Establishment Of A Radioresistant Lung Adenocarcinoma Cell Line And Preliminary Study On The Mechanisms Of Radiation Resistance

BACKGROUNDLung cancer,one of the most serious life-threatening malignancies,remains the leading cause of cancer-related mortality worldwide.Histologically,lung adenocarcinoma,which accounts for 40%of all cases,is the most common subtype of lung cancer.Radiation therapy is a mainstay treatment for lung cancer.Due to technological advancement and innovation,the accuracy of radiation treatment has been improved.However,radioresistance remains the major reason for the failure of radiotherapy and tumor recurrence.The exact mechanisms regarding radiation resistance have not been clearly elucidated.Tumor cells are affected by different genetic backgrounds,therefore,establishing isogenic radioresistant cell lines and analyzing their biological properties are of vital importance for thoroughly investigating the molecular mechanism underlying radiation resistance.A growing body of research suggests that the key enzymes of the ubiquitin-proteasome system were implicated in radioresistance.Targeting these may contribute to unraveling the mechanism driving radioresistance,as well as providing robust radiosensitization strategies for clinical radiation therapy.OBJECTIVEThe present study aimed to establish isogenic radioresistant cell lines of lung adenocarcinoma.A comprehensive analysis was performed on the morphological and biological properties of the radioresistant and parental cells.Finally,the potential mechanism of USP9X in lung adenocarcinoma and radiation resistance was preliminarily explored.METHODSThe isogenic radioresistant cell line of lung adenocarcinoma A549R was established based on parental cell line A549 by employing fractionated dose-escalation irradiation approach,and the radiosensitivity of cells were measured by clonogenic survival assay.The two distinct cell lines were then morphometrically analyzed by optical microscopy,scanning electron microscopy,and transmission electron microscopy.The CCK8 assay,cell cycle experiments,and transwell migration assay were utilized to evaluate the biological properties including cell proliferation,cell cycle distribution,and cell migration ability in the two groups of cells.The mRNA and protein expression levels of USP9X in lung adenocarcinoma,as well as the prognostic value,were analyzed by bioinformatics databases.Western blot assay was performed to detect the protein expression of USP9X in isogenic radioresistant and parental cell lines,as well as homologous recombination repair proteins in two groups of cells in which USP9X was knocked down by specifically designed siRNA.RESULTSAn isogenic radioresistant cell line of lung adenocarcinoma A549R was successfully established.A549R showed an increased survival fraction at different ionizing radiation doses compared with A549(P<0.05),which illustrated that A549R cells show increased resistance to X-ray.Morphologically,A549R cells were irregular with abundant organelles,mitochondria aggregation,and marked nuclear atypia.In biological properties,the cell proliferation of the A549R group was significantly slowed down(P<0.05)compared with parental A549 cells,and the cell cycle was redistributed with increased percentages of cells in the G1 phase(P<0.01)and decreased percentages of cells in the G2/M phase(P<0.01),while no significance was found in the percentages of cells in the S phase.The enhanced migration ability was also determined in A549R cells(P<0.01).The mRNA and protein expression levels of USP9X were upregulated in lung adenocarcinoma compared with normal tissues(mRNA P<0.001;protein P<0.001).High USP9X expression was significantly related to poor overall survival in lung adenocarcinoma patients compared with low expression ones(P=0.046).Protein expression of USP9X was elevated in A549R cells,and expression levels of homologous recombination repair proteins(BRCA1,RAD51)were markedly reduced in USP9X knock-down A549R and A549 cells.CONCLUSIONSFractionated dose-escalation irradiation is a viable approach to establishing an isogenic radioresistant cell line.USP9X was overexpressed in lung adenocarcinoma and correlated with prognosis,it may affect the radiosensitivity of lung adenocarcinoma by regulating the homologous recombination pathway.Thus,USP9X is an extremely promising biomarker and a potential target for radiosensitization in the clinical treatment of lung adenocarcinoma.