The Role And Mechanism Of Long-chain Acyl-CoA Synthetase 1 In Lung Cancer Resistance To Radiotherapy

Objective:For the past few years,the incidence and mortality of lung cancer in our country are increasing.However,about 40%-50%of lung cancer patients will have uncontrolled or recurrent local lesions after conventional dose radiotherapy,and radiotherapy resistance is the main factor leading to local treatment failure.Therefore,it is of great clinical value to explore the occurrence reason of radiotherapy resistance and find new treatment strategies.Long-chain acyl-Co A synthetase 1(ACSL1)is an isoenzyme of the long-chain acyl-Co A synthetase family,which promotes lipid synthesis and fatty acid oxidation.Previous studies have found that ACSL1 is closely related to the development of various types of tumors,but the role of ACSL1 in radioresistance of non-small cell lung cancer(NSCLC)is still unclear.This study is calculated to explore the function and mechanism of ACSL1 in radioresistance of NSCLC and provide new ideas for lung cancer treatment.Methods:(1)The effect of ACSL1 on the growth and proliferation of lung cancer cells was detected by clonogenesis assay,Ed U assay and CCK8 assay.(2)The effect of ACSL1 in radiotherapy sensitivity of lung cancer cells was determined by neutral comet assay,γH2AX focus formation assay and cell clonal formation assay.(3)The effect of ACSL1 knockdown on the growth of the transplanted tumor and the effect on the transplanted tumor after radiotherapy were investigated in the subcutaneous transplanted tumor model of nude mice.(4)Construct lung cancer cell lines resistant to radiation and detect the expression of ACSL1.(5)Oil red O staining and BODIPY 493/503 staining were used to detect the number and size of lipid droplets in cells.(6)Western blot assay was used to detect the expression of key enzymes in lipid metabolism pathway after ACSL1 knockdown.(7)Lipidomic detection of lung cancer cells was performed by Liquid Chromatograph Mass Spectrometer(LC-MS).(8)ATP assay,Seahorse assay,mitochondrial activity staining and mitochondrial DNA copy number assay were used to determine the effect of ACSL1 depletion on mitochondrial function.(9)The intracellular NAD+/NADH levels were detected by enzyme label,and intracellular ROS levels were detected by flow cytometry.(10)RNA sequencing technology to explore the downstream mechanism of ACSL1.(11)The protein-protein interaction was verified by co-immunoprecipitation.(12)The expression of downstream related proteins after down-regulated ACSL1 was detected by immunohistochemical staining.Results:(1)The knockdown of ACSL1 by siRNA and sgRNA inhibited the growth of lung cancer cells in vitro and vivo.(2)Increased expression of ACSL1 in radio-resistant cell lines.(3)Comet tailing assay,γH2AX focus-forming assay and post-radiotherapy clonogenesis assay confirmed that knockdown of ACSL1 could increase the radiosensitivity of lung cancer cells.(4)knockdown of ACSL1 can increase the radiosensitivity of lung cancer grafts.(5)Oil red O staining and BODIPY 493/503 assay showed that the accumulation of lipid droplets in lung cancer cells decreased after ACSL1 knockdown.(6)Lipidomics analysis showed that down-regulating ACSL1 could reduce cardiolipin levels in lung cancer cells after radiotherapy.(7)Western blot assay confirmed that the expression of Cardiolipin synthase 1(CRLS1)was down-regulated after ACSL1 knockdown.(8)Knockdown of ACLS1 expression can reduce the level of intracellular ATP induced by radiotherapy,and this effect is partly dependent on CRLS1.(9)Seahorse mitochondrial stress assay,mitochondrial activity staining assay and mitochondrial DNA assay confirmed that ACSL1knockdown could inhibit mitochondrial function and reduce mitochondrial ATP production after radiotherapy.(10)After knockdown of ACSL1,the ratio of NAD~+/NADH increased,ROS increased,and the level of oxidative phosphorylation decreased.(11)The results of RNA sequencing and western blot showed that ACSL1 induced the expression of CRLS1by activating PI3K/mTOR signaling pathway.(12)Western blot assay showed that the intracellular mTOR signaling pathway was activated after exogenous palmitic acid supplementation.(13)Co-IP assay demonstrated the interaction between intracellular expression of ACSL1 and Phosphoinositide-3 kinase(PI3K).(14)Immunohistochemical staining confirmed that the expression of p-mTOR and CRLS1 was down-regulated in lung cancer tissues after ACSL1 knockdown.Conclusions:ACSL1 is highly expressed in radioresistant lung cancer cells and plays roles in promoting the growth and multiplication of lung cancer cells in vitro and vivo.In mechanism,ACSL1 can activate mTOR signaling pathway by promoting lipid synthesis and interaction with PI3K,upregulate the expression of the downstream gene CRLS1,and then increase the production of mitochondrial ATP to provide energy for the repair of DNA damage after radiotherapy,and ultimately lead to the radiotherapy resistance in lung cancer.This study suggests that ACSL1 may be a new potential target for radiotherapy resistance in lung cancer.