The Identification And Functional Study Of The Super Enhancer Of HIF1α In Human Cancer Cell Lines

Background and ObjectiveTumor treatment and related research have been the research areas of great concern.The"abnormal energy metabolism"of tumor cells is considered to be one of the main characteristics of malignant tumors.The reprogramming of tumor metabolism causes a series of abnormal metabolic phenotypes of tumor cells to meet the needs of tumor cells for energy and material anabolic metabolism.Hypoxia-inducible factor HIF1α is one of the main regulators of energy metabolism reprogramming.This project predicts and validates a new super enhancer of HIF1α in tumor cells.It can promote the transcription and expression of HIF1α,and then affect the biological behaviors of tumor cell metabolism,growth,and apoptosis.The reason for reprogramming will provide more effective and reliable targets for tumor treatment.Methods and ResultsThrough bioinformatics analysis,we predicted the HIF1α superenhancer HSE and verified it experimentally.The spatial interaction and activity of HSE in tumor cell lines were verified by 3C and ChIP experirments.CRISIR Cas9 gene editing technology was used to 対 A549 cells for HSE knockout,and quantitative effects of HSE sequence knockout on HIF1α expression were detected by quantitative PCR and Western blot.,Prove that the non-coding sequence here is indeed a super enhancer of HIF1α.We examined the effects of HSE on the survival and metabolism of A549 cells,and the results showed that the expression of multiple metabolic-related genes downstream of HIF1α decreased after HSE knockout.We used CCK-8 to detect the regulation of HSE on the activity of A549 cells;flow cytometry to detect the effects of HSE on cell cycle,ROS,mitochondrial membrane potential and quality;plate clones to detect the effects of HSE on the ability to form clones;ECAR to detect the enhancer HSE The effect on the level of glycolysis of cells,the results showed that the viability of A549 cells decreased after HSE knockout:A549 cells were inhibited from proliferating and their proliferation was slowed down;inhibition of cell transformation from S to G2;and the ability of A549 cells to form clones Decreased;A549 cells’ ability to form solid tumors decreased.We explored the extensiveness of the effects of HSE on tumor survival and metabolic regulation,and verified the universality of this superenhancer in tumors by knocking out HSE in other tumor cell lines;then we selected one of the cells obtained in the previous experiment,SKOV3.Corresponding functional experiments were performed,including CCK-8,plate cloning,and ECAR to detect the effects of HSE on cell survival and metabolism;the results showed that HSE is widely present in a variety of tumor cell lines and promotes HIF1α expression.Finally,we predicted the upstream transcription factor STAT3 bound to HSE,and preliminary verified that the transcription factor STAT3 regulates the expression of HIF1α by interacting with the enhancer HSE using CRISIR/Cas9 gene editing technology,quantitative PCR and ChIP experiments.ConclusionWe predicted and verified the expression of HIF1α by the superenhancer sequence HSE through bioinformatics analysis;HSE promoted the survival and glycolysis of tumor cells under hypoxic environment and widely’ existed in many tumor cell lines;after HSE knockout The expression levels of HIF1α decreased and cell viability decreased in multiple tumor cell lines.Finally,we investigated possible molecular mechanisms that produce these phenotypes,and predicted and preliminary verified that the transcription factor STAT3 regulates HIF1α through the distal enhancer sequence HSE expression.