Effects Of DADS On Proliferation, Migration And Invasion In BGC823Cells After LIMK1Silenced

ObjectiveOur studies reported that diallyl disulfide (DADS) inhibited proliferation, migrationand invasion in human Gastric cancer cells via down-regulation of LIMK1. In thisstudy, we will investigate the effects of DADS on proliferation, migration andinvasion in BGC823cells after LIMK1silenced.MethodsAccording to LIMK1gene sequence design miRNA1, miRNA2, miRNA3,miRNA4interference sequence, and design a non-specific sequence as a negativecontrol.the miRNA containing plasmid targeting LIMK1pcDNATM6.2-GW/EmGF-PmiR-LIMK1miRNA-containing-plasmid targeting LIMK1pcDNATM6.2-GW/EmGFPmiR-LIMK1transfected human Gastric cancer cells BGC823to establish astable cell lines. The expression of LIMK1inhibition was verified by western blot.MTT colorimetric assay observed the change of BGC823cells proliferation aftereffects of DADS and interference with LIMK1. Transwell experiment and scratch testobserved the change of BGC823cell invasion and migration effects of DADS andinterference with LIMK1. Immunohistochemistry detected LIMK1expression inBGC823cells after the role of DADS and interference with LIMK1. RT-PCR andwestern blot detected mRNA and protein expression of LIMK1in BGC823cells aftereffects of DADS and interference with LIMK1. Western blot were used todetect Rac1, Rock1, Pak1and cofilin1protein expression.Results1pcDNA6.2/LIMK1-miRNA/BGC823cells with stable low expression of LIMK1pcDNA6.2-GW/EmGFPmiR-LIMK1interfering plasmid were extracted frominterfering vector. Human gastric cancer BGC823cells were transfected with fiveplasmids and can be seen with green fluorescent hair under the inverted fluorescencemicroscope after treated with blasticidin2weeks. Western blot showed that the LIMK1protein expression of transfected LIMK1-miRNA1-4of BGC823cellscompared with negative transfection group decreased by70.11%,15%,25.12%and72.32%. The most obvious protein expression inhibition was the fourth transfectedgroup (P<0.05). Follow-up experiments are used LIMK1-miRNA4plasmid assubjects.2The change of BGC823cells proliferation after LIMK1silencedMTT assay result showed that LIMK1silenced inhibited proliferation in BGC823cells. LIMK1-miRNA/BGC823cells were treated with DADS for24,48h,72h and96h, the inhibition ratio was much higher than the untransfected and transfectedgroups which hinted that LIMK1silenced increase the inhibiton effects of DADS onproliferation in BGC823cells.3Effect of DADS on migration and invasion in BGC823cells after LIMK1wassilenced by RNA interference.Scratch test and the results show that the LIMK1-miRNA3BGC823cellstransfected group and the untransfected cells DADS treatment group, taking0h,24hand48h observation of healing scratches, no scratches transfected Healing wassignificantly faster than the transfected group and DADS treatments proved migrationof BGC823cells was inhibited by transfected BGC823cells and DADS treated(P<0.05).Transwell experiment showed that, DADS treatment and the LIMK1genesilencing in human gastric cancer can reduce the invasive ability of BGC823cells.4LIMK1expression in Human gastric cancer cells BGC823after DADS andLIMK1silencedRT-PCR and Western blot and immunohistochemistry results showed thatLIMK1-miRNA4transfection group, by30mg L-1DADS were treatednon-transfected group, negative transfection group and group24h after transfectionof LIMK1expression were significantly lower than the negative transfection groupand untransfected group (P<0.05), which indicated that DADS and the LIMK1genesilencing can suppress the expression of LIMK1. 5Effect of LIMK1Silenced on Rac1-Rock1/Pak1-LIMK1-cofilin1signalingpathwayWestern blot results showed that the expression of Rac1, Rock1, Pak1andP-cofilin1in the silenced LIMK1was significantly lower than the negativetransfection group and untransfected group (P<0.05). Wheras the protein expressionsof cofilin1were not affected. LIMK1silenced inhibited the expression of P-cofilin1,Rac1, Rock1and Pak1.Conclusions1. LIMK1silenced can inhibit proliferation, migration and invasion in BGC823cells.2. LIMK1silenced increases the inhibiton effects of DADS on proliferation,migration and invasion in BGC823cells.3. DADS inhibites proliferation, migration and invasion in BGC823cells viadownregulating the expression of LIMK1and P-cofilin1.LIMK1silenced can inhibitRac1-Rock1/Pak1pathway.