Lei Xuhui, Chang Liang, Ye Wei, Jiang Chuanlu, Zhang Zhiren
Department of Neurosurgery, The Second Affiliated Hospital of Harbin Medical University Harbin, China.
Department of Neurosurgery, The Tumor Hospital of Harbin Medical University Harbin, China.
Int J Clin Exp Pathol. 2015 Nov 1;8(11):14214-20. eCollection 2015.
To investigate the effects and the potential mechanisms of RKIP on cell migration, invasion and proliferation in human glioma cell lines in vitro.
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.
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.
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.
探讨RKIP对人胶质瘤细胞系体外迁移、侵袭及增殖的影响及其潜在机制。
利用RKIP过表达和RKIP敲低的人U87胶质瘤细胞,揭示RKIP对人胶质瘤细胞迁移、侵袭及增殖的影响。通过脂质体法将重组质粒pcDNA3.0-RKIP或RKIP-shRNA转染至U87细胞系后,采用划痕愈合实验、Transwell实验和MTT实验检测细胞的迁移、侵袭及增殖能力。然后,通过RT-qPCR检测RKIP、MMP-3、MMP-9和HMGA2 mRNA转录水平,利用蛋白质免疫印迹法测定蛋白表达水平。
MTT实验结果表明,PKIP对胶质瘤细胞增殖的抑制作用较小(P>0.05)。本文显示,与pcDNA3.0-RKIP组和对照组相比,RKIP-shRNA组的迁移距离明显增加。同样,结果表明,RKIP-shRNA组的侵袭细胞数量比pcDNA3.0-RKIP组和对照组显著增加。蛋白质免疫印迹法和RT-qPCR表明,RKIP过表达降低了MMP-2、MMP-9和HMGA2的表达,然而,降低RKIP表达则显示出相反的效果。
RKIP抑制细胞迁移和侵袭。同时,RKIP可能通过抑制MMPs和HMAG2表达来抑制胶质瘤细胞。因此,我们证明RKIP是治疗胶质瘤的潜在靶点。