lncRNA GAS6-AS1调节miR-326/E2F1轴对神经母细胞瘤细胞增殖、迁移和侵袭影响的实验研究OA

Objective To investigate the effects of long non-coding RNA(lncRNA)growth retardation specific gene 6 antisense RNA1(GAS6-AS1)on the proliferation,migration,and invasion of neuroblastoma(NB)cells by regulating the microRNA-326(miR-326)/E2F transcription factor 1(E2F1)axis.Methods Tumor tissue and peritumor tissue of 23 NB children treated in He-bei Children's Hospital from August 2021 to January 2023 were collected.The expression levels of lncRNA GAS6-AS1,miR-326 and E2F1 mRNA in NB tissue and cell were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and their relationship among the three was detected by double luciferase assay.SK-N-SH cells were randomly divided into control group,short hairpin RNA negative control(sh-NC)group,GAS6-AS1 short hairpin RNA(sh-GAS6-AS1)group,sh-GAS6-AS1+inhibitor negative control(anti-NC)group and sh-GAS6-AS1+miR-326 inhibitor(anti-miR-326)group.The level of lncRNA GAS6-AS1 and miR-326 in cells were detected by qRT-PCR.The proliferation,migration and invasion of NB cells and the protein expression of E2F1,Cyclin D1,matrix metalloproteinase(MMP)-2 and MMP-9 were detected by clonal forma-tion assay,scratch assay,Transwell assay and western blotting,respectively.The nude mouse transplanted tumor model was con-structed,and the mice were randomly divided into sh-NC group and sh-GAS6-AS1 group.The lncRNA GAS6-AS1 and miR-326 levels in the transplanted tumor tissues were detected by qRT-PCR,and the expression of E2F1 protein in the transplanted tumor tissues was detected by immunohistochemistry.Results Compared with peritumor tissues and human normal nerve cells retinal pigment epithelial cells D-407,the expression of lncRNA GAS6-AS1 and E2F1 mRNA were increased in NB tissues and cells,while the expression of miR-326 were decreased,and the differences were statistically significant(t=7.221～53.271,all P<0.05).There was a targeting relationship between lncRNA GAS6-AS1 and miR-326,and between E2F1 and miR-326.Compared with the sh-NC group,the number of cell clones formed,scratch healing rate and number of invasive cells in the sh-GAS6-AS1 group were decreased,the expression of lncRNA GAS6-AS1,E2F1,Cyclin D1,MMP-2 and MMP-9 were decreased,the expression of miR-326 was increased,the differences were statistically significant(q=8.706～16.489,all P<0.05).Compared with the sh-GAS6-AS1+anti-NC group,the number of cell clones formed,scratch healing rate,and number of invasive cells in the sh-GAS6-AS1+anti-miR-326 group were increased,the expression of miR-326 were reduced,the expression of E2F1,Cyclin D1,MMP-2 and MMP-9 were increased,the differences were statistically significant(q=4.173～13.407,all P<0.05).In the nude mouse experi-ment,compared with the sh-NC group,the tumor mass and volume in the sh-GAS6-AS1 group were decreased,while lncRNA GAS6-AS1 and E2F1 were downregulated and miR-326 was upregulated,the differences were statistically significant(t=8.684～13.494,all P<0.05).Conclusions Inhibiting the expression of lncRNA GAS6-AS1 may inhibit proliferation,migration and invasion of NB cells by targeting miR-326/E2F1 axis.