Raf kinase inhibitor protein (RKIP) inhibits the cell migration and invasion in human glioma cell lines in vitro

Abstract

Objective: To investigate the effects and the potential mechanisms of RKIP on cell migration, invasion and proliferation in human glioma cell lines in vitro.

Methods: The RKIP over-expressing and RKIP knockdown human U87 glioma cells were used to reveal the effects of RKIP on human glioma cells migration, invasion and proliferation. After the recombinant plasmid pcDNA3.0-RKIP or RKIP-shRNA was transfected into the cell lines U87 by the means of liposome assay, the cells migration, invasion and proliferation were detected by wound healing, Transwell and MTT assay. Then, the levels of RKIP, MMP-3, MMP-9 and HMGA2 mRNA transcription were measured by means of RT-qPCR and levels of proteins expressions were determined using Western blot.

Results: The results of MTT assay suggested that the PKIP have little inhibitive effects on glioma cells proliferation (P>0.05). The present paper showed that the migration distances in the group of RKIP-shRNA were markedly increased compared to the pcDNA3.0-RKIP and control. Similarly, the results showed that the numbers of invasion cells in RKIP-shRNA were remarkably increased than the pcDNA3.0-RKIP group and control group. Western blot and RT-qPCR suggested that over-expressions of RKIP lessened the MMP-2, MMP-9 and HMGA2 expression, however, turning down the RKIP expression showed the inverse effects.

Conclusion: RKIP inhibits the cells migrations and invasions. Meanwhile, RKIP might inhibit the glioma cells through inhibiting MMPs and HMAG2 expression. Therefore, we demonstrated that RKIP is an underlying target for the treatment of glioma.

Keywords: HMGA2; Raf kinase inhibitor protein (RKIP); glioma; invasion; migration.

Figure 1

Effects of RKIP on cells migration in human U87 glioma cell by means of wound healing assay. The relative distance was ratio of experimental group distance vs. the distance of the cell-blank scratch at 12 h, 14 h and 48 h after transfection. *P<0.05, **P<0.01.

Figure 2

Effects of RKIP on cells invasion in human U87 glioma cell by means of Transwell assay. The number of glioma cells cross through the membrane was recorded. *P<0.05, **P<0.01.

Figure 3

The effects of RKIP on the proliferation of U87 glioma cells. To determine the effects of RKIP on the ability of proliferation in U87 cells, the MTT assays were carried out to detected the proliferations of cells that over-expressing or down regulating of RKIP. The results showed that RKIP had little effects on the proliferation of U87 glioma cells.

Figure 4

Effects of over-expressing and knockdown RKIP on mRNA expressions in human U87 glioma cells. A. The RKIP mRNA relative expressions (vs. GAPDH), B. The MMP-2 mRNA relative expression (vs. GAPDH), C. The MMP-9 mRNA relative expressions (vs. GAPDH), D. The HMGA2 mRNA relative expression (vs. GAPDH), *P<0.05, **P<0.01.

Figure 5

Effects of over-expressing and knockdown RKIP on protein expression in human U87 glioma cells. A. The RKIP protein relative expressions (vs. GAPDH), B. The protein relative expression of MMP-2, MMP-9 and HMGA2, *P<0.05, **P<0.01.