Screening Of CD90~+ Cells And Investigate The Role Of PI3K/AKT Signaling Pathway In Their Stem Cell-Like Characteristics

Objective:At present, the incidence of liver cancer ranked fifth in the world, and first in China. As a global high-risk malignant tumor, the main treatment included surgical resection, radiotherapy and chemotherapy. Over the past decade, new concept of tumor treatment had come up and cancer stem cell theory was identified in vitro and vivo experiment by a variety of parenchymal cells. A growing number of evidence pointed out that heterogeneity existed in tumor, and cancer stem cells might be the root. Cancer stem cells were derived from a tiny amount of extremely vigorous growth cells which possessed the ability of self-renewal, infinite proliferation potential, differentiation and high tumorigenicity. These cells played an important role in tumor occurrence and progress and resistance to drug. The most cancer cells did not have these trait, however they almost followed a brief maturity, finally headed towards death. In present research, we aim to verify there exist liver cancer stem cells by sphere-forming culture combined fluorescence-activated cell sorting to explored the potential influence of PI3K/AKT signaling on the stem cell-like characteristics of a CD90+ subpopulation derived from MHCC97L cell line.Methods:1. Using complete medium and serum-free medium culture liver cancer cell line MHCC97L, respectively make it adherent or suspending into sphere-forming.2. Detecting spherical cell sources by Glycogen staining reagent.3. Flow cytometry testing the expression of CD90 marker in adherent cells and sphere-forming cells.4. After treating with LY294002, flow cytometry are used to analyze the change of CD90 expression in sphere-forming cells.5. MTT detecting cell proliferation rate of CD90+subpopulation and CD90-subpopulation after treating with LY294002, a PI3K/AKT pathway inhibitor.6. qRT-PCR analyze expression of CD90 and SHP2 in CD90+ subpopulation and CD90- subpopulation after treating with LY294002.7. Western blot testing the expression of CD90, SHP2, P-AKT and AKT protein after treating with LY294002.8. Tablet clone formation experiment testing the effects of cloning in CD90+ subpopulation and CD90- subpopulation after treating with LY294002.9. Compare the subcutaneously tumorigenic ability of CD90+ subpopulation and CD90- subpopulation in nude mice with different grade cells number, and the change after treating with LY294002.10. Compare the expression of CD90, SHP2, P-AKT and AKT protein in subcutaneou transplantation tumor formed after CD90+ subpopulation and CD90- subpopulation cells injection into nude mice by immunohistochemistry; Compare the change of expression after use of LY294002.Results:1. Liver cancer cell MHCC97L lines can stably passage in the complete medium 5 days. In serum free medium, sphere-forming cells also can stably passage 7 to 9 days.2. Glycogen dyeing shows that sphere-cells sources are derived from liver cancer cells.3. Flow cytometry testing result:the expression of CD90 in sphere-forming cells> adherent cells (P< 0.05).4. After treating with the PI3K/AKT pathway inhibitor (LY294002), the expression of CD90 in sphere-forming cells are markedly declined(P< 0.05), whereas expression of CD90 in adherent cells are not (P> 0.05).5. The proliferation rate of CD90+ subpopulation> the proliferation rate CD90" subpopulation.6. The expression of liver cancer stem cell associated protein CD90, SHP2 and PI3K/AKT signaling pathways downstream of specific protein P-AKT are significantly decreased after treating with LY294002 in CD90+ subgopulation (P < 0.05), but not in CD90- subpopulation. The expression of AKT protein has no change both in CD90+subpopulation and CD90- subpopulation after treating with LY294002.8. The ability of clone formation CD90+ subpopulation are superior to CD90" subpopulation [(93.13±4.72)% vs (20.59±3.23)%] (P< 0.05). LY294002 can obviously reduced the ability of clone formation in CD90+ subpopulation [(93.13±4.72)% vs (51.32±6.27)%] (P< 0.05), but no significant change in CD90- subpopulation [(20.59±3.23)% vs (18.37±3.21)%] (P> 0.05).9. The ability of tumor-formation in CD90+ subpopulation cells was significantly stronger than CD90- subpopulation with the same number cells. LY294002 can reduce the tumorigenic ability of CD90+ subpopulation, but not for CD90’subpopulation.10. The expression of CD90, SHP2 and PI3K/AKT protein are significantly decreased after treating with LY294002 in subcutaneou transplantation tumor formed by CD90+subpopulation, but no obviously change in subcutaneou transplantation tumor formed by CD90- subpopulation. The expression of AKT protein has no change both in subcutaneou transplantation tumor formed by CD90+ subpopulation and CD90- subpopulation after treating with LY294002.Conclusion:Sphere-forming culture can enrich liver cancer stem cells, CD90+cells possess the characteristic of liver cancer stem cells, and their stem cell-like characteristics may be impacted by PI3K/AKT signaling. These results lay the groundwork for further study of stem cells in HCC, and suggest the possible therapeutic targets.