Researches Of G3BP1in The In Vitro Migration Of Breast Cancer And Lung Cancer Cells

Object:To study whether GTPase activating protein-(Src homology domain3)-binding protein1(G3BP1), which is overexpressed in human breast cancer line MDA-MB-231and human non-small cell lung cancer cell line A549, is involved in in vitro migration process of those two cancer cell lines. And continually argue possible theoretical mechanisms if the results are positive.Methods:Part1:Western Blot examined whether G3BP1expression in MDA-MB-231agrees with reports. G3BP1expression was inhibited by transfection of small interfering RNA (siRNA) targeted at the mRNA of G3BP1gene into MDA-MB-231. Boyden Chamber Assay examined, after inhibition of G3BP1expression in MDA-MB-231, cell number of passing through8μm polycarbonate membrane, Akt, pPKCζThr410/103and pAktSer473when stimulated with10ng/ml epidermal growth factor (EGF) by30seconds,1minute,2minutes and5minutes have been examined by Western Blot. Immunoprecipitation examined whether G3BP1interacts with PKCζ in MDA-MB-231.Part2:G3BP1expression in human non-small cell lung cancer cell line A549was examined by Western Blot with a positive control of MDA-MB-231. G3BP1expression was inhibited by transfection of siRNA targeted at the mRNA of G3BP1gene into A549. Boyden Chamber Assay, in vitro Invasion Assay and Wound Healing Assay examined respectively chemotactic and invasive cell numbers and directional moving distance without extracellular chemoattractants. Finally, G3BP1expression in3other lung cancer cell lines:GLC-82, H1299and NCI-H358were detected by Western Blot with positive controls of A549and MDA-MB-231.Results:Part1:G3BP1is overexpressed in MDA-MB-231, which agrees with reports. Western Blot testified that intracellular G3BP1expression was temporarily and totally suppressed after transfection of siRNA. Boyden Chamber Assay and Wound Healing Assay separately showed that chemotactic cell number and directional moving distance without extracellular chemoattractants have been greatly reduced (P<0.05). Intracellular expression of G3BP1, PKCζ_and Akt merely changed under stimulation of EGF, but pPKCζThr410/403and pAktSer473expressions continually raised and reached the peak at the point of5minutes. Immunoprecipitation showed an interaction of G3BP1and PKCζ.Part2:Western Blot showed that expression of G3BP1in A549is almost the same as that in MDA-MB-231, so G3BP1is probably overexpressed in A549. siRNA transfection temporarily and totally suppressed A549intracellular G3BP1expression, which was testified by Western Blot. Boyden Chamber Assay, in vitro Invasion Assay and Wound Healing Assay have separately showed great reduction of chemotactic cell number, invasive cell number, and directional moving distance(P<0.05). Finally, G3BP1expression is not the same among GLC-82, H1299and NCI-H358: overexpression in the former two but low expression in the last one.Conclusions:G3BP1is overexpressed in MDA-MB-231, and its suppression could greatly impair cancer cell MDA-MB-231’s chemotaxis, and movement, so G3BP1could play an important role in migration of MDA-MB-231. Because phosphorylation of PKCζ and its down-stream molecule Akt raised when extracellular chemoattractant stimulation lasted, so G3BP1could be involved in migration of MDA-MB-231though PKCζ-Akt pathway. G3BP1is also overexpressed in A549, and its suppression also inhibited A549cells’ chemotaxis, movement and in vitro invasion, so G3BP1could also play an important role in A549’s migration. But since G3BP1is not overexpressed among all lung cancer cell lines, so more works are needed on researching whether G3BP1functions in all migration of tumours.