The Function And Mechanism Of PcG Family Member CBX7in Regulating Gastric Cancer Stem-like Cells’ Properties

Objective:We conducted this study to investigate the function and mechanism of PcG(Polycomb Group) family protein CBX7(Chromobox Protein Homolog7) in regulating gastric cancer stem-like cells’ propertis, including the ability of self-renewal and clonogenic proliferation.Methods:Gastric cancer stem-like cells were seperated from gastric cancer cell lines via serum-free culture in ultra-low attachment plates. The expression of CBX7and stem cell-related factors. Oct4, CD44, CD24in gastric cancer stem-like cells and adherent cells were detected by western blot. The ability of self-renewal was assessed using serum-free culture, and anchorage independent growth was analyzed through colony formation assay. CBX7interfering plasmid (CBX7i-GFP) and its control plasmid (Ctrli-GFP) were respectively tranfected into gastric cancer cells using LipofectamineTM2000. After transfection, CBX7expression level, its well-known downstream target p16, E-cadherin and probable downstream targets pERK, pAkt were determined by Western blot, and expression level of miR-21was detected by real time PCR. Meanwhile, the expression of CBX7, p16, pERK, pAkt and miR-21in gastric cancer floating spheres and adherent cells were assayed using western blot and real time PCR. After downregulating the expression of p16in gastric cancer cells, the changes of gastric cancer stem-like cells’ properties were observed. CBX7interfering plasmid (CBX7i) and p16interfering plasmid (pl6i) were co-tranfected into gastric cancer cells, and then the changes of gastric cancer stem-like cells’ properties were assayed.Results:1. MKN28, SGC-7901and NCI-N87gastric cancer cell lines could form floating spheres in serum-free medium, while MKN45, HGC-27gastric cancer cell lines couldn’t. Floating spheres expressed obviously high level of Oct4and CBX7. Spheroid colonies originated from MKN28cell line expressed high level of CD24, while spheroid colonies originated from SGC-7901cell line expressed high level of CD44.2. After knockdown of CBX7in gastric cancer cells by interfering plasmid transfection, both sphere-forming efficiency and colony-forming efficiency were decreased.3. After knockdown of CBX7in SGC-7901cell line by transfecting interfering plasmid, the expression of p16and E-cadherin increased, and pERK, pAkt, miR-21were downregulated. In MKN28and AGS cell line, we also found that downregulating CBX7could inhibit the expression of pAkt and pERK.4. There was no significant difference of pERK and pAkt in gastric cancer floating spheres and adherent cells. Gastric cancer stem cells seperated from SGC-7901cell line expressed low level of p16, whlie MKN28cell line didn’t express p16. Floating spheres derived from MKN28cell line didn’t expressed high level of miR-21compare to adherent cells.5. Floating spheres derived from MKN28cell line expressed low level of ATM/p53, but there was no significant change in SGC-7901spheres.6. The ability of self-renewal and clonogenic proliferation were increased after the inhibition of p16in gastric cancer cells. Morever, when both CBX7and p16were downregulated in gastric cancer cells, the properties of gastric cancer stem-like cells were restored, which related to the the ability of self-renewal and clonogenic proliferation.Conclusion:1. CBX7plays significant roles in regulating gastric cancer stem cells’ self-renewal and clonogenic proliferation.2. The surface markers of gastric cancer stem-like cells derived from different cell lines are not always the same.3. pERK and pAkt are newly discovered CBX7downstream target genes, and CBX7also regulate the expression of miR-21.4. In p16positive gastric cancer cell lines, CBX7regulates gastric cancer stem cells’ self-renewal and clonogenic proliferation through inhibiting the expression of p16. However, further studies are needed to investigate the mechanism how CBX7regulate gastric cancer stem cells’ properties in P16negative cell lines.5. Due to different biologic characteristics of different cancer cell lines, there maybe some difference in regulation mechanisms of cancer stem cells.