Research On The Effect Of Histone Demethylase KDM4B On Tumor Cell Signaling Pathways And Its Antibody Preparation

Background:Histone demethylase mutations or abnormal expressions play a pivotal role in the occurrence and development of tumors.Among them,KDM4 family is highly expressed in various types of cancers.As a member of JMJD2/KDM4 demethylase family,KDM4B can specifially demethylate H3K9me3/2,H3K36me3/2,H4K20me3 and H1.4K26me3.The expression of KDM4B is affected by numerous cellular stimulus,such as DNA damage,hormones and hypoxia.Previous studies have shown that KDM4B is overexpressed in breast cancer,lung cancer,colon cancer and other tumors.But how KDM4B influences the signaling pathway in tumor progression still remains elusive.Objectives:To systematically explore the effect of histone demethylase KDM4B on tumor cell signaling pathways,and explore the molecular mechanisms of actions preliminarily.Methods:Two stable transfected cell lines overexpressing and knocking down KDM4B were constructed in breast cancer cells MDA-MB-231 and MDA-MB-468.The expression level of KDM4B was detected by Western blot to identify whether the transfected cell lines were constructed successfully.Stable cell lines were treated with cytokine EGF to explore the signaling pathway of cancer cells.RNA-seq transcriptome analysis was carried out on KDM4B knock-down MDA-MB-468 stable transfected cell line,and enriched metabolic pathways and differentially expressed proteins were explored.Tumor cells were treated with cytokine TGF-β,and changes in proteins and nucleic acids level of downstream genes were detected by RT-PCR and Western blot.Serum starvation and rapamycin,an antagonist of mTOR pathway were used to induce autophagy.Besides,chloroquine diphosphate was applied to inhibit autophagy.The effect of autophagyon the KDM4B expression was detected when using agonist and inhibitor of autophagy respectively,the effect of alteration s of autophagy on the expression of KDM4B were also detected by Western blot.In addition,rabbit anti-KDM4B was also performed.Results:MDA-MB-231 and MDA-MB-468 stable transfected cell lines were successfully constructed.No significant effect had been revealed on the signaling pathways in breast cancers when knocking down KDM4B in non-stimulated conditions.Gene expression analysis in transcriptome sequencing RNA-seq data indicated that KDM4B was able to regulate many genes in cells.Functional enrichment analysis had integrated many signaling pathways,such as purine metabolism,protein digestion and absorption pathway,PPAR pathway,complement and coagulation cascade,extracellular matrix receptor interaction etc.GO category showed that ERK1/2 signaling pathway was upregulated at the supression of expression of KDM4B.Many autophagy related genes were enriched in differential expression of analysis in transcriptome sequnencing RNA-seq data,including ATG16L,TP53INP2,RNF152,S100A8,LEPR,TAB3 etc.Autophagy was activated when KDM4B was overexpressed in breast cancer cells,and the repression of expression of KDM4B curbed autophagy.In Hela,L02 and MDA-MB-231 cells as well as MDA-MB-231 stable transfected cell line,KDM4B was enhanced when autophagy was activated by autophagy agonist,while the expression of KDM4B was inhibited at the repression of autophagy by inhibitor of autophagy.The anti-KDM4B antibody confirmed by Western blot worked well and could be used for subsequent research.Conclusion:The histone demethylase KDM4B promotes tumor cells autophagy.When autophagy is suppressed,the expression of KDM4B is also reduced.KDM4B and cell autophagy might promote each other.Our study might offer a new clue for epigenetic modification of autophagy.