Effects Of DADS On Migration And Invasion In MGC803Cells After RNA Interference LIMK1

Objective LIMK(Lim kinase), one of the important molecules that regulate structureof cytoskeletal, plays a vital role in tumor cell invasion and migration. Our studiesreported that diallyl disulfide (DADS) inhibited migration and invasion in humanGastric cancer MGC803cells via down-regulation of LIMK1. In this study, we willinvestigate the effects of DADS on migration and invasion in MGC803cells afterRNA interference LIMK1to elucidate that LIMK1is one of the important moleculesinhibiting the migration and invasion of tumor cells.Methods Interference efficiency was examined with RT-PCR and Western blot afterRNA interference LIMK1in MGC803cells. Migration and invasion potentials wereexamined by scratch test and transwell assay. The protein of Rac1, Rock1, Pak1,LIMK1, p-cofilin1and cofilin1in MGC803cells were determined by Western blot.Results1pcDNA6.2/LIMK1-miRNA/MGC803cells with stable low expression of LIMK1Four pcDNA6.2-GW/EmGFPmiR-LIMK1interfering plasmid and pcDNA6.2-GW/EmGFPmiRNA-negative interfering plasmid running agarose electrophoresisgel results indicated high purity. Human gastric cancer MGC803cells weretransfected with five plasmids and can be seen with green fluorescent hair under theinverted fluorescence microscope. Western blot showed that the LIMK1proteinexpression of transfected with LIMK1-miRNA1-4of MGC803cells compared withuntransfected group decreased by30%,28%,72%,75%. The most obvious proteinexpression inhibition was the fourth transfected group (P<0.05). RT-PCR showed thatthe obvious mRNA expression inhibition was the first and fourth transfected groups.In conclusion, Follow-up experiments used LIMK1-miRNA4plasmid as subjects.2Effects of DADS on migration and invasion in MGC803cells after LIMK1wassilenced by RNA interference Scratch test showed that scar distance of LIMK1-miRNA4/MGC803group waswider than untransfected group and negative transfected group at24h (P<0.05). Nodifference was found between untransfected group and negative transfected group(P>0.05).Treated with30mg·L-1DADS，scar distance of LIMK1-miRNA4/MGC803group was wider than untransfected group and negative transfected group at24h(P<0.05). No difference was found between untransfected group and negativetransfected group(P>0.05). There was no difference betweenLIMK1-miRNA4/MGC803and LIMK1-miRNA4/MGC803treated with DADS. It isindicated that LIMK1silenced inhibited migration ability of MGC803cells.Transwell assay showed that the number of cancer cells which passed throughthe matrigel coated membrane was reduced in the DADS treatment group andtransfected group compared with untransfected group and negative transfected group(P<0.05). No difference was found between untransfected group and negativetransfected group (P>0.05). It is indicated that LIMK1silenced inhibited invasiveability of MGC803cells and increased the inhibiton effects of DADS on invasion inMGC803cells.3Effects of LIMK1silenced and DADS on Rac1-Rock1/Pak1-LIMK1-cofilin1signaling pathwayWestern blot showed that the protein expressions of Rac1, Rock1, Pak1andcofilin1were not affected in transfected group.Wheras the protein expressions ofLIMK1and p-cofilin1were reduced. No difference was found between untransfectedgroup and negative transfected group(P>0.05). The protein expressions of Rac1,Rock1and Pak1were reduced in transfected group treated with30mg·L-1DADS fortwenty-four hours. Wheras the protein expressions of LIMK1, p-cofilin1and cofilin1were not affected. No difference was found between untransfected group and negativetransfected group(P>0.05).Conclusions1LIMK1silenced can inhibit migration and invasion in MGC803cells.2LIMK1silenced increases the inhibiton effects of DADS on invasion in MGC803cells. 3LIMK1silenced downregulates p-cofilin1and inhibites migration and invasion inMGC803cells.