Downregulation Of LIMK1 Inhibit Migration And Invasion In Human Colon Cancer SW480 Cells Induced By Diallyl Disulfide

Objective To study the effects of LIMK1 expression and migration and invasion in human colon cancer SW480 cells by DADS and silencing LIMK1. To observe influences on the expression of uPA and uPAR by DADS and silenced LIMK1. And to investigate the molecular mechanism of the inhibiting effect on migration and invasion in SW480 cells by DADS.Methods The proliferation of SW480 cells by DADS was examed with MTT assay. A pcDNA6.2/LIMK1-miRNA/SW480 with stable low expression of LIMK1 was constructed by microRNA interference. The expression of LIMK1 and uPA/uPAR in SW480 cells with DADS and silencing LIMK1 was detected by RT-PCR, western blot and Immunocytochemistry. Migration and Invasion potentials were examined by Wound Healing assay and transwell chamber assays. The mRNA and Protein of genes in the Rac1-LIMK1 signal pathway were determined by RT-PCR and Western blot.Results1. The migration and invasion were inhibited in SW480 cells by DADS.Apparently growth inhibition in SW480 cells treated by DADS could be seen in a dose-time dependent model (P<0.05). Wound Healing assays showed that migration rate was significantly reduced in a dose dependent model (P<0.05). Transwell invasion assays showed that the number of cancer cells which passed through the Matrigel coated membrane was reduced gradually as a dose dependent manner after treated with DADS(P<0.05).2. LIMK1 expression was Downregulated in SW480 cells by DADS.RT-PCR showed that expression of LIMK1 mRNA was inhibited in SW480 cells after treated with DADS in a time-dose dependent model (P<0.01). LIMK1 protein was overexpressed in SW480 cells detected by Immunocytochemistry. And its expression was remarkably downregulated after treatment with DADS (P<0.05). Moreover, Western Bolt showed that expressions of LIMK1 protein was reduced in a time-dose dependent manner (P<0.05).3. Effect of DADS on migration and invasion in SW480 cells after LIMK1 was silenced by RNA interference.pcDNA6.2/LIMK1-miRNA/SW480 with stable low expression of LIMK1 was successfully constructed identificated by Fluorescent microscope, Western blot and Immunocytochemistry. Western blot showed that expression of LIMK1 and p-LIMK1 was downregulated in the LIMK1-miR and DADS treatment group, compared with untreated group and empty vetor group (P<0.05). There was no difference between LIMK1-miR and LIMK1-miR treated with DADS (P>0.05). It is suggested that DADS can not only downregulate the expression of LIMK1, but also inhibit the phosphorylation of LIMK1. Wound Healing assays showed that migration rate of LIMK1-miRNA/SW480 group and DADS treatment group was significantly reduced in comparision with the control and vector group at 48h (P<0.05). And no difference was found between the control group and vector group (P>0.05). It is indicated that LIMK1 is associated with migration ability of SW480 cells, mechanism of DADS inhibiting the migration ability of SW480 cells was related with downregulation of LIMK1, migration ability was the biggest inhibited in the LIMK1-miR group treated with DADS. Transwell invasion assays showed that the number of cancer cells which passed through the Matrigel coated membrane was reduced in the DADS treatment group and LIMK1-miR group (P<0.05). No difference was found between the control and vector group (P>0.05). It is indicated that invasive ability was inhibited in the LIMK1-miR group, which exhibited LIMK1 gene has positive relation with invasive ability of SW480 cells, the mechanism of DADS inhibiting the invasion in SW480 cells was related with downregulation of LIMK1 expression and phosphorylation of LIMK1, and invasive ability was biggest inhibited in the LIMK1-miR group treated with DADS.4. DADS downregulate Rac1-LIMK1 pathway.RT-PCR and Western blot showed that DADS inhibited the expression of Rac1, Rock1, Pak1, LIMK1 and Destrin mRNA and protein in SW480 cells in a time- dependent model (P<0.05), respectively, while did not affect the expression of cofilin1 gene and protein (P>0.05), and expression of p-LIMK1 and p-cofilin1 was ruduced in a time-dependent model (P<0.05).5. DADS downregulated the expression of uPA and uPAR directly or through LIMK1 silenced indirectly.Western blot demonstrated that the expression of uPA and uPAR in SW480 cells was significantiy downregulated in DADS treatment group and LIMK1-miR/SW480, the latter than more obviously the former (P<0.05). It is indicated that DADS and silenced LIMK1 can downregulat the expression of uPA and uPAR. DADS may downregulate the expression of uPA and uPAR directly or through LIMK1 silenced indirectly.Conclusions1. DADS can inhibit migration and invasion in SW480 cells through downregulate of expression and phosphorylation of LIMK1.2. LIMK1-miRNA can inhibit migration and invasion of SW480 cells .3. The Rac1-LIMK1 pathway can be downregulated by DADS.4. DADS and silenced LIMK1 can regulate uPA/uPAR system.5. DADS downregulated the expression of uPA and uPAR directly or through LIMK1 silenced by RNA Interference indirectly.