Fig 2: MiR-124 suppresses metastatic phenotypes of CC cells by suppressing its target iASPP.(a) Morphology of Hela cells transfected with miR-124 mimic (Mimic 124) or negative control mimic (Mimic Ctr). (b) Left: Representative image of Hela cell migration in cell migration assay. Right: Relative migration of Hela cells after transient overexpression or Siha cells after knockdown of miR-124. Invasion (c), proliferation (d), sphere formation (e) and qPCR analysis of indicated mRNAs (f,g) in CC cells after transient overexpression or knockdown of miR-124. Mimic 124 or mimic Ctr was transfected into Hela cells, together with (or without) iASPP cDNA vector lacking the 3′-UTR region. Anti-miR-124 inhibitor or anti-miRNA control was transfected into Siha cells, together with (or without) iASPP-Si. The cells were assayed for cell invasion (h), proliferation (i), and sphere formation (j). Data are shown as mean ± SEM; n = 3; **P < 0.01.

Fig 3: iASPP interacts with p53 to suppress its transcriptional activities toward target genes, and represses the expression of p53-responsive miR-34a.(a) Co-immunoprecipitation (Co-IP) assays reveal associations between endogenous iASPP and p53 in CC cells. Cell lysates were labeled as input. IgG was used as the control. (b) Chromatin immunoprecipitation (ChIP)-qPCR assays confirmed the occupancy of p53 at p21, Bax and MMP-2 loci, but not at the GAPDH promoter. (c) ChIP-qPCR assays. p21, Bax and MMP-2 promoter regions containing p53-binding sites were quantified by qPCR in CC cells after overexpression or knockdown of iASPP. Fold change of enrichment was determined relative to IgG controls and GAPDH. (d) Siha cells were transfected with p21-, Bax-, or MMP-2-promoter luciferase plasmids, together with (or without) p53 expression vector, in the presence (or absence) of increasing amounts of iASPP expression vector. Luciferase activities were corrected for transfection efficiency by normalization to Renilla luciferase activity. (e) Hela cells were transfected with the indicated promoter luciferase plasmids, together with (or without) p53 siRNA, in the presence (or absence) of increasing amounts of iASPP-Si. Relative reporter activities were assessed. (f) Protein expression of p53 in CC cells transfected with control or p53 expression vector. qPCR analysis of miR-23b and miR-34a expression in CC cells expressing either the control or p53 vector (g), or in CC cells after overexpression or knockdown of iASPP (h). (i) Immunoblot of the indicated proteins in CC cells after knockdown or overexpression of iASPP. (j) qPCR analysis of the indicated mRNAs in Hela cells following iASPP knockdown, or in Siha cells after iASPP overexpression. The data are shown as the mean ± SEM; n = 3; **P < 0.01.

Fig 4: Low miR-124 expression is associated with worse outcome and inversely correlated with iASPP overexpression in CCs.The expression levels of miR-124 (a), iASPP (b), Survivin (c), Snail (d) and MMP-9 (e) were assessed by qPCR analysis of 40 paired cancerous and normal tissue samples from CC patients. Data are shown as mean ± SEM; n = 3; **P < 0.01. Analysis of iASPP (f), Survivin (g), Snail (h) and MMP-9 (i) mRNA expression using microarray (Oncomine) on normal cervical versus CC patient data sets. (j) Correlations between miR-124 expression and clinicopathological features of patients with CC. Kaplan-Meier analysis of overall (k) and disease-free survival (l) in 40 CC patients according to high and low miR-124 expression.

Fig 5: Oncogene iASPP is a direct target of miR-124 in CC cells.(a) Summary of the number of miRNAs that were predicted to bind to the 3′-UTR of iASPP by TargetScan and microRNA.org (upper panel). The 6 predicted miRNAs were common to these two algorithms. Schematic representation of the 3′-UTR of iASPP with the predicted target site for miR-124 (lower panel). (b) In silico prediction and molecular pathway enrichment analysis was performed on predicted target genes regulated by miR-124. The top 10 ranking KEGG pathways are listed. (c) qPCR analysis of miR-124 expression in CC cell lines and a normal cell line. The results are presented as the fold-change in expression compared with normal cell. (d) Expression of the iASPP mRNA and protein in CC cells after the transient overexpression or knockdown of miR-124. Hela (e) and Siha (f) cells were cotransfected with reporter plasmids containing wild-type iASPP or a mutant iASPP 3′-UTR together with a miR-124 mimic, miR-124 inhibitor, or respective negative controls. The relative luciferase activity was assayed. Data are shown as mean ± SEM; n = 3; **P < 0.01.