05; data not shown). We selected the most promising candidate to be recombined into the RABEX-5-siRNA lentiviral vector, which was then transfected Tipifarnib chemical structure into MCF-7 cells (MCF-7/KD). MCF-7/KD cells showed a significant decrease in RABEX-5 mRNA and protein expression levels compared with MCF-7 cells (CON) or negative control-transduced cells (MCF-7/NC) (Figure  2A, Figure  2B). Table 2 siRNA sequence-specific to RABEX-5 Marker Gene Targetseq pLVT540 RABEX-5 CCCTCACATTCTCCAAGTT PLX4032 research buy pLVT541 RABEX-5 CCTTCCATAAACCGGCAAA pLVT542 RABEX-5 GGATGCAAACTCGTGGGAA pLVT543 RABEX-5 GCATCACCAAGTGCAGCAA pLVT7 NC TTCTCCGAACGTGTCACGT Figure 2 Downregulation of RABEX-5 in MCF-7 cell and effects of RABEX-5 on

the colony formation and cell proliferation of breast cancer cells. (A),

RABEX-5 mRNA levels were analyzed by real time-PCR. MCF-7 cells were transfected with pMAGic-siR lentiviral plasmid (MCF-7/KD) and pMAGic-siR-neg lentiviral control plasmid (MCF-7/NC). (B), RABEX-5 protein levels in MCF-7/KD and MCF-7/NC were analyzed by western blot. GAPDH was used as an internal control. P<0.05 compared with normal control (MCF-7) or MCF-7/NC. (C), CCK-8 cell proliferation assay for vector- and RABEX-5-transfecetd MCF-7 cells, curves PKA activator indicate a significant level of proliferation compared to controls(P <0.05). (D), Representative colony formation assay, the numbers of colonies in MCF-7/NC were set to 100%. Values are expressed as mean±SD from three experiments, and the asterisks indicate statistical 4-Aminobutyrate aminotransferase significance compared to controls (P<0.05). Downregulation of RABEX-5 inhibits colony formation and breast cancer cell proliferation A CCK-8 assay was used to further explore the ability of RABEX-5 to modulate breast cancer cell proliferation. The MCF-7/KD group displayed significantly decreased proliferation at 24, 48, 72 and 96 h after incubation compared with the MCF-7/NC group (P<0.05, Figure  2C). Meanwhile, the colony formation assay further revealed the effects of RABEX-5 knockdown on the growth of MCF-7 cells. Downregulation of RABEX-5 markedly suppressed

the colony formation ability of MCF-7 cells. The MCF-7/KD group had reduced positive colony formation than the MCF-7/NC group (P<0.05, Figure  2D). These data suggest that downregulation of RABEX-5 suppresses breast cancer cell proliferation. Downregulation of RABEX-5 inhibits the migration of breast cancer cells To investigate the role of RABEX-5 in breast cancer metastasis, we investigated the migratory and invasive capacity of MCF-7/KD and MCF-7/NC cells. To test whether downregulation of RABEX-5 could inhibit tumor cell migration, a wound healing assay was performed. The migration of MCF-7/KD cells across the wound edges was remarkably slower than that of the MCF-7/NC cells at 54 h (Figure  3A).