Study On The Mechanism Of Target KDM5B Enhancing Chemosensitivity Of Lung Cancer Cells To Doxrubicin

Post-translational modifications of histones plays an important role in the formation of transcriptional activation or inhibition modes,which including methylation,acetylation,ubiquitination,phosphorylation,etc..Histone methylation can occur on arginine and lysine residues,which is accomplished under the action of histone methyl transferase（HMT）.Its specific demethylases can be divided into FAD-dependent monoamine oxidase LSD1 and JMJD protein family containing Jmj C domain.JMJD family proteins rely on Fe²⁺andα-ketoglutarate（2-OG）to catalyze demethylation modification,including KDM2A/B,KDM3A/B,KDM4A-E,KDM5A-D（or JARID1A-D）,KDM6A-B and many other sub-families,selectively catalyze different substrates.1.Down-regulation of KDM5B inhibits lung cancer cell proliferation in vivo and in vitroWestern Blot detection showed that among several common lung cancer cell lines,KDM5B has the highest expression in H1299 and PC-9 cell lines.Further lentivirus transfection was used to establish H1299 and PC-9 KDM5B knockdown（KDM5B KD）cell lines.The clone formation ability of the KDM5B-KD groups were significantly reduced,and the growth curve was consistent with above result.KDM5B selective inhibitor CPI-455 was used to act on H1299 and PC-9 and the cell proliferation ability was also significantly inhibited.At the same time,an immunodeficiency mouse xenograft tumor model was established and found that the tumor-forming ability of KDM5B-KD cells was significantly inhibited.These findings prove that KDM5B plays an important role in lung cancer cell proliferation.2.Down-regulation of KDM5B promotes the chemotherapy sensitivity of DOX to lung cancer cellsAfter pretreating cells with CPI-455 and then acted with DOX,the cytotoxicity of DOX was significantly increased.Under the same DOX concentration,cell growth inhibition was observably higher than that of the DOX alone group,the apoptotic rate of cells generally increased by 10%and promoted the nuclear damage.In vivo experiments found that knockdown or inhibition of KDM5B with CPI-455 could inhibit tumor growth,increase chemotherapy sensitivity of DOX to tumors and promote tumor cell apoptosis.3.Inhibition of KDM5B increases DNA damage and intracellular drug accumulation in lung cancer cellsComet experiment analysis results showed that the cells exhibited tailing phenomenon after DOX treatment,while the DNA damage was significantly aggravated after the inhibitor CPI-455 treatment.Under the microscope,the single cell tail was more obvious,the DNA content of the head was reduced,and the tail became longer and brighter.Then the KDM5B-KD cell line was detected,and it was found that the DNA damage was inversely proportional to the content of KDM5B.The lower the content of KDM5B,the more serious the DNA damaged.In addition,the ABC family protein as a drug efflux pump is one of the main causes of drug resistance,which can efflux drugs or molecules outside the cell.Quantitatively detect the intracellular drug accumulation and found that the drug concentration of the cell group pretreated with CPI-455 was much higher than that of without-pretreatment group,and the expression levels of ABC family proteins ABCC1 and ABCG2 also decreased.The KDM5B-KD cell line has the same phenomenon.4.Inhibition of KDM5B increased drug sensitivity through up-regulating hsa-mi RNA-105-5pInhibition of KDM5B could down-regulate ABCC1 and ABCG2 simultaneously.Increasing evidence shows that the efflux pump of the ABC transporter family is regulated by mi RNA-mediated gene regulation.Through database analysis,it is found that there are 12 mi RNAs that co-regulate ABCC1 and ABCG2.RT-PCR result indicated that knockdown of KDM5B could obviously up-regulate mi RNA-105-5p.Further treated cells with mi RNA-105-5p mimics and inhibitors,and detected the changes of ABCC1 and ABCG2 from the protein level and transcriptome level,respectively.It was discovered that overexpression of mi RNA-105-5p can markedly down-regulate the expression of ABCC1 and ABCG2,while inhibition of mi RNA-105-5p promoted the expression of ABCC1 and ABCG2.In vivo and in vitro researches in this study showed that inhibition of KDM5B could suppress the expression of ABCC1 and ABCG2 through upregulating hsa-mi RNA-105-5p,increasing intracellular drug accumulation and aggravating DNA damage,thus promoting the chemotherapy sensitivity of DOX to lung cancer cells.