β2-Spectrin Involved In Hepatocarcinogenesis By Infecting The Differentiation Of Liver Cancer Stem Cells

Objectives: To isolate the Ep CAM positive cells in SNU398 cell line, then identifying its features of cancer stem cell, and to study the effect of β2-Spectrin on the differentiation of the Ep CAM positive SNU398 cancer stem cells.Methods: Flow cytometry(FCM) was used to detect the expression of Ep CAM, CD133,CD90 in SNU398 cell line. And to obtain Ep CAM+ cells and Ep CAM- cells, anti-Ep CAM microbeads antibody was used by Magnetic-activated cell sorting( MACs).The purity of sorted cells was investigate by FCM. A series of tests which included DOX chemoresistance assay, spheroid formation assay, tumor formation in NOD/SCID mice and cell invasion assay were conducted using the Ep CAM+ cells and Ep C AM- cells to authenticate that the Ep CAM+ cells is the liver cancer stem cells. The recombined plasmid of pc DNA3.1(-)-β2SP and the control plasmid of pc DNA3.1(-) was respectively transfected into Ep C AM+ cells. The gene expression of β2SP in the Ep CAM+ cells was detected by Real-time RT-PCR. Other data including the expression of liver function protein gene ALB and HCSCs surface marker gene Ep C AM, CK19 was also detected.Results: 1. The CSCs marker Ep CAM was detected in(4.00±0.18)% cell of SNU398 cell line, while CD133 in(1.20±0.28)%, and CD90 in(98.26±0.92)%; 2. The part of cells expressing Ep CAM in Ep CAM+ cells was about(82.3±3.8)% and in Ep CAM- cells was about(3.95±0.21)%. 3. Ep CAM+ cells formed more tumorspheres than the Ep CAM- cells(93.33±10.41 vs 39.00 ±3.60)in the serum- free medium. 4. Ep CAM+ cells possessed higher colony formation efficiency than Ep C AMcells in colony formation assay(328.00 ±39.60 vs 133.00 ±28.28); and Ep CAM+ cells initiated more tumors in NOD/SCID mice. 5. Ep CAM+ cells possessed more powerful invasive ability than Ep CAM- cells(136.00±32.62 vs 20.00 ±7.07) in cell invasion assay; 6. Ep CAM+ cells resisted more tough than Ep CAM- cells(cell viability: 0.58±0.05 vs 0.46±0.03)in DOX chemoresistance assay. 8. The expression of β2SP significantly increased after pc DNA3.1(-)-β2SP was transfected into Ep CAM+ SNU398 cells. 9. The gene expression of liver function protein ALB significantly increased, and the gene expression of LCSCs surface markers Ep CAM, CK19 clearly decreased after β2SP transfected into Ep CAM+ cells.Conclusions: Ep CAM+ SNU398 cells have the biological characteristics of CSCs, and β2SP could induce the differentiation of Ep CAM+ CSCs...