Explore The Roles Of Sox2in Invasion And Metastasis Of Breast Cancer

Objective：To explore the biological roles of Sox2in breast cancer cell line ZR7530, thensearch out the differentially expressed miRNAs, which are related by SOX2gene, andfurther discuss the molecular mechanisms that may be involved in the malignant eventof breast cancer.Methods：1、We adopted Western blotting to detect the expression level of Sox2protein, toverify the established cell lines which ZR7530with SOX2konckdown, SOX2overexpression or scramble.2、The cell proliferation, tumor formation and migration of human breast cancercell line ZR7530with SOX2konckdown or overexpression were assessed by cellcount, colony formation and wound healing assay, respectively. Nude mice werexenografted with cancer cells each subcutaneously. Then the tumor volume wasmeasured with caliper every three days. The metastatic capality of the breast cancercell was further analyzed by in vivo study in nude mice which were injected via thetail vein with tumor cells. Nude mice were sacrificed eight weeks after the tail veininjection, and the lung was sliced for HE staining to examine for metastasic focus.3、The Human miRNA Microarray, miRCURY LNA Expression Array(v.18.0), was carried out on cell line ZR7530with SOX2knockdown oroverexpression compared with scramble in this study. We used SAM software,following the criteria that the differentially expressed miRNAs at least two-foldchange. Screened out the miRNAs, and the target genes of these differentiallyexpressed miRNAs were predicted by TargetScan, miRDB, PicTar and miRandadatabase. Results：1、Compared with the control, the expression level of Sox2protein wasdecreased significantly in cell line ZR7530with SOX2knockdown (P=0.0480), andincreased in cell line ZR7530with SOX2overexpression (P=0.0296).2、 The cell count assay: After72h, compared with ZR7530Scramble, theproliferation ability of cell line ZR7530pCDH-Sox2was increased, and the difference wasstatistically significant (P=0.0106,P0.05). The statistical significance also wasdifference at96h(P=0.0021,P0.01). However, the count of cell line ZR7530withSOX2knockdown decreased only a little and the difference was no statisticallysignificant until96hours(P=0.1179,P0.05). Colony formation assay: Compared withcontrol group, the numble of cell colony-forming, which diameter greater than0.5mm,reduced siglificantly in ZR7530Sox2-KDgroup（P=0.0377,P0.05）, and enhanced inZR7530pCDH-Sox2group（P=0.4401,P0.05）. Wound healing assay: No significantdifference was found in both cell line ZR7530with SOX2knockdown andoverexpression before24hours. Results at48hours, the data showed that migrationability enhanced in cell line ZR7530with SOX2overexpression compared with thecontrol group(P=0.0379,P0.05). But there is no statistical difference in cell lineZR7530with SOX2knockdown (P=0.9738,P0.05). Xenograft transplantation assay：the tumor sizes in the ZR7530pCDH-Sox2xenograft mice increased significantly atday48when compared with the control（P=0.0057，P0.01）. Meanwhile, the tumorsizes reduced in SOX2knockdown group(P=0.0220，P0.05). In vivo metastasisstudy: Lung metastatic area was larger in nude mice which were injected withZR7530pCDH-Sox2than the ones which were injected with ZR7530Scramble(P=0.0055,P0.01). Yet the data in nude mice which were injected ZR7530Sox2-KDisquite opposite (P=0.3894,P0.05).3、According to the results of miRNA microarray, compared with ZR7530Scramble,91differentially expressed miRNAs in ZR7530Sox2-KDincluding49up-regualted miRNAs and42down-regulated miRNAs were identified. Among251differentiallyexpressed miRNAs in cell line ZR7530with SOX2overexpression,136miRNAs wereup-regualted and115miRNAs were down-regulated.Conclusions：1、The cell line ZR7530with Scramble, SOX2knockdown or overexpression wassuccessfully constructed, respectively.2、Sox2promotes cell proliferation, colony formation and migration of breastcancer cell line ZR7530in vitro.3、Sox2contributes to breast cancer cell line ZR7530growth and metastasis invivo, too.4、The Sox2transcription factor, affect the malignant process of cancer, may beoperated by interaction with some miRNAs.