MiR-34a Inhibits Proliferation Ability And Induces Apoptosis Of Human Neuroblastoma Cells Via Wnt/β-catenin Signaling

Objective:Look for clues that miR-34a targets on Wnt/β-catenin signaling by literaturesearching and bioinformatics. Use molecular biology techniques to over-express miR-34ain human neuroblastoma cell line SK-N-SH, then analyze the activity of Wnt/β-cateninsignaling and investigate the effects on proliferation and apoptosis of SK-N-SH cells. Ourstudy will contribute to further research about the function of miR-34a and Wnt/β-cateninsignaling during neuroblastoma pathogenesis. Our study also can provide a new line and anexperimental basis for the molecular mechanism involved in neuroblastoma developmentand progression and gene therapy of neuroblastoma.Methods:1. Four online softwares for bioinformatics analysis, i.e., PicTar, TargetScan,microRNA.org and RNA22, were used for searching miRNAs which might target on Wnt/β-catenin signaling, and for genes which gene may be miR-34a’s potential target genes.2. SK-N-SH cells were transfected with miR-34a mimics. Then the expression ofmiR-34a was verified by semiquantitative reverse transcription-polymerase chainreaction(RT-PCR) and real-time quantitative reverse transcription-polymerase chainreaction(Real-time PCR).3. The transcriptional levels of Wnt1mRNA, catenin-δ-1mRNA and β-catenin/TCF were valued by Luciferase reporter assay. Then the protein expressions levelsof some key proteins in Wnt/β-catenin signaling(Wnt1、β-catenin、CyclinD) were valuedby Western blot. Meanwhile the nuclear translocation of β-catenin was studied with laserconfocal microsopy.4. Cell proliferation ability was measured by MTT and flow cytometry（FCM） wasapplied to analyze the apoptosis of SK-N-SH cells. Results:1. After literature searching and bioinformatics we had a preliminary result thatmiR-34a might target on Wnt/β-catenin signaling. We found that miR-34a had target onthe sites in the3’-UTR of Wntl and catenin-δ-1mRNA by miRNA binding site predictionprogram.2. RT-PCR and Real-time PCR confirmed that compared with the control group, themiR-34a were over-express in test group which were transfected with miR-34a mimics.3. In test group, Luciferase reporter assay showed that the transcriptional levels ofWnt1mRNA, catenin-δ-1mRNA and β-catenin/TCF decreased, compared with thoseof control groups. Western blotting analysis showed that the expressions of some keyproteins in Wnt/β-catenin signaling(Wnt1、β-catenin、CyclinD) were decreased inSK-N-SH cells treated with miR-34a mimics compared with the control groups. It wasconfirmed by laser confocal microsopy that the phenomena of β-catenin nucleartranslocation was less obvious in SK-N-SH transfected with miR-34a mimics.4. Cell proliferation was determined with MTT assay, and lower rates of MTTreduction was observed in SK-N-SH transfected with miR-34a mimics group than controlgroup, and the cell growth inhibition rate was40.8%,20.9%and18.2%, respectively (p <0.05) at24h,48h, and72h. Flow cytometry assay showed that apoptosis rates of SK-N-SHtransfected with miR-34a mimics and control group were2.09,2.25and1.77(p<0.05),respectively.Conclusions:Our results show that miR-34a can efficiently inhibit proliferation ability of humanneuroblastoma cells and induce apoptosis of human neuroblastoma cells by regulatingcanonical Wnt/β-catenin signaling.