[Investigation of self-renewal mechanism about CD133+ cancer stem cells in human laryngeal carcinoma Hep-2 cell line].
OBJECTIVE
To investigate the self-renewal mechanism of CD133+ cancer stem cells from Hep-2 cell line.


METHODS
The CD133+ cells were sorted by flow cytometry from Hep-2 cell line. Then the sorted CD133+ cells were cultured in RPMI1640. The ability of self-renewal of CD133+ cells were tested by MTT assay. mRNA and protein expression of self-renewal related genes were detected by western blot and RT- PCR.


RESULTS
(3.10 ± 0.21)% of Hep-2 cells expressed the membrane antigen CD133. CD133+ fraction was raised to (90.20 ± 5.51)% by flow cytometry. In vitro culture and growth curve showed CD133+ cells had more active proliferation ability than CD133- cells, which showed statistically significant difference between these two group (P < 0.01). RT- PCR and western blot results showed upregulated mRNA and protein expression of Fas, c-myc, survivin in CD133+ group (P < 0.01). In the same time, the ratio of Bcl-2/Bax gene expression was obviously increased in CD133+ group. Self-renewal related gene such as β-catenin, SHH, SMOH and Bmi-1,Gli-1 were all up-regulated in CD133+ group both in mRNA and protein. On the contrary, PTCH gene was down-regulated.


CONCLUSIONS
CD133 positive cells are a small proportion of a Hep-2 cell line. The results of this experiment verified that CD133 positive cells owned the properties of cancer stem cells. Upregulated anti-apoptotic gene is the foundatiom of self-renewal mechanism of CD133+ cells. Cancer stem cells related signal pathways such as Hedgehog, Wnt and Bmi-1 pathway are in state of activation. The identification of self-renewal mechanism about cancer stem cell provides a powerful tool to investigate the tumorigenic process in the larynx and to develop therapies targeting to these signal pathways.
Xudong
X
Wei
Jian
J
He
Jiangxia
J
Gao
Jing
J
Chen
Jingyu
J
Wang
D000071916:AC133 Antigen; D015703:Antigens, CD; D017209:Apoptosis; D002468:Cell Physiological Phenomena / Q000502:physiology; D015536:Down-Regulation; D005434:Flow Cytometry; D006023:Glycoproteins; D006801:Humans; D007822:Laryngeal Neoplasms; D014411:Neoplastic Stem Cells / Q000502:physiology*; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D010455:Peptides; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051176:beta Catenin / Q000235:genetics
D000071916:AC133 Antigen; D015703:Antigens, CD; D006023:Glycoproteins; C000606023:PROM1 protein, human; C000606236:PTCH1 protein, human; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D010455:Peptides; D011956:Receptors, Cell Surface; D051176:beta Catenin