| Background and objectiveCervical cancer is regarded as one of the most common malignant tumors in women.For the past few years,the incidence of cervical cancer has been increasing and presenting a trend of younger age.Some patients especially who suffered from advanced cervical cancer are calling for a better clinical prognosis.Aerobic glycolysis is crucial metabolic characteristics of tumor cells,where pyruvate dehydrogenase complex(PDHc)links glycolysis process with tricarboxylic acid cycle.PDHA1 is the initial enzyme catalyzed by PDHc,regulating the metabolic flow of glycolysis pathway.According to previous study,PDHA1 and AP-2α are closely related to the occurrence and development of various kinds of tumors.In ours preliminary work,we predicted that AP-2α binds to the promoter sequence of PDHA1.For further investigation,this study will explore the effects of PDHA1 on cercical cancer from several angles of tumor proliferation,invasion and aerobic glycolysis in vitro and in vivo and to evaluate the relationship and interaction between AP-2α and PDHA1.We hope to lay a theoretical foundation for the individual treatment and biological targeted treatment of advanced cervical cancer.MethodsPart 1:(1)Detected PDHA1 and transcription factor NRF1 level in cervical cancer tissues by qRT-PCR and WB;(2)detected PDHA1 and transcription factor NRF1 level in cervical cancer cells lines by qRT-PCR,WB and IF.Part 2:(1)Established overexpressing PDHA1 lentivirus plasmid and transfected plasmid into cervical cancer cell lines.(2)Investigated the effects of overexpression of PDHA1 in cervical cancer cells in vitro:① proliferation and activity of cells accessed by CC-K8 and EdU;② cell invasion and migration accessed by Transwell and Wound healing assay;③ OCR and ROS level in cells;④cells apoptosis accessed by TUNEL staining and flow cytometry.(3)Injected overexpressing PDHA1 cervical cancer cells into mice to construct xenograft mouse model.(4)Investigated overexpression PDHA1 function on cervical cancer mice in vivo:① calculated tumor volume and weight;② detected the expression of PDHA1 in tumor tissues by qRT-PCR and WB;③ detected the proliferation and apoptosis of tumor tissues by immunohistochemical analysis.Part 3:(1)Analysed the relationship and interaction between AP-2α and PDHA1:① predicted the binding sites of PDHA1 transcription factor using UCSC database;② detected the expression of AP-2α in cervical cancer tissues by qRT-PCR and WB;③ detected the expression of AP-2α in cervical cancer cells by qRT-PCR,WB and IF;④verified the binding effect of AP-2α on PDHA1 promoter by ChIP-qPCR and EMSA;(2)Established silenced AP-2α and silenced AP-2α+PDHA1 plasmids and transfected them into cervical cancer cells;(3)Detected the function of AP-2α and PDHA1 on cervical cancer cells in vitro:① activity and proliferation accessed by CC-K8 and EdU assays;② cells invasion and migration accessed by Transwell and Wound healing assay;③ OCR and ROS level in cells;④ apoptosis accessed by flow cytometry and TUNEL staining.ResultsPart 1:(1)Compared to normal tissues and cells,PDHA1 mRNA and protein were significantly low in expression of cervical cancer.(2)The expression of PDHA1 transcription factor NRF1 significantly decreased in cervical cancer tissues and cells.Part 2:(1)Overexpression PDHA1 inhibited proliferation,invasion and migration of cervical cancer cells,increased cancer cells apoptosis,and promoted OCR as well as ROS level.(2)Overexpression PDHA1 inhibited tumor growth in cervical cancer cell transplantation mice and increased cancer cells apoptosis.Part 3:(1)AP-2α binds to PDHA1,interacted with each other.(2)AP-2α was in high level in cervical cancer tissues and cells,negatively correlated with PDHA1 expression.(3)AP-2α promote proliferation,migration,invasion,ROS level,OCR level and decrease the apoptosis of cancer cells by suppressing PDHA1.ConclusionAP-2α negatively associated with PDHA1 to regulated cervical cancer proliferation,migration,invasion,apoptosis and aerobic glycolysis. |
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