Luciferase reporter experiments The 3′UTR segments from the WT1 a

Luciferase reporter experiments The 3′UTR segments from the WT1 and Bcl-2 gene were amplified by PCR from cDNA and inserted into the pGL3 control vector (Promega), using the XbaΙ site immediately downstream from the stop codon of luciferase. Bcl-2 is one of known targeted gene by miR-15a/16-1[9]. Selleckchem PI3K inhibitor The following primer set was used to generate specific fragments: Bcl-2UTRF, 5′-CTA GTC TAG AGC CTC AGG GAA CAG AAT GAT CAG-3′; Bcl-2UTRR, 5′-CTA GTC TAG AAA GCG TCC ACG

TTC TTC ATT G-3′[9]. WT1UTRF, 5′-CTA GTC TAG GTA GAC CCA AAG GTC CTT AAG TT-3′; WT1UTRR, 5′-CTA GTC TAG GAT ACC GGT GCT TCT GGA A-3′. The cells were cotransfected in 24 well plate using Lipofectamine™ LTX and PLUS™ Reagents (Invitrogen) according to the manufacturer’s protocol using 0.1 ug of the firefly luciferase report vector and 0.1 ug of the Selleckchem CHIR99021 control vector pRL-TK (Promega, WI, USA) containing Renilla luciferase. For each well 0.5 ug pRS-15/16 or negative control pRS-E were used. Firefly and Renilla luciferase activities were measured by dual luciferase reporter assay (Promega) after transfection. Firefly luciferase activity was normalized to Renilla luciferase

activity. Statistical Analysis The significance of the difference between groups was determined by Student’s t-test. A P value of less than .05 was considered statistically significant. Relationships between miR-15a/16-1 and WT1 expression were explored by Pearson’s correlation coefficient. All statistical analyses were performed with SPSS HSP90 software (version 13). Results miR-15a/16-1 suppress the proliferation of K562 and HL-60 cells In order to explore the functional role of miR-15a/16-1 in leukemic cells, we examined the effect of miR-15a/16-1 over-expression

on the proliferation of K562 and HL-60 cell lines. The cells were transfected with either pRS-15/16 or negative control plasmid (pRS-E) for 24, 48, and 72 h. The qRT-PCR analysis confirmed that the expression of miR-15a and LBH589 in vivo miR-16-1 was obviously increased in cells transfected wth pRS-15/16 compared with negative control (Figure 1A and 1B). CCK-8 assay and direct cell count showed that over-expression of miR-15a/16-1 significantly inhibited the proliferation of both K562 (*P < 0.05, Figure 1C and 1D) and HL-60 cells (* P < 0.05, Figure 1E and 1F). In a word, our data indicate that miR-15a/16-1 may play an important role in the proliferation of leukemic cells in vitro. Figure 1 Effects of miR-15a/16-1 on the proliferation of K562 and HL-60 cells. K562 and HL-60 cells were transfected with pRS-15/16 or pRS-E vector (negative control) for 24, 48 and 72 hours, then the relative expressions of miR-15a/16-1 were measured by qRT-PCR (A and B). CCK-8 assay (C and E) and direct cell count (D and F) were performed when K562 and HL-60 cells were transfected with pRS-15/16 or pRS-E vector at different time periods. Data are shown as mean ± SD from three independent experiments. *P < 0.05 versus negative control.

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