Fig 1: miR-193b inhibits proliferation via KRAS and suppresses migration and invasion via STMN1. (A) Transfection efficiency was verified after F4 and F5M2 cells were transfected with siKRAS or pMSCV-KRAS, respectively. (B) Transfection efficiency was verified after F4 and F5M2 cells were transfected with siSTMN1 or pMSCV-STMN1, respectively. (C) Co-transfection with pMSCV-KRAS reversed miR-193b-mimic induced cell cycle arrest at G1 phase in F5M2 cells, whereas co-transfection with siKRAS reversed the miR-193b inhibitor-induced increase in percentage of cells at G2 phase in F4 cells. (D) Transwell cell invasion assay (magnification ×40). (E) wound-healing assay (magnification ×20) revealed that co-transfection of F5M2 cells with pMSCV-STMN1 and miR-193b mimic or co-transfection of F4 cells with siSTMN1 and miR-193b inhibitor reversed the regulatory effects of miR-193b mimic or inhibitor. Data are presented as the mean ± SD of at least three independent experiments. *P<0.05. miR-193b, microRNA-193b; NC, negative control; STMN1, stathmin 1; si, small interfering RNA.
Fig 2: Expression, methylation and functional role of SAM target genes as determined by QPCR, pyrosequencing and shRNA depletion(A) Normalized mRNA expression quantified by RT-QPCR for genes whose expression is altered by SAM treatment in both HepG2 and SKhep1: NFIL3, CDT1, HAT1, RAN, DYNC1H1, MYC, TRIB3, RRM2, MCM3, CBS, PEG10 and E2F1. (B) Normalized mRNA expression quantified by RT-QPCR for genes whose expression is altered by SAM treatment in SKhep1 cells: SLC2A1, STMN1, PBK, DDIT3, ITGA6, CLIC4, NFIB, TAF15, MTHFD2, MIA and PDK1. (Experiments were performed in triplicate and expression levels were normalized to 18S rRNA values). (C-D) Average methylation levels at indicated CpG sites as determined by pyrosequencing in the promoters of DYNC1H1, and PEG10 (C) in both HepG2 and SKhep1 cell lines and NFIB, TAF15, STMN1 (D) in SKhep1 (see Supplementary Figure 12 for additional methylation analysis). Positions (relative to TSS) of CpGs that were pyro sequenced are indicated above the chart. (E) Expression of shRNA depleted genes in SKhep1 cells was quantified by qPCR and western blot analysis after infection with STMN1, TAF15 and scrambled shRNA lentiviral vectors. (F-G) anchorage independent growth was measured by soft-agar assay and invasiveness using ECM550 invasion assay kit after depletion of STMN1 and TAF15 as described in “Material and Methods”. All results represent mean ±SD of three determinations in either two independent experiments; ****, P<0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.
Fig 3: miR-193b suppresses KRAS and STMN1 expression through their 3′-UTRs. (A) Schematic diagram of KRAS and STMN1 3′-UTRs with the locations of predicted conserved miRNA-targeting sequences highlighted. (B) Expression levels of KRAS and STMN1 were higher in F5M2 cells compared with F4 cells. (C) Luciferase reporter assay revealed that miR-193b suppressed luciferase activities of all WT constructs. (D and E) miR-193b negatively regulated the expression of KRAS and STMN1. (F) Immunofluorescence staining revealed that tumor tissue from nude mice injected with miR-193b-upregulated F5M2 cells exhibited lower expression levels of KRAS and STMN1 compared with the control group (magnification ×20). Data are presented as the mean ± SD of at least three independent experiments. *P<0.05. LV, lentivirus; miR-193b, microRNA-193b; MUT, mutant; NC, negative control; STMN1, stathmin 1; UTR, untranslated region; WT, wild-type.
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