The Research Of Oct4B1 Expression And Its Relevance With Twist In Colorectal Cancer Stem Cells

Objective: To Explore the Oct4B1 role in colorectal cancer stem cells and its action mechanism whether have the relevant with the adjustment of Twist for epithelial to mesenchymai transition(EMT).Methods: 1: SW480 colorectal cancer cell line(parental cells) was cultivate in serum free medium that contain EGF, b FGF, B-27 growth factor,to concentrat 3D microspheres;2:Use differentiation, soft AGAR cloning experiments and flow cytometry technology detect the expression of cancer stem cell marker(CD133, CD44), to assess whether 3D microspheres enrich the colorectal cancer stem cells(SW480CSCs);3:Use real-time fluorescent quantitative PCR(q PCR) technology to detect the expression of Oct4B1 m RNA in SW480 CSCs and SW480;4:Use Western blott technology and cellular immunity fluorescence detect the expression of epithelial markers(E-cadherin) and mesenchymal cells markers(N-cadherin,Vimentin) in SW480 CSCs and parental cells;5:Use q PCR technology to detect the expression of Twist m RNA in SW480 CSCs and SW480, analying the correlation with Oct4B1.Results: 1:parental cells was cultivate in serum free medium that contain EGF, bFGF, B-27 growth factor can form 3D microspheres,which can suspended growth; 2: ①3D microspheres can differentiate into the similar shape with the parental cells in the 10% FBS medium,which can continue to grow after wedding, and the form has no obvious change; ② in vitro cloning formation ability of 3D microspheres was obviously higher than the parental cells; flow cytometry technology found that the expression of cancer stem cell ③marker CD133 and CD44 is significantly higher in 3D microspheres than SW480 cells, respectively 2 times and 4 times that of parental cells(P < 0.01 and P < 0.02). 3:Oct4B1 m RNA expression was significantly higher in the SW480 CSCs compared with parental cells, respectively 27 times that of parental cells(P < 0.01); 4:The expression of epithelial markers(E-cadherin) in SW480 CSCs significantly below parental cells(P<0.01), while mesenchymal cells markers( N-cadherin,Vimentin) was significantly higher than that of parental cells(P < 0.01); Cell immunofluorescence technique found E-cadherin fluorescence intensity significantly below parental cells; N-cadherin and Vimentin are significantly higher than that of parental cells;4: Twist m RNA expression was significantly higher in the SW480 CSCs compared with parental cells, respectively 185 times that of parental cells(P < 0.01), the factor has the positive relation with Oct4B1.Conclusions:1:Serum free medium that contain EGF, bFGF, B-27 growth factor can make ordinary tumor cells form 3D microspheres,which can suspended growth; 2:3D microspheres enrich the characteristicsa of CSCs compared with parental cells(the 3D microspheres is called as SW480CSCs);3:SW480CSCs happened EMT process compared with parental cells;4:Oct4B1 participate in SW480 CSCs stemness adjustment and its regulation mechanism may be through the interaction with Twist adjust epithelial mesenchymal transition.