SND1 Acts Upstream Of SLUG To Regulate The Epithelial–mesenchymal Transition (EMT) In SKOV3 Cells

Objectives: Tumor metastasis is a sophisticated process,in which different signal pathways and various regulatory molecules interact with each other and play pivotal roles.This biological behavior is caused by the changes from cell morphology to molecular characteristics.After these cells acquire mesenchymal characteristic,they are more likely to invade and migrate to the other parts.This change is called epithelial to mesenchymal transformation,also known as EMT.After tumor cells attach to the new site and start to expand,they will return to epithelial type which is called MET.SND1 protein,also called Tudor-SN or p100 protein,is widely expressed and has been proved to be a oncoprotein involved in the development and metastasis of various tumors.Previous results showed that SND1 could interact with TGF? signaling pathway in breast cancer cells in promoting tumor metastasis.Compared with ovarian cystadenoma samples,the expression of SND1 protein was strongly positive in epithelial ovarian cancer and partially expressed in borderline samples.Surprisingly,the expression level of SND1 in epithelial cells with invasive tendency in benign samples was significantly higher than that in adjacent normal sites.Meanwhile,in borderline samples,tumors with malignant characteristic showed papillary growth and the expression of SND1 was significantly higher than that in opposite site.These results suggest that SND1 may function in promoting tumor metastasis in ovarian cancer cells but the regulatory mechanism remains unclear.Here,we will investigate whether SND1 is involved in the metastasis of ovarian cancer cells and the underlying molecular mechanism.Methods: 1.SND1 promotes epithelium to mesenchymal transformation(EMT)in SKOV3 cells.i.To construct the stable cell lines with different SND1 levels in SKOV3 cells and monitor the morphological changes,then detect the expression of SND1 by Western Blot.ii.To test the abilities of migration and invasion in these cells by using wound-healing and transwell assays.iii.To detect the effect of SND1 on metastasis in vivo by using luciferase system in SND1-knockdown and control cells and compare the differences of tumor growth and dissemination.iv.To compare the expression profiles between control and SND1-knockdown groups and verify the results by q PCR.v.To detect the localization of EMT markers in control and SND1-knockdown cells by immunofluerence and their expression levels by Wetstern Blot.vi.To rescue the expression of SND1 in knockdown cells by infecting the lentivirus and then verify the expressing changes of EMT moleculars by Western Blot;To test the abilities of migration and invasion in these cells by using wound-healing and transwell assays;To detect the localization of epithelial and mesenchymal markers in these cells by immunofluorescence.2.SND1 promotes EMT in SKOV3 cells by regulating SLUG expression.i.To verify SLUG expression is correlated to SND1 in SKOV3 cells with different SND1 levels.ii.To construct over-expressed SLUG plasmid with HA tag and crisprcase 9 plasmid to knock out SLUG.iii.To construct the stable cell lines with different SLUG levels in SKOV3 cells and monitor the morphological changes and then,detect the expression of EMT markers and test the abilities of migration and invasion in these cells.iv.To modify the expression of SLUG in SKOV3 cells with different SND1 expression levels and detect the expression profiles of the related markers by Western Blot.Also,to test the abilities of migration and invasion in these cells by using wound-healing and transwell assays.3.SND1 activates SLUG transcription by increasing chromatin accessibility.i.To verify SND1 can enrich and activate the SLUG promoter region by Ch IP and Gluc-promoter assays.ii.To detect the interaction between SND1 and histone acetyltransferases and their downstream targets by Co-IP and verify whether SND1 has an important role in the enrichment of histone acetyltransferases and their downstream sites on the SLUG promoter region by q Ch IP.Result: 1.SND1 promotes epithelium to mesenchymal transformation(EMT)in SKOV3 cells.i.The morphological feature of SND1-knockdown cells changed to round or cobblestone shape and distributed in clusters.ii.Knockdown SND1 inhibited the abilities of invasion and migration in SKOV3 cells.iii.Knockdown SND1 inhibited cell growth and dissemination in vivo.iv.Knockdown SND1 in SKOV3 cells affected the m RNA levels of genes and signaling pathways involved in the EMT activation.v.Knockdown SND1 restored the expression of CDH1 which only located on cell membrane and CDH2 in the cytoplasm was down-regulated.Knockdown SND1 protein increased the expression of epithelial markers CDH1/CLDN1 and decreased the expression of mesenchymal markers CDH2/VIMENTIN.vi.The restoration of SND1 in knockdown cells led to the decreased expression of epithelial markers CDH1/CLDN1 and the increased expression of mesenchymal markers CDH2/VIMENTIN.2.SND1 promotes EMT in SKOV3 cells by regulating SLUG expression.i.SLUG was positively correlated with SND1 protein in both m RNA and protein levels in SKOV3 cells.ii.SLUG overexpression led to the decreased expression of epithelial markers CDH1/CLDN1 and the increased expression of mesenchymal markers CDH2/VIMENTIN;the epithelial markers were up-regulated and the mesenchymal markers were down-regulated after SLUG was knockdown.iii.The ability of SKOV3 cell migration and invasion were enhanced by overexpressing SLUG protein.Knockout SLUG inhibited cell migration and invasion.iv.Overexpression of SLUG in SND1-knockdown cells led to the decreased expression of epithelial proteins and increased mesenchymal ones compared with SND1-knockdown cells.Also,the migration and invasion of SLUG-restored cells were enhanced compared with SND1-knockdown cells.v.Knockout SLUG in SND1-overexpression cells led to the increased expression of epithelial proteins and decreased mesenchymal ones compared with SND1-overexpression cells.Also,the migration and invasion of SLUG-knockout cells were inhibited compared with SND1-overexpression cells.3.SND1 activates SLUG transcription by increasing chromatin accessibility.i.The fluorescence value of medium was slightly increased after transfected with Gluc reporter plasmids containing SLUG promoter region.The fluorescence value was significantly increased when the Gluc reporter plasmids were co-transfected with FLAGSND1 plasmids;SND1 can combine with the SLUG promoter sequence.ii.SND1 interacted with histone acetyltransferases GCN5/p300 and also their downstream modification sites H3K9/H3K18.Knockdown of SND1 protein can reduce the enrichment intensity of GCN5/p300 and their modification sites on SLUG promoter region.Conclusion: We identified SLUG as a novel downstream target of SND1 in the SKOV3 cell line.SND1 regulates the gene transcriptional activation of SLUG by recruiting CBP/p300 and GCN5 for acetylation of histone,thus influencing chromatin remodeling and consequently promotes the metastatic ability of SKOV3 cells via SLUG mediated EMT process.