MiR-34a Inhibits The Proliferation, Metastasis And Autophagy Of Neuroblastoma Cells In Children By Targeting ATG5

Neuroblastoma is the most common malignant tumor in infants and young children,characterized by a highly malignant tendency,which is the main cause of high mortality.Although neuroblastoma has a variety of treatments,patients with recurrent or metastatic neuroblastoma still have poor prognosis,with a survival rate of less than 40%.Up to now,the molecular mechanism of the occurrence,development and metastasis of neuroblastoma are not very clear.Therefore,it is still essential to explore new therapeutic targets and improve prognosis for neuroblastoma.As a group of endogenous single-stranded non-coding RNA fragments,the length of microRNAs is about 18-25 nucleotides.These non-coding RNAs play a key regulatory role in tumor formation and progression.Recently,these studies have shown that these miRNAs can inhibit target gene translation into proteins or directly degrade target gene mRNA fragments by complementary sequence pairing to the 3’-UTR or coding region of mRNA targets.These data suggest that miRNAs can be used as biomarkers in the diagnosis of tumors and play a carcinogenic or anticancer role.It is reported that many miRNAs,such as miR-26 a,miR-27,miR-92,and miR-338-3p,can participate in the growth and metastasis of neuroblastoma.Previous studies have shown that miR-34 a is involved in the occurrence and development of multiple tumors.Although miR-34 a has been extensively studied in recent years,little is known about its function and molecular mechanism in the genesis and development of neuroblastoma.The research includes three parts.The first part is that miR-34 a regulates proliferation,metastasis and autophagy in children neuroblastoma cells.The second part is that miR-34 a targeted regulation of ATG5.The last part is the mechanism of ATG5 reversing miR-34 a regulating NB cell function.Part One miR-34 a regulates proliferation,metastasis and autophagy in children neuroblastoma cells.Methods:1.The expression level of miR-34 a in neuroblastoma tissues and cell lines(SH-SY5 Y cells,SK-N-SH cells)were measured by using qPCR,and analyzed the correlation between miR-34 a and clinicopathological features and survival rate.2.MTT assay was used to detect the effects of miR-34 a overexpression or knockdown on the proliferation of neuroblastoma cells.3.Cell clone assay was used to detect the effect of overexpression or inhibition of miR-34 a on the cell clone of neuroblastoma cells.4.The effect of miR-34 a on migration and invasion of neuroblastoma cells was detected by Transwell method.5.The effect of miR-34 a expression on the percentage of GFP-LC3 positive cells and autophagy-associated proteins(LC3 II,LC3 I,p62)were detected by quantitative analysis and Western blot.Results:1.The expression of miR-34 a in neuroblastoma tissues and cell lines are significantly down-regulated and correlated with tumor grade.2.The increase of miR-34 a expression significantly increases the survival rate of neuroblastoma patients.3.Overexpression of miR-34 a inhibite the proliferation and cloning of neuroblastoma SH-SY5 Y cells,promote the proliferation and cloning of SK-N-SH cells by inhibiting the expression of miR-34 a.4.Overexpression of miR-34 a inhibite migration and invasion of neuroblastoma SH-SY5 Y cells,and promote migration and invasion of SK-N-SH cells by inhibiting the expression of miR-34 a.5.Overexpression of miR-34 a decrease the percentage of GFP-LC3 positive cells,inhibite the expression of miR-34 a increased the percentage of GFP-LC3 positive cells.6.miR-34 a overexpression inhibite the level of LC3 II/I protein and increase p62 protein in neuroblastoma SH-SY5 Y cells,increase the level of LC3 II/I protein and decrease the level of p62 protein in neuroblastoma SK-N-SH cells by inhibiting the expression of miR-34 a.Part Two miR-34 a targeted regulation of ATG5 Methods:1.Using bioinformatics analysis,the target genes of miR-34 a were predicted.2.Dual luciferase report experiments and RIP assay were used to identify the target genes of miR-34 a.3.The expression of ATG5 in neuroblastoma tissues and cell lines(SH-SY5 Y cells,SK-N-SH cells)was detected by qPCR,and the correlation between ATG5 and miR-34 a was analyzed.4.The expression of ATG5 in cells by overexpression and knockdown of miR-34 a were detected by Western blot.Results:1.Bioinformatics analysis by using miRcode suggests that miR-34 a was predicted to target the ATG5.2.Luciferase reporter and RIP assays confirm that ATG5 is a direct downstream target of miR-34 a.3.The expression of ATG5 is up-regulated in neuroblastoma tissues and cell lines,and the expression of ATG5 is negatively correlated with the expression of miR-34 a.4.Overexpression of miR-34 a inhibits the expression of ATG5 in neuroblastoma cell line SH-SY5 Y,and knockdown of miR-34 a promote the expression of ATG5 in SK-N-SH cells.Part Three The mechanism of ATG5 reversing miR-34 a regulating neuroblastoma cell functionMethods:1.Western blot was used to detect the effect of overexpression miR-34 a and ATG5 on expression levels of ATG5 in cells.2.The effect of restored ATG5 expression on the proliferation of neuroblastoma cell lines(SH-SY5 Y and SK-N-SH)were detected by MTT assay.3.The effect of restored ATG5 expression on the cloning of neuroblastoma cell lines(SH-SY5 Y and SK-N-SH)were detected by clone formation assay.4.The effect of restored ATG5 expression on migration and invasion of neuroblastoma cell lines(SH-SY5 Y and SK-N-SH)were detected by Transwell assay.5.The positive ratio of GFP-LC3 and the expression of autophagy-associated proteins(LC3 II,LC3 I,p62)were detected by quantitative analysis and Western blot.Results:1.Overexpression of miR-34 a and ATG5 co-transfection significantly increase the expression of ATG5 protein in SH-SY5 Y cells;knockdown of miR-34 a and ATG5 co-transfection significantly decrease the expression of ATG5 protein in SK-N-SH cells.2.Restoration of ATG5 expression significantly reverses the inhibitory effect of miR-34 a on proliferation,cloning,invasion,migration and autophagy of neuroblastoma cells.Conclusion1.The expression of miR-34 a in neuroblastoma is down-regulated and correlated with clinical tumor grade.2.The up-regulation of miR-34 a expression significantly increases the survival rate of the patients.3.Overexpression of miR-34 a inhibits the proliferation,cloning,invasion,migration and autophagy of neuroblastoma cell lines(SH-SY5 Y and SK-N-SH).4.ATG5 is a direct target gene of miR-34 a.5.The expression of ATG5 is up-regulated in neuroblastoma and negatively correlated with the expression of miR-34 a.6.miR-34 a inhibits the malignant biological characteristics of neuroblastoma cells by regulating ATG5.