Histone Lactylation Drives The Expression Of USP39 To Promote The Progression Of Endometrial Cancer Via De-ubiquitinized PGK1

Objective: This study intends to explore the modification level of histone lactylation in endometrial cancer and its effect on regulating the malignant biological behaviors of endometrial cancer cells;To explore the specific molecular mechanism that histone lactylation regulates the proliferation and migration of endometrial cancer cells by promoting the transcription of ubiquitin-specific protease 39(USP39)and that USP39 regulates the PI3K/AKT/HIF-1α pathway by binding with phosphoglycerate kinase 1(PGK1).Methods: The lactate level in endometrial cancer tissue and cell lines were detected with the lactate detection kit;Immunohistochemistry(IHC)staining and Western Blot(WB)assays were used to verify the modification levels of total lactylation and histone lactylation sites(H3K9,H3K14,H3K18,H3K27,and H3K56)in endometrial cancer tissues and cell lines;Ishikawa cells and KLE cells were treated with histone lactylation inhibitors 2-deoxy-D-glucose(2-DG)and oxamate;Cell counting kit-8(CCK-8)test,clone formation test,Transwell test,and flow cytometry were used to detect cell biological behaviors.The downstream regulatory genes of histone lactylation were predicted by transcriptome sequencing;The chromatin immunoprecipitation(ChIP)experiment was to verify the binding of H3K18 la to USP39 promoter region;Quantitative real-time polymerase chain reaction(qRT-PCR)and Western Blot assay were used to detect the mRNA and protein expression changes of USP39 in cells treated with 2-DG;To construct USP39 small interfering RNA(siRNA)and overexpression plasmid,and to explore the effect of histone lactylation and USP39 on the proliferation and migration of endometrial cancer cells;The effect of histone lactylation and USP39 on the proliferation and metastasis of endometrial cancer in vivo was investigated by subcutaneous tumor transplantation and lung metastasis models in nude mice.Western Blot test was used to detect the changes in the PI3K/AKT/HIF-1α pathway and the protein expression levels of five glycolytic-related proteins(GLUT-1,HK2,LDHA,MCT-1,and MCT-4).The kits were used to detect the content of lactate,glucose,and ATP.Co-immunoprecipitation(Co-IP)experiment and MS/MS mass spectrometry detection were to obtain the protein binding to USP39;Co-IP and Western Blot experiments were to verify the interaction between USP39 and PGK1 in cells,and how USP39 regulates the ubiquitination level of PGK1.Results: The overall lactylation levels in endometrial cancer tissue and cell lines were increased,and the modification level of Kla site H3K18 la was most significant;Inhibiting the level of histone lactylation,the proliferation and migration of endometrial cancer cells were weakened,the cell cycle was blocked,and the rate of apoptosis was increased.Mechanically,the Kla modification of H3K18 la in the USP39 promoter region promoted the transcription of USP39,therefore,participated in the malignant progression of endometrial cancer;Down-regulation or over-expression of USP39 could change the activity of PI3K/AKT/HIF-1α pathway and the level of cell glycolysis;USP39 stabilized PGK1 protein by de-ubiquitination in a proteasome-dependent manner,thus targeting PI3K/AKT/HIF-1α pathway.Conclusions: This study finds that the modification level of histone lactylation in endometrial cancer is significantly higher than that in adjacent normal tissues,and is closely related to the malignant progression of endometrial cancer.Histone lactylation can promote the stability of PGK1 mediated by USP39,activate PI3K/AKT/HIF-1α pathway,accelerate the rate of glycolysis,and form a positive feedback loop,thus promoting the proliferation and metastasis of endometrial cancer.Inhibition of histone lactylation can effectively inhibit the progression of endometrial cancer.