Objective To investigate the effects and mechanisms of LINC00657 on oxidative glu-cose deprivation(OGD)-induced injury in mouse hippocampal neurons.Methods Mouse hippocam-pal neuron cell line HT22 was given OGD treatment to establish an injury model,with normally cul-tured HT22 cells as controls.The si-NC,si-LINC00657,microRNA(miR)-NC,and miR-224-3p mimics were transfected into HT22 cells,followed by OGD treatment.Co-transfection of si-LINC00657 and anti-miR-NC,or co-transfection of si-LINC00657 and anti-miR-224-3p,was performed in HT22 cells before OGD treatment.Real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect the relative expression levels of LINC00657 and miR-224-3p.CCK-8 assay and flow cytometry were used to detect cell viability and apoptosis rate,respectively.Kits were used to detect the activities of lactate dehydrogenase(LDH),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and the level of malondialdehyde(MDA).Dual-luciferase reporter gene assay was used to detect the effect of miR-224-3p overexpression on the luciferase activity of wild-type LINC00657 vector(WT-LINC00657)and mutant LINC00657 vector(MUT-LINC00657).Results Compared with controls,the expression of LINC00657 was upregulated and the expression of miR-224-3p was downregulated in OGD-induced HT22 cells(P＜0.05).Compared with trans-fection of si-NC or miR-NC,transfection of si-LINC00657 or miR-224-3p mimics resulted in in-creased cell viability,SOD activity,and GSH-Px activity,as well as decreased apoptosis rate,LDH activity,and MDA level(P＜0.05).Overexpression of miR-224-3p reduced the luciferase activity of WT-LINC00657(P＜0.05).Compared with cells co-transfected with si-LINC00657 and anti-miR-NC,cells co-transfected with si-LINC00657 and anti-miR-224-3p showed decreased cell viabili-ty,increased apoptosis rate,increased LDH activity and MDA level,and decreased SOD and GSH-Px activities(P＜0.05).Conclusion Interference with LINC00657 can promote cell proliferation,inhibit apoptosis and oxidative stress response by upregulating miR-224-3p,thereby alleviating OGD-induced injury in mouse hippocampal neurons.

Objective To investigate the effects and mechanisms of LINC00657 on oxidative glu-cose deprivation(OGD)-induced injury in mouse hippocampal neurons.Methods Mouse hippocam-pal neuron cell line HT22 was given OGD treatment to establish an injury model,with normally cul-tured HT22 cells as controls.The si-NC,si-LINC00657,microRNA(miR)-NC,and miR-224-3p mimics were transfected into HT22 cells,followed by OGD treatment.Co-transfection of si-LINC00657 and anti-miR-NC,or co-transfection of si-LINC00657 and anti-miR-224-3p,was performed in HT22 cells before OGD treatment.Real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect the relative expression levels of LINC00657 and miR-224-3p.CCK-8 assay and flow cytometry were used to detect cell viability and apoptosis rate,respectively.Kits were used to detect the activities of lactate dehydrogenase(LDH),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and the level of malondialdehyde(MDA).Dual-luciferase reporter gene assay was used to detect the effect of miR-224-3p overexpression on the luciferase activity of wild-type LINC00657 vector(WT-LINC00657)and mutant LINC00657 vector(MUT-LINC00657).Results Compared with controls,the expression of LINC00657 was upregulated and the expression of miR-224-3p was downregulated in OGD-induced HT22 cells(P＜0.05).Compared with trans-fection of si-NC or miR-NC,transfection of si-LINC00657 or miR-224-3p mimics resulted in in-creased cell viability,SOD activity,and GSH-Px activity,as well as decreased apoptosis rate,LDH activity,and MDA level(P＜0.05).Overexpression of miR-224-3p reduced the luciferase activity of WT-LINC00657(P＜0.05).Compared with cells co-transfected with si-LINC00657 and anti-miR-NC,cells co-transfected with si-LINC00657 and anti-miR-224-3p showed decreased cell viabili-ty,increased apoptosis rate,increased LDH activity and MDA level,and decreased SOD and GSH-Px activities(P＜0.05).Conclusion Interference with LINC00657 can promote cell proliferation,inhibit apoptosis and oxidative stress response by upregulating miR-224-3p,thereby alleviating OGD-induced injury in mouse hippocampal neurons.

Resistance of tumor cells is a complex biological process involving multiple mechanisms and factors, in which anti-apoptosis is the most important cause of drug resistance. Previous studies have shown that the DNA binding activity of Runt related transcription factor 3 (RUNX3) increased prominently in Herceptin resistant gastric cancer cells (NCI N87R) while the relevance of which to drug resistance has not yet been confirmed. In this study, we employed CRISPR/Cas9 to establish RUNX3 knock-out cell line (△RUNX3/NCI N87R) to investigate the functions of RUNX3 in Herceptin resistance of NCI N87R cells and its potential mechanisms. We investigated proteomics profiling of △RUNX3/NCI N87R cells based on label free quantitative proteomics. Differentially expressed proteins were screened out according to fold change and significance level between △RUNX3/NCI N87R and NCI N87R cells. Pathway enrichment analysis was done using GeneAnalytics database, and gene ontology analysis was conducted by DAVID Bioinformatics Resources database. Protein-protein interaction networks were constructed based on STRING database. The results showed that △RUNX3/NCI N87R cells increased the sensitivity to Herceptin. Proteomic data demonstrated that the expression of 577 genes changed significantly in △RUNX3/NCI N87R cells, among which 191 genes were up-regulated while 386 ones down-regulated comparing with NCI N87R cells. Pathway analysis showed that autophagy, cell cycle, apoptosis, mitochondrial fatty acid β oxidation, neurogenic locus notch homolog protein 1 (NOTCH1), mammalian target of rapamycin (mTOR), Hedgehog and DNA damage response pathways exhibited notable changes based on pathway enrichment ratio and significance level (P < 0.05). These results indicated that RUNX3 knock-out altered multiple signaling pathways of NCI N87R cells. Western blotting manifested that the expression of autophagy regulatory molecules autophagy-related protein (ATG) 13, 7 and BECN1 increased remarkably while cell cycle molecules serine/threonine-protein kinase Chk2 (CHEK2) and apoptosis regulator Bcl-2 (BCL2) decreased prominently in △RUNX3/NCI N87R cells. The p-AKT expression decreased significantly in △RUNX3/NCI N87R cells compared with NCI N87R cells (P < 0.01) and was suppressed by Herceptin. These results indicated that RUNX3 knock-out altered cell cycle, increased inhibition to p-AKT by Herceptin, promoted autophagy and induced cell apoptosis of NCI N87R cells. These results suggested that RUNX3 may be a potential therapeutic target for reversing or reducing Herceptin resistance in gastric cancer cells.