Background: Recent studies have attempted to elucidate the function of super enhancers by means of microRNAs. Although the functional outcomes of miR-1301 have become clearer, the pathways that regulate the expressions of miR-1301 remain unclear. Objective: The objective of this paper was to consider the pathway regulating expression of miR- 1301 and miR-1301 signaling pathways with the inhibition of cell proliferation. Methods: In this study, we prepared the cell clones that the KLF6 super enhancer was deleted by means of the CRISPR/Cas9 system-mediated genetic engineering. Changes in miR-1301 expression after the deletion of the KLF6 super enhancer were evaluated by RT-PCR analysis, and the signal pathway of miR-1301 with inhibition of the cell proliferation was examined using RNA interference technology. Results: The results showed that miR-1301 expression was significantly increased after the deletion of the KLF6 super enhancer. Over-expression of miR-1301 induced by deletion of the KLF6 super enhancer also regulated the expression of p21 and p53 in human hepatoma cells. functional modeling of findings using siRNA specific to miR-1301 showed that expression level changes had direct biological effects on cellular proliferation in Human hepatoma cells. Furthermore, cellular proliferation assay was shown to be directly associated with miR-1301 levels. Conclusion: As a result, it was demonstrated that the over-expression of miR-1301 induced by the disruption of the KLF6 super enhancer leads to a significant inhibition of proliferation in HepG2 cells. Moreover, it was demonstrated that the KLF6 super enhancer regulates the cell-proliferative effects which are mediated, at least in part, by the induction of p21and p53 in a p53-dependent manner. Our results provide the functional significance of miR-1301 in understanding the transcriptional regulation mechanism of the KLF6 super enhancer.
The Klf6 gene is a tumor suppressor gene belonging to the family of the Klf gene and closely associated with tumor formation. Recently, super enhancers(SEs) have been shown to play a particularly important role in regulating cell identity and are attempting to evaluate the in vivo function of SEs. But direct functional evidence of SE associated with Klf6 is lacking. Using genomic editing technology, we have attempted to identify super enhancer associated with the expression of Klf6 and clarify its function in human hepatoma (HepG2) cells. As a result, it identified the Klf6 related SE and demonstrated that the Klf6related SE is responsible for more than 80% of Klf6 gene expression. It also revealed the hierarchical structure of the Klf-6 related SE and the function of individual enhancers. Our results provide the functional significance of the super enhancer in understanding the transcriptional regulation mechanism of Klf6.
Background: The Klf6 gene, belonging to Krüppel-like family of C2H2 zinc finger transcription factors, is strongly associated with tumor formation through high somatic mutations in carcinomas of the prostate, liver, colon, stomach, lung, neck, pituitary gland and nervous system. Recently, Klf6 super-enhancer which strongly regulates Klf6 gene expression has been identified, and the function of Klf6 super-enhancer which regulates cell growth studied. Objective: The development of inhibitors targeting BRD4-binding super-enhancers is a potential target therapeutic strategy for tumor therapy. However, the suppression of Klf6 super-enhancer function by BRD4 inhibitors is not known. Methods: CRISPR-Cas9 editing technique was used for Klf6 super-enhancer deletion experiment, and the expression levels of several genes for cell clones were detected by qRT-PCR analysis and Western blotting. Cell proliferation assay was applied to evaluate the functional role of Klf6 super-enhancer using several BRD4 inhibitors. And, the interaction of several BRD4 inhibitors against the target protein was analyzed by molecular docking simulation. Results: As a result, JQ-1, a human BRD4 inhibitor, inhibits Klf6 gene expression and its activity in HepG2 cells in a time and dose-dependent manner while simultaneously inhibiting cell growth. In addition, BETd-246, a human BRD4 inhibitor, strongly inhibited Klf6 gene expression, significantly inhibited cell growth, and exhibited higher efficacy than JQ-1. Molecular docking studies revealed that some key residues were critical for ligand-receptor interactions by forming hydrogen bonds with ligands (JQ-1: ASN140, BETd-246: ASN140, TYR106, LYS65, GLN58, MET105, and MET53.) Conclusions: Our findings suggest that KLF6 is regulated by Klf6 super-enhancer and the targeting of Klf6 super-enhancer by BRD4 inhibitors may be an effective therapeutic strategy for liver cancer therapy.
Background: The Klf6 gene, which belongs to Krüppel-like family of C2H2 zinc finger transcription factors, is greatly related to tumorigenesis via a high rate of somatic mutation in the carcinomas of prostate, liver, colon, stomach, lung, neck, pituitary, and nervous system: Furthermore, the pathways regulating the expressions of Klf6 splice variants termed Klf6-SV1,-SV2, and-SV3 remain obscure although their functional outcomes have been clear. In this study, the functional roles of Klf6 variants in the inhibition of cell proliferation induced by the disruption of Klf6related super enhancer in human hepatoma (HepG2) cells were evaluated. Results: As a result, the disruption of Klf6-related super enhancer not only induced the upregulation of Klf6-SV2 but also led to a significant reduction of proliferation in HepG2 cells. In addition, the disruption of Klf6-related super enhancer led to the induction of p21 and Bax genes mediated by the upregulation of Klf6-SV2. Conclusion: In conclusion, it was demonstrated that Klf6-related super enhancer modulates cell proliferation via the regulation of Klf6-SV2 expression in human hepatoma (HepG2) cells. The results provide the functional significance of Klf6-related super enhancer in understanding the transcriptional regulation mechanism of Klf6.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.