Inhibitio Effect Of GKN2 Overexpression On Gastric Cancer Stem Cells

Objective To study the role of GKN2 over-expression in proliferation,migration,invasion,cell cycle and apoptosis of CD44?+?SGC7901 gastric cancer stem cells?GCSCs?,then elucidate the inhibitory effect of GKN2 on GCSCs,which may provide a theoretical basis for the clinical effective treatment strategy of gastric cancer.Methods1.Accumulating the CD44?+?cells from human gastric cancer cell line SGC7901 by magnetic cell sorting?MACS?technique,and detecting the positive efficiency using immunofluorescence.Adenovirus vector of GKN2 will be transfected into GCSCs,then verifying the transfection is successful by using Western Blot.The recombinant adenovirus vector with GKN2 overexpression was tranfected into GCSCs,and Western Blot was used to verify the success of the tranfection.2.The role of GKN2 over-expression in proliferation,cell cycle,apoptosis,migration and invasion of GCSCs was observed by tumor sphere formation assay,CCK-8 analysis,flow cytometry and Transwell migration and invasion experiment.3.Western Blot was performed to show protein expression of Sonic Hedgehog in GCSCs and SGC7901 cells induced by over-expressed GKN2.Results1.Immunofluorescence analysis showed that CD44 positive cells expressed green fluorescence on the membrane surface.The number of CD44?+?cells had been increased greatly after magnetic cell sorting.It was proved that the separation of GCSCs from SGC7901 cells was successful.The number of CD44?+?cells significantly increased after immunomagnetic beads sorting,suggesting that GCSCs were successfully accumulated from SGC7901 cells.2.The positive cell rate was 80%in GKN2 overexpression adenovirus transfected GCSCs using immunofluorescence analysis,and Western Blot also had the same result,indicating GKN2 overexpressed in GCSCs successfully.3.Tumor sphere formation assay demonstrated that the number of tumor sphere in GKN2 overexpression group?9.1±3.59?was much lower than untransfection group?30.7±3.13?and the empty vector group?25.8±3.47??P<0.05?,and the diameter of sphere was smaller,the transmittance was higher.All above showed GKN2 overexpression can inhibit the ability of tumor sphere formation in GCSCs.4.CCK8 assay revealed that the OD₄₅₀ value of GKN2over-expressiongroupreducedaftertransfectedwithGKN2overexpression vector for 48h,72h or 96h,compared with untransfection group and the empty vector group.Furthermore,the inhibition ratio of GKN2 overexpression group enhanced in time-dependent manner.This indicated GKN2 can inhibit proliferation of GCSCs.5.Transwell migration experiment found the number of migration cells in GKN2 over-expression group?31±4.6?was much less than those in untransfection group?198±9.8?and in the empty vector group?1172±8.4?.Transwell invasion experiment also showed the number of invasion cells in GKN2 over-expression group?14±2.7?decreased sharply vs untransfection group?155±9.9?and the empty vector group?131±8.4?.This suggested the migration and invasion ability of GCSCs can be suppresseded by GKN2 remarkably.6.Flow Cytometry verified there is no significant difference between GCSCs with or without GKN2 transfection.It also revealed the apoptosis ratio of GKN2 over-expression group?32.90%?upregulated obviously compared with untransfection group?18.65%?and the empty vector group?19.24±5.4%?.These results proved that GKN2 may promote the apoptosis of GCSCs.7.Western Blot displayed that SHH expression level of GCSCs was higher than that of SGC7901,and GKN2 overexpression downregulated SHH expression in both two cell lines significantly.ConclusionGKN2 overexpression can inhibit growth,migration and invasion of GCSCs and induce apoptosis,which is related to downregulation of SHH expression.