Intracellular Hypoxia Induces G1/S Cell Cycle Arrest In Esophageal Cancer Cells And The Induced Circular RNA CircPUM1 Promotes Oxidative Phosphorylation

Esophageal cancer ranks eighth among the most common cancers in the world,and sixth in terms of the worst prognosis.About 400,000 people worldwide die from esophageal cancer each year.According to the pathological type,esophageal cancer can be divided into esophageal squamous cell carcinoma(ESCC)or esophageal adenocarcinoma(EAC).In China,esophageal squamous cell carcinoma is the main type.As a solid tumor,esophageal cancer,like many cancers,experiences a hypoxic tumor microenvironment.As the most important hypoxia-inducing factor in the tumor microenvironment,HIF1? is involved in various aspects of tumor malignant progression.For example,HIF1? can directly or indirectly participate in the Gl/S phase arrest of tumor cells,thereby assisting tumor cells to evade the killing effect of radiochemotherapy,causing radiochemotherapy resistance.In addition,HIF1? also plays an important role in regulating tumor cells to adapt to the hypoxic microenvironment.Based on this,we constructed an intracellular hypoxia model using cobalt chloride in vitro to stabilize the expression of HIF1? for the aim of exploring the mechanism of cell cycle G1/S arrest and how does the circRNA downstream of HIF1? regulates cell energy metabolism to promote the survival of tumor cells.Just like the expression pattern of HIF1?,we found that YES2,and KYSE30 cells undergo cell cycle G1/S phase arrest under condition of treatment of 200 ?M CoCl2 for 18 hours.Based on the sequencing results,and combined with the reported HIF1? targeting genes,we selected ZNF292 as the candidate.ChIP experiments verified that HIF1? could bind the promoter element of ZNF292.ZNF292 can affect the expression of SKP2 that degrades P27 via the ubiquitin proteasome pathway to regulate cell cycle distribution.ChIP and luciferase reporter assay were performed to confirme that ZNF292 was capable of suppressing SKP2 expression at the transcriptional level.Moreover,knocking down ZNF292 can significantly promote the proliferation rates and colony formation ability of esophageal cancer cell lines.We thought this might be related to the involvement of ZNF292 in inhibiting cell cycle progression.The proposal of HIF1?/ZNF292/SKP2/P27 pathway may explain the phenomenon of cell cycle Gl/S phase arrest under hypoxic condition to a certain extent.Circular RNA(circRNA)is a new type of RNA widely found in the transcriptome of eukaryotes.Unlike linear RNA,circRNA's most prominent feature is the formation of covalently closed continuous loops.circRNA has a variety of biological functions.In this study we firstly identified the nuclear derived and mitochondrial localized circPUM1.In addition,circPUMl can bind to UQCRC2,a constitutive protein of mitochondrial complex?,and provide a scaffold for the proteins interaction in mitochondrial complex III.circPUM1 is highly expressed in esophageal cancer cell lines and plays the role of an oncogene.Knocking down circPUM1 will cause a decrease of intracellular oxygen content,decrease in mitochondrial membrane potential,and decrease of cellular oxidative phosphorylation.In addition,we also found that knockdown of circPUM1 can cause pyroptosis in esophageal cancer cells that expresses GSDME protein.The discovery of circPUM1 and an explanation of its mechanism of action will help us understand how esophageal cancer cells adapt to environmental stimuli under hypoxic conditions.At the same time,propose of the circPUM1/Caspase3/GSDME/pyroptosis pathway provides theoretical support for the clinical treatment of esophageal cancer.