Research On The Regulation Of CUL4B On Gemcitabine-resistance In Lung Cancer Cells

Background:Chemotherapy is one of the most important treatments for patients with lung cancer.In clinic,the most commonly used chemotherapy drugs for lung cancer include gemcitabine(GEM),platinum,etoposide and paclitaxel.GEM exerts its anti-tumor activity through its triphosphate metabolite,which arrests the DNA replication/synthesis and eventually leads to cell apoptosis.GEM is the first-line chemotherapy drug for non-small cell lung cancer.However,patient develops resistance to GEM seriously affects its efficacy.It is of great significance to clarify the mechanism of GEM resistance in lung cancer to improve the survival rate of lung cancer patients.Cullin 4B(CUL4B)is a scaffold protein of the Cullin-Ring ubiquitin E3 ligase complex involved in the regulation of various growth and developmental processes.Studies have found that CUL4B is involved in regulating the proliferation,migration,and invasion of various tumors.Our previous studies showed that in gastric cancer and bladder cancer cells,CUL4B knockdown can increase the sensitivity of tumor cells to chemotherapy drugs,suggesting that CUL4B may be involved in the regulation of tumor chemotherapy resistance.However,it is not clear whether CUL4B regulates GEM resistance in lung cancer.Objective:To clarify the role of CUL4B in regulating GEM resistance in lung cancer cells and explore its related mechanism.Methods:CUL4B knockdown and overexpression cell lines were established in A549 and H1299 lung cancer cells,respectively.MTT,EdU incorporation,clone formation and wound healing assays were used to evaluate the effects of changing CUL4B expression on the growth,proliferation and migration of tumor cells after GEM treatment.Western blot was used to detect the effect of changing CUL4B expression on the expression of various drug-resistant proteins.Microarray detection combined with software analysis was used to determine the candidate miRNAs that may be regulated by CUL4B and participate in the regulation of drug-resistant protein expression.Real-time fluorescent quantitative PCR and ChIP experiments were used to investigate the expression of miRNAs and analyze the regulatory mechanism.Results:(1)The expression level of CUL4B correlated with the sensitivity of lung cancer cells to GEM,Firstly,we analyzed the expression of CUL4B in lung cancer cell lines H1299 and A549.We found that the expression level of CUL4B in H1299 was significantly higher than A549 cells,and the GEM IC50 of H1299 cells was significantly higher than that of A549 cells,By altering the expression level of CUL4B in A549 and H1299 cells,we was found that knockdown of CUL4B significantly increased the sensitivity of A549 cells and H1299 cells to GEM,while overexpression of CUL4B reduced the sensitivity of cells to GEM.(2)CUL4B positively regulates the expression of drug-resistant protein ABCB1.We analyzed the expression of drug-resistant proteins in lung cancer cells with different expression levels of CUL4B,and found that CUL4B positively regulates the expression of ABCB1 protein,which occurrs at the post-transcriptional level.(3)CUL4B regulates ABCB1 expression through miR-7-1-3p.Based on the results of our previous analysis of miRNA expression in CUL4B knocked-down A549 cells and control cells,combined with software prediction,6 candidate miRNAs that may be involved in the regulation of ABCB1 by CUL4B were identified.Real-time fluorescent quantitative PCR analysis showed that CUL4B negatively regulated these candidate miRNAs transcription.Cells were transfected with mimics/inhibitors for these miRNAs,we found that only miR-7-1-3p mimics and miR-7-1-3p inhibitor transfection could change ABCB1 expression level.In addition,transfection of the miR-7-1-3p inhibitor significantly attenuated the increased GEM sensitivity caused by CUL4B knockdown,while transfection of miR-7-1-3p mimics restored the decreased GEM sensitivity caused by CUL4B overexpression.The dual-luciferase reporter gene assays confirmed that miR-7-1-3p regulates the expression of ABCB1.(4)The CUL4B complex,coordinating with PRC2 and HDAC complex,represses miR-7-1-3p transcription.Using qChIP,we found that CUL4B can bind to the-3.0 kb～-2.0 kb region upstream of the transcription start point of miR-7-1-3p.Further analysis demonstrated that knockdown of CUL4B significantly reduced the enrichment of H2AK119ub1,EZH2,H3K27me3,HDAC1,HDAC3 on the promoter of miR-7-1-3p.Conclusions:CUL4B is involved in the regulation of GEM resistance in lung cancer cells.CUL4B cooperates with PRC2 complex and HDAC complex to inhibit the transcription of miR-7-1-3p by affecting histone modification,thereby promoting the expression of drug-resistant protein,ABCB1.