Biological Role And Mechanism Of Exosomal CircKIF20B Regulating Gefitinib Resistance In Non-small Cell Lung Cancer

Background:Lung cancer is one of the malignant tumors with high morbidity and mortality in our country and even the world.As a first-line treatment drug for non-small cell lung cancer(NSCLC)with EGFR-mutated,gefitinib improved the overall survival of patients.However,gefitinib-acquired resistance to NSCLC is one of the critical reasons that severely limits the prognosis of patients.Both exosomes and circular RNA play an essential role in the occurrence,development,and treatment of tumors.However,there are few reports on the involvement of exosomal circular RNA in the mechanism of acquired resistance to gefitinib.Objective:1.To explore the differential expression profile of circ RNA in exosomes of gefitinib-sensitive cells and gefitinib-resistant cells from NSCLC by high-throughput sequencing.2.To explore the expression of circ KIF20 B in serum exosomes and NSCLC tissues of gefitinib-resistant patients.To analyze the correlation of circ KIF20 B in clinical data of patients with NSCLC.3.To study the biological functions of circ KIF20 B in NSCLC cell lines,including gefitinib resistance,cell proliferation,cell cycle,and apoptosis.4.To investigate the function of circ KIF20B/mi R-615-3p/MEF2 A signaling axis.5.To explore the research of circ KIF20 B based on exosome loading system for the treatment of gefitinib resistance.Methods:1.We extracted exosomes from relevant cells and serum samples by ultra-height gradient centrifugation and exosome extraction kit and verified exosomes using an electron microscope,particle size analyzer,and Western blot.2.We researched the circ RNA differential expression profile in the exosomes of gefitinib-resistant cells and gefitinib-sensitive cells of NSCLC by high-throughput sequencing.We screened out the circ KIF20 B and performed characterization and identification.3.The expression level of circ KIF20 B in serum exosomes and NSCLC tissues was detected by q RT-PCR,and the clinical correlation of circ KIF20 B was analyzed in combination with clinical baseline data.4.Using PC9,PC9 GR,HCC827 and HCC827 GR cell lines as the target cells,we constructed circ KIF20 B knockdown and overexpression stably transfected cell lines and detected cell cycle and apoptosis-related proteins by Western blot;CCK-8,Edu cell proliferation assay,flow cytometry analysis,and other methods were used to verify the biological effects of circ KIF20 B on resistance to gefitinib,cell proliferation,cell cycle progression,and apoptosis levels in NSCLC.5.To verify the biological function of circ KIF20 B in NSCLC by xenograft tumor model in vivo.6.We researched the interaction of circ KIF20 B and mi R-615-3p and the interaction of mi R-615-3p and MEF2 A by bioinformatics analysis,RNA pull-down,RNA immunoprecipitation,luciferase reporter gene,and fluorescence in situ hybridization.We detected the expression levels of mi R-615-3p and MEF2 A in serum exosomes and NSCLC tissues of gefitinib-resistant patients by q RT-PCR;we performed overexpression and knockdown experiments to verify the effect of mi R-615-3p and MEF2 A effect of gefitinib IC50 values.7.We up-regulated and down-regulated the expression of circ KIF20 B,mi R-615-3p,and MEF2 A in cell lines and constructed the rescue experiment groups.We detected the biological role of the circ KIF20B/mi R-615-3p/MEF2 A axis in NSCLC cell lines through CCK-8,Edu cell proliferation experiment,flow cytometry,and Western blot.8.We investigated the regulation of circ KIF20B/mi R-615-3p/MEF2 A axis on the oxidative metabolism of NSCLC cells by ATP synthesis detection,mitochondrial membrane potential detection,and oxygen consumption rate measurement(OCR).9.We studied the biological regulation of exosomal circ KIF20 B on recipient cells by exosome co-culture.Results:1.The serum exosomes and cell supernatant exosomes were extracted and identified successfully.2.376 significantly differently expressed exosome-derived circ RNAs were screened by sequencing,and circ KIF20 B was one of the significantly low-expressed molecules.Characterization experiments demonstrated the circular structure of circ KIF20 B,the enrichment of circ KIF20 B in the cytoplasm,and resistance to the Rnase R and actinomycin D.3.Circ KIF20 B was low expressed in serum exosomes of gefitinib-resistant patients(n= 24)and tumor tissues of NSCLC patients(n = 85).Circ KIF20 B expression was negatively correlated with tumor size,TNM stage,and degree of differentiation.4.Knockdown of circ KIF20 B up-regulated CDK4 protein expression,down-regulated BAX protein expression,increased gefitinib IC50 value,accelerated cell cycle progression,enhanced cell proliferation,and inhibited cell apoptosis.Overexpression of circ KIF20 B down-regulated CDK4 protein expression,up-regulated the expression of BAX protein,reduced the IC50 value of gefitinib,blocked the process of the cell cycle,weakened the ability of cell proliferation,and promoted cell apoptosis.5.Knockdown of circ KIF20 B promoted the development of NSCLC while overexpression of circ KIF20 B inhibited the tumor progression in vivo.6.We confirmed the interaction between circ KIF20 B and mi R-615-3p,mi R-615-3p and MEF2 A,and demonstrated the endogenous existence of circ KIF20B/mi R-615-3p/MEF2 A signaling axis in NSCLC.Mi R-615-3p has highly expressed in serum exosomes of gefitinib-resistant patients and tumor tissues of NSCLC patients;MEF2A has lowly expressed in serum exosomes of gefitinib-resistant patients and tumor tissues of NSCLC patients.Overexpression of mi R-615-3p or knockdown of MEF2 A increased the IC50 value of gefitinib,and knockdown of mi R-615-3p or overexpression of MEF2 A decreased the IC50 value of gefitinib.7.Knockdown of circ KIF20 B enhanced the activity of mi R-615-3p,inhibited the expression of MEF2 A,enhanced cell viability,increased the IC50 value of gefitinib,accelerated cell cycle progression,enhanced cell proliferation,and inhibited cell apoptosis;overexpression of circ KIF20 B inhibited the activity of mi R-615-3p,enhanced expression of MEF2 A,inhibited cell viability,reduced the IC50 value of gefitinib,blocked cell cycle progression,weakened cell proliferation,and promoted cell apoptosis.8.Knockdown of circ KIF20 B enhanced activity of mi R-615-3p,inhibited MEF2 A expression,increased ATP,enhanced mitochondrial function and oxidative metabolism;overexpression of circ KIF20 B inhibited activity of mi R-615-3p,enhanced MEF2 A expression,reduced ATP,weakened mitochondrial function and oxidative metabolism.9.Exosomes loaded circ KIF20 B targeted recipient cells,reversed gefitinib resistance of recipient cells,weakened cell proliferation,and blocked cell cycle and oxidative metabolism.Conclusion:This study reveals a novel mechanism by which the circ KIF20B/mi R-615-3p/MEF2 A signaling axis is involved in gefitinib resistance and tumor progression in NSCLC.Exosomal circ KIF20 B has the potential to become an important body fluid biopsy marker and therapeutic target in the early screening,detection and late treatment of acquired resistance to gefitinib in NSCLC.