| Objective: urokinase-type plasminogen activator receptor (uPAR) is highly expressed in all human tumors. Upregulation of uPAR correlates with increased proliferation, migration and invasion of cancer. Diallyl disulfide (DADS) could inhibit proliferation, migration and invasion in human gastric cancer cells, in which downregulated uPAR expression was involved. But its specific mechanism is not clear. In this study, after the specific miRNA targeting uPAR was constructed to interfer uPAR expression, it was observed that the effect on proliferation, migration and invasion inhibition induced by DADS were brought about from uPAR expression silence. Therefore, molecular mechanism was explored in inhibition of gastric cancer cells induced by DADS.Methods:1. The pcDNATM6.2-GW/EmGFPmiR-uPAR plasmid which contain the specificmiRNA targeting uPAR and the negative plasmid pcDNATM6.2-GW/EmGFPmiR-negative were transfected respectively into human gastric cancer MGC803 cell by lipofectin medium. Expression of uPAR was anaylized by RT-PCR and Western Blot.2. In this study, RT-PCR, IHC , Scratch test and Transwell assay were employed to observe the migration and invasion on cultured human MGC803 cells induced by DADS in vitro after inhibition of uPAR with miRNA.Results:1. The pcDNATM6.2-GW/EmGFPmiR-uPAR plasmid was showed by DNA sequencing analysis that constructing successful, the green fluorescence was observed in transfected cells by fluorescence microscopy. The MGC803 cell were transfected with pcDNATM6.2-GW/EmGFPmiR-uPAR plasmid resulted in dramatic down-regulation of uPAR as demonstrated by RT-PCR and western-blot analysis (P<0.05).2. RT-PCR indicated that DADS inhibits uPAR expression in gastric cancer cells. The gray value of uPAR were lower in human gastric cancer MGC803 cells exposed to DADS 30mg·L-1 for 12h,24h,48h than control, which exhibited a time-dependent model (P<0.05), indicating that DADS had effect on the inhibition of uPAR expression by a time-dependent manner.3. Observe the migration results on 0h and 24h of MGC803 cells exposed to 30mg·L-1 DADS, scratch test showed that migration lenth of miR-uPAR transfection groups was decreased, which showed knockdown of uPAR inhibited migration of MGC803 cells. After treated with DADS in all groups, migration lenth decreased which means that cell migration was inhibited. However, the effect in two miR-uPAR transfection groups were weaker than other groups after treatment with DADS (P<0.05), which suggest that knockdown of uPAR interfered function of migration inhibition by DADS.4. Transwell experiments revealed that, MGC803 cells were exposed to 30 mg·L-1 DADS for 24h, and cell counting was performed in hight power field. The cell counting in miR-uPAR transfection group is less than other groups (P<0.05), which showed knockdown of uPAR inhibited invasion ability of MGC803 cell. After treated with 30mg·L-1 DADS for 24h, cell counting decreased, was less than untransfected cell. Cell counting of miR-uPAR transfection groups were less than untransfection groups. This displayed that gene silencing uPAR could enhance the invasion inhibition effect induced by DADS.Conclusion:1. uPAR expression was obviously suppressed after transfected by pcDNATM6.2-GW/Em GFPmiR-uPAR-miRNA in MGC803 cells.2. Knockdown of uPAR by miRNA interference inhibited proliferation of MGC803 cells, moreover, enhanced DADS-induced growth inhibition and migration and invasion arrest in MGC803, which suggested that down-regulation of uPAR expression is involved in molecular mechanism of DADS-induced proliferation,migration and invasion inhibition in MGC803 cells.
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