MiRNA-20a-5p Targeted Regulation Of NFKBIB On Proliferation,Invasion And Migration Of Neuroblastoma SH-SY5Y Cell Line

Objective Neuroblastoma(NB)is the most common extracranial solid malignant tumor in children.It has the characteristics of high malignancy,early onset age,atypical symptoms,and early migration.Therefore,the treatment of NB is facing great challenges worldwide.In recent years,more and more studies have found that miRNA is abnormally expressed in NB,and confirmed that it is closely related to the occurrence and development of NB.Therefore,this study will explore the effect of miR-20a-5p on the proliferation,invasion and migration of NB cell line SH-SY5 Y and its regulatory mechanism.Methods The miR-20a-5p mimics and inhibition genes were transfected into SH-SY5 Y cells respectively,which were divided into miR-20a-5p mimics group,NC mimics group,miR-20a-5p inhibition group,NC inhibition group,according to the experimental needs.The effects of miR-20a-5p on the proliferation,invasion and migration of NB cell line SH-SY5 Y were studied through in vitro experiments.Preliminary prediction of NFKBIB according to bioinformatics prediction software(Iκ B β)it may be the downstream target gene of miR-20a-5p.Double luciferase reporting experiment,quantitative reverse transcription PCR(q RT-PCR)and Western Blot were used to verify that miR-20a-5p targets NFKBIB gene.The biological functions of miR-20a-5p in proliferation,invasion and migration of SH-SY5 Y cells were studied by cell colony formation experiment,CCK-8 experiment,cell invasion experiment,cell scratch experiment and flow cytometry.At the m RNA level,the expression of NFKBIB m RNA in each group of cells was detected by q RT-PCR;Expression variation of NFKBIB and NF-κB in each group were detected by Western blot.Results The number of colony forming cells in miR-20a-5p mimics group were(90 ± 8),which was significantly higher than that in NC mimics group(16 ± 12)(P<0.05).CCK-8 results showed that miR-20a-5p mimics group significantly promoted SH-SY5 Y cell proliferation compared with NC mimics group.The results of cell invasion experiment showed that the number of cell invasions in miR-20a-5p mimics group were(594 ± 8),which was significantly higher than that in NC mimics group(440 ±9)(P<0.001).The results of cell scratch test showed that the cell healing rate of miR-20a-5p mimics group was(0.56 ± 0.05)%,and that of NC mimics group was(0.40 ± 0.08)%,the difference was statistically significant(P<0.05).The results of flow cytometry showed that after inhibiting miR-20a-5p,the number of SH-SY5 Y cells with early apoptosis increased compared with NC inhibition group.After inhibiting the expression of miR-20a-5p,the above experimental results were contrary to those of overexpression of miR-20a-5p,and the difference was statistically significant(P<0.05).After overexpression of miR-20a-5p,q RT-PCR showed that the expression of NFKBIB m RNA in miR-20a-5p mimics group was(0.36 ± 0.24),significantly lower than that in NC mimics group(1.00);Western blot showed that the expression of NFKBIB in miR-20a-5p mimics group was(1.15 ± 0.30),which was significantly lower than that in NC mimics group(1.92 ± 0.31);The expression of NF-κB was(4.17 ± 0.45),which was significantly higher than that of NC mimics group(1.64 ± 0.39),and the difference was statistically significant(P<0.05).Conclusions miR-20a-5p promotes the proliferation,invasion and migration of NB cells,which may activate NFKBIB by inhibiting the expression of NF-κB pathway can be used as a potential target to promote the proliferation,invasion and migration of NB cells.