriptional complexes. Histone lysine methyltransferase 2D (KMT2D) is widely present in many cancers. It is known to epigenetically stimulate the expression of genes associated with tumorigenesis and metastasis. Our analyses show that KMT2D possesses two distinct low-complexity domains (LCDs) capable of driving the assembly of membrane-less condensates. The dependence of the mechanisms underlying monomethylation of H3K4 on the LLPS microenvironment derived from KMT2D LCDs is unclear in tumor. METHODS: KMT2D LCD-depletion cells were used to investigate tumor cell proliferation, apoptosis, and migration. We identified some core proteins, including WDR5, RBBP5, and ASH2L, which are involved in the KMT2D-associated catalytic complex in KMT2D LCD-deficient cells to further elucidate the mechanism that decreases monomethylation of H3K4. We also evaluated the viability of KMT2D LCD-deficient cells in vivo. Finally, using 1,6-hexanediol (HD), an inhibitor of LLPS, we determined cell activities associated with KMT2D function in wild-type PANC-1 cells. RESULTS: Without the LLPS microenvironment in KMT2D LCD-deficient cells or wild-type PANC-1 cells treated with HD, the WDR5 protein was significantly less stable and the protein-protein interactions between the components of the KMT2D-enzyme complex were attenuated, impairing the formation of the complex. Moreover, with the decrease in H3K4me1 level at enhancers, transcription factors such as LIFR and KLF4 were markedly downregulated, effectively inhibiting tumor progression. In xenograft tumor models, PANC-1 cells lacking the KMT2D LCDs showed effectively suppressed tumor growth compared to normal cells. CONCLUSIONS: Our data indicate that the two low-complexity domains of the KMT2D protein could form a stable LLPS microenvironment, promoting the KMT2D catalysis of H3K4 monomethylation through stabilization of the WDR5 protein and KMT2D-enzyme complex. Therefore, finding ways to regulate the LLPS microenvironment will be benefitial for new cancer treatment strategies.
