| Objective:The aim of study was to identify the expression pattern of mi RNA molecules in autophagy derived from TOCP-induced neuroblastoma SK-N-SH cells,obtaining mi RNAs differentially expressed information in autophagy of neural cells induced by TOCP.Preliminary study on the role and mechanism of mi RNAs in SK-N-SH cells autophagy induced by TOCP,further clarify the relationship between mi RNA and autophagy,provide experimental and theoretical basis for further study of autophagy induced by TOCP.Methods:1.Human neuroblastoma SK-N-SH cells were cultured in vitro,treated with TOCP at different concentrations(0,0.25,0.5,0.75,1.0 m M)for 24 h,and the effect of TOCP on the proliferation of SK-N-SH cells was detected by MTT assay.The dose of TOCP in the autophagic cell model was determined.2.SK-N-SH cells treated with TOCP(0,0.25,0.5 m M)for 24 h were collected as the experimental groups and Hank? s treated cells as the control group,then extract the total RNA of these cells for small RNA sequencing,obtain mi RNA expression profile of SK-N-SH cells autophagy induced by TOCP.3.Using bioinformatics software to analyse significantly different expressed mi RNAs(top 20)target genes network;predict the interest mi RNAs associated with key autophagy-related genes;real-time PCR was performed to validate the results of small RNA sequencing.4.The target of mi R-381-3p was predicted by bioinformatics methods,3? UTR fragments of target gens were synthesized by PCR and the luciferase expression vector containing 3? UTR fragments were constructed,and dual-luciferase reporter assay was used to demonstrate the binding of mi R-381-3p and targets.Results:1.The results of the MTT assay showed that the cell proliferation was significantly inhibited after TOCP treatment for 24 h,showing a certain dose-dependent manner,and the IC50 was 0.65 m M.2.Based on analysis of small RNA sequencing,we found that significantly altered mi RNA expression profiles in cells treated with TOCP and Hank's solution compared to the 0 m M group.Among these,there were 10 up-expressed mi RNAs(such as hsa-mi R-1285-5p,hsa-mi R-192-3p,hsa-mi R-7976)and 25 down-expressed mi RNAs(such as hsa-mi R-326,hsa-mi R-4448,hsa-mi R-31-3p)in 0.25 m M groups with more than two-fold changes;19 mi RNAs were up-expressed(such as hsa-mi R-2355-3p,hsa-mi R-127-3p,hsa-mi R-7978),and 22 mi RNAs were down-expressed(such as hsa-mi R-362-3p,hsa-mi R-153-5p,hsa-mi R-6866-5p)in 0.5 m M groups with more than two-fold changes;45 mi RNAs were up-expressed(such as hsa-mi R-1468-5p,hsa-mi R-20a-3p,hsa-mi R-7978),and 44 mi RNAs were down-expressed(such as hsa-mi R-122-5p,hsa-mi R-3195,hsa-mi R-148a-3p)in Hank? s groups with more than two-fold changes.3.Through the analysis of the network of target-gene,and based on the previous researches,selected 6 mi RNAs(mi R-1285-5p,mi R-20a-3p,mi R-210-5p,mi R-155-5p,mi R-6866-3p,mi R-381-3p)to do q RT-PCR,it was reveal that most of the small RNA sequencing results credible.mi R-381-3p was significantly down-regulated in 0.25 m M and 0.5 m M group,and it was used as the target mi RNA for further research.4.Bioinformatics tools(Target Scan,mi RDB,mi Randa,CLIP-seq and mi RBase)were used to predict the putative target genes of mi R-381-3p,3? UTR sequences of autophagy related genes RPS6KA3,take on complimentary binding sites with mi R-318-3p seed.PCR was used to acquire partial 3? UTR fragment of RPS6KA3 gene and pmir GloRPS6KA3 WT and pmir Glo-RPS6KA3 Mut luciferase vector wereconstructed.All the expression vectors were confirmed by sequencing.mi R-381-3p mimics(or NC)and luciferase vector were co-transfected into 293 T cells,and PC-mi R-424 inhibit sponge was used as internal control.Pmir Glo-RPS6KA3 luciferase activity decreased to 54%(P? 0.05),but not in the mutant 3? UTR vector.Conclusions:1.TOCP induced significant changes in mi RNA expression profiles in SK-N-SH cells.2.RPS6KA3 is one of the target genes of mi R-381-3p,which may be involved in the occurrence and development process of TOCP-induced autophagy of SK-N-SH cells as an autophagy-related gene. |
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