Objective To investigate the effects and molecular mechanism of non-coding RNA-activated DNA damage(NORAD)induced autophagy on oxaliplatin resistance in adenocarcinoma of esophagogastric junction(AEG).Methods Four pairs of surgical samples of AEG and para-carcinoma normal tissues from patients with advance AEG treated in Anyang Tumor Hospital from January to June 2023 were collected.The expression of NORAD in AEG and para-carcinoma tissues was analyzed by long non-coding RNA microarray chip.The primary tumor cell line of AEG(PDC)was derived from fresh AEG tissues.Oxaliplatin-resistant cell lines of PDC and AEG cell line OE19(PDC-R and OE19-R)were established.NORAD expression knockdown PDC-R and OE19 cell lines(shNORAD PDC-R and shNORAD OE19-R)were prepared by transfection.The target of NORAD,the correlation and interaction between microRNA-433-3p(miR-433-3p)and NORAD were predicted using Starbase v3.0 and DIANA-lncBase v3.0.PDC,PDC-R,OE19 and OE19-R cells were co-transfected with miR-144-3p and wild-type NORAD(NORAD-WT)or mutant NORAD(NORAD-Mut)plasmid,respectively.Dual-luciferase reporter assay was used to verify the correlation between NORAD and miR-433-3p.The expression levels of NORAD and miR-433-3p in normal gastric mucosal cell line GES-1 and AEG cell lines PDC,PDC-R,shNORAD PDC-R,OE19,OE19-R and shNORAD OE19-R were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR).The expression of p62 protein and microtubule-associated protein 1 light chain 3B-Ⅱ(LC3B-Ⅱ)was determined by Western blotting.The half inhibitory concentration(IC50)of PDC,PDC-R,shNORAD PDC-R,OE19,OE19-R and shNORAD OE19-R cells was measured by cell counting kit-8(CCK-8)assay.Independent sample t-test was used for statistical analysis.Results The results of microarray analysis showed that NORAD was significantly up-regulated in AEG compared with that in para-carcinoma tissues(fold change ≥2.0,P＜0.05).Bioinformatics studies found that miR-433-3p was the potential target of NORAD.The results of dual-luciferase reporter assay indicated that the relative luciferase activity of the NORAD-WT group was lower than that of NORAD-Mut group in PDC and PDC-R cells(0.441±0.104 vs.0.928±0.204,0.449±0.112 vs.0.947±0.201),and the differences were statistically significant(t=-14.74 and-14.94,both P＜0.001).The results of dual-luciferase reporter assay of OE19 and OE19-R cell lines were the same as those of PDC cell lines.The results of qRT-PCR showed that the expression of NORAD in GES-1 cells(1.016±0.213)was lower than that of PDC cells(2.194±0.322)and PDC-R cells(4.040±0.336),and the differences were statistically significant(t=-14.94 and-37.21,both P＜0.001).Furthermore,the expression level of NORAD in PDC was also found to be lower than that in PDC-R cells,and the difference was statistically significant(t=-19.43,P＜0.001).Additionally,shNORAD PDC-R cells exhibited lower expression level of NORAD(0.290±0.165)compared with PDC-R cells,and the difference was statistically significant(t=-49.05,P＜0.001).The expression level of miR-433-3p in GES-1 cells(1.017±0.248)was higher than that in PDC cells(0.470±0.156)and PDC-R cells(0.203±0.045),and the differences were statistically significant(t=9.15 and 15.85,both P＜0.001).Moreover,the expression level of miR-433-3p was found to be higher in PDC cells compared with PDC-R cells,and the difference was statistically significant(t=8.11,P＜0.001).Additionally,the expression level of miR-433-3p in shNORAD PDC-R cells(0.699±0.256)was also higher than that in PDC-R cells(t=9.37,P＜0.001).The results of Western blotting showed that the expression of LC3B-Ⅱ in PDC-R was higher than that in PDC cells(0.426±0.060 vs.0.212±0.041),the expression of LC3B-Ⅱ in shNORAD PDC-R cells(0.155±0.029)was lower than that in PDC cells,and the differences were statistically significant(t=8.70 and-79.45,both P＜0.001).However the expression of p62 protein in each cell line showed an opposite trend,with a lower relative expression in PDC-R than PDC(0.205±0.031 vs.0.311±0.400),and the expression in shNORAD PDC-R(0.504±0.084)was higher than that in PDC,and the differences were statistically significant(t=-31.19 and 62.80,both P＜0.001).The expression patterns of NORAD,miR-433-3p,LC3B-Ⅱ and p62 proteins in OE19,OE19-R and shNORAD OE19-R cells were similar to those in PDC.The results of CCK-8 assessment of target cell viability showed that the IC50 values of PDC,PDC-R and shNORAD PDC-R cell lines were 14.28,22.27 and 2.51 µg/mL,respectively;and the IC50 values of OE19,OE19-R and shNORAD PDC-R cell lines were 3.95,8.12 and 1.89 μg/mL,respectively.Conclusions NORAD is highly expressed in AEG tissues and cells.NORAD is overexpressed in oxaliplatin-resistant cell lines and increase the autophagy activity of cells.After NORAD is knockdown,autophagy activity is inhibited and the sensitivity of AEG cells to oxaliplatin is significantly enhanced.
Adenocarcinoma of esophagogastric junctionLong non-coding RNANORADMicroRNA-433-3pOxaliplatinDrug resistanceAutophagy activity
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