10.3969/j.issn.2095-4344.2013.25.001

Objective To investigate the effect of down?regulation of insulin?like growth factor 2 (IGF2) gene on the biological characteristics of HCT116 colon cancer stem cells (CSCs). Methods Flow cytometry sorting technology was used to isolate CSCs from colon cancer cell line HCT116 by a monoclonal antibody against CD133; serum free floating culture assay was used for the enrichment of CSCs. The proportion of CD133+ cells was analyzed by flow cytometry; CSCs were identified by sphere culturing, immunofluorescence analysis and soft agar clone formation. RT?qPCR method was used to examine transcriptional level of IGF2 gene in CSCs. Western blotting was used to examine IGF2 protein expression in CSCs. siRNA was used to establish IGF2 transient knock down model in CSCs. Cell proliferation array, cell cycle and apoptosis analysis, cell invasion array and colony forming assay were used to further examine the role of IGF2 on the biological characteristics of colon CSCs. Results CSCs were successfully isolated from HCT116 cell lines, which were cultured to form cell spheres in serum?free stem cell culture medium. We found that the morphology of sphere?forming?like cells after several passages maintained the same characteristics as that of the first passage. The results of immunofluorescence showed that CSC markers including CD133 and ALDH continued positively expressing on the cell surface of CSCs, and flow cytometry analysis showed that more than 90% of the spheroid cells remained CD133 positive. The clone formation rate of non?CSCs group and CSCs group were ( 28.10 ± 2.66 )% and ( 43.73 ± 2.30 )% respectively, with significant difference (P＜0.01). The RT?qPCR results showed that the transcriptional level IGF2 gene in non?CSCs group and CSCs group were ( 1.06 ± 0.24) and ( 2.17 ± 0.51) respectively, with significant difference (P＜0.05). The western blot results showed that the protein expression of IGF2 in CSCs group and non?CSCs group were (1.10±0.55) and ( 2.14±0.23) respectively, with significant difference (P＜0.05). Knockdown of IGF2 significantly decreased the percentage of CD133+ cells in CSCs and cell proliferation (P＜0.01). Knockdown of IGF2 increased the percentage of G2／M phase (23.46% of siNC group vs 60.14%of siIGF2 group) and cell apoptosis (2.80% of siNC group vs 40.70% of siIGF2 group), while decreased the percentage of G0／G1 phase (40.77% of siNC group vs 17.73% of siIGF2 group). The invasion results showed that the number of cells penetrating into the basement surface in siNC group and siIGF2 group was (109.00±16.37) and (54.00±8.19) respectively, with significant difference (P＜0.01). The rate of sphere?forming of colon CSCs in siNC group and siIGF2 group were (51.70±7.42)%and (21.27±2.35)% respectively, with significant difference (P＜0.01). The clone formation rate of siNC group and siIGF2 group were (37.20± 3.87)% and (18.23±2.25)% respectively, with significant difference (P＜0.01). Conclusion IGF2 gene plays an important role in maintaining the biological characteristics of colon cancer stem cells and promoting self?renewal and stemness of colon CSCs.

Objective To investigate the effect of down?regulation of insulin?like growth factor 2 (IGF2) gene on the biological characteristics of HCT116 colon cancer stem cells (CSCs). Methods Flow cytometry sorting technology was used to isolate CSCs from colon cancer cell line HCT116 by a monoclonal antibody against CD133; serum free floating culture assay was used for the enrichment of CSCs. The proportion of CD133+ cells was analyzed by flow cytometry; CSCs were identified by sphere culturing, immunofluorescence analysis and soft agar clone formation. RT?qPCR method was used to examine transcriptional level of IGF2 gene in CSCs. Western blotting was used to examine IGF2 protein expression in CSCs. siRNA was used to establish IGF2 transient knock down model in CSCs. Cell proliferation array, cell cycle and apoptosis analysis, cell invasion array and colony forming assay were used to further examine the role of IGF2 on the biological characteristics of colon CSCs. Results CSCs were successfully isolated from HCT116 cell lines, which were cultured to form cell spheres in serum?free stem cell culture medium. We found that the morphology of sphere?forming?like cells after several passages maintained the same characteristics as that of the first passage. The results of immunofluorescence showed that CSC markers including CD133 and ALDH continued positively expressing on the cell surface of CSCs, and flow cytometry analysis showed that more than 90% of the spheroid cells remained CD133 positive. The clone formation rate of non?CSCs group and CSCs group were ( 28.10 ± 2.66 )% and ( 43.73 ± 2.30 )% respectively, with significant difference (P＜0.01). The RT?qPCR results showed that the transcriptional level IGF2 gene in non?CSCs group and CSCs group were ( 1.06 ± 0.24) and ( 2.17 ± 0.51) respectively, with significant difference (P＜0.05). The western blot results showed that the protein expression of IGF2 in CSCs group and non?CSCs group were (1.10±0.55) and ( 2.14±0.23) respectively, with significant difference (P＜0.05). Knockdown of IGF2 significantly decreased the percentage of CD133+ cells in CSCs and cell proliferation (P＜0.01). Knockdown of IGF2 increased the percentage of G2／M phase (23.46% of siNC group vs 60.14%of siIGF2 group) and cell apoptosis (2.80% of siNC group vs 40.70% of siIGF2 group), while decreased the percentage of G0／G1 phase (40.77% of siNC group vs 17.73% of siIGF2 group). The invasion results showed that the number of cells penetrating into the basement surface in siNC group and siIGF2 group was (109.00±16.37) and (54.00±8.19) respectively, with significant difference (P＜0.01). The rate of sphere?forming of colon CSCs in siNC group and siIGF2 group were (51.70±7.42)%and (21.27±2.35)% respectively, with significant difference (P＜0.01). The clone formation rate of siNC group and siIGF2 group were (37.20± 3.87)% and (18.23±2.25)% respectively, with significant difference (P＜0.01). Conclusion IGF2 gene plays an important role in maintaining the biological characteristics of colon cancer stem cells and promoting self?renewal and stemness of colon CSCs.

Objective: To investigate the effect of down-regulation of insulin-like growth factor 2 (IGF2) gene on the biological characteristics of HCT116 colon cancer stem cells (CSCs). Methods: Flow cytometry sorting technology was used to isolate CSCs from colon cancer cell line HCT116 by a monoclonal antibody against CD133; serum free floating culture assay was used for the enrichment of CSCs. The proportion of CD133⁺ cells was analyzed by flow cytometry; CSCs were identified by sphere culturing, immunofluorescence analysis and soft agar clone formation. RT-qPCR method was used to examine transcriptional level of IGF2 gene in CSCs. Western blotting was used to examine IGF2 protein expression in CSCs. siRNA was used to establish IGF2 transient knock down model in CSCs. Cell proliferation array, cell cycle and apoptosis analysis, cell invasion array and colony forming assay were used to further examine the role of IGF2 on the biological characteristics of colon CSCs. Results: CSCs were successfully isolated from HCT116 cell lines, which were cultured to form cell spheres in serum-free stem cell culture medium. We found that the morphology of sphere-forming-like cells after several passages maintained the same characteristics as that of the first passage. The results of immunofluorescence showed that CSC markers including CD133 and ALDH continued positively expressing on the cell surface of CSCs, and flow cytometry analysis showed that more than 90% of the spheroid cells remained CD133 positive. The clone formation rate of non-CSCs group and CSCs group were (28.10±2.66)% and (43.73±2.30)% respectively, with significant difference (P<0.01). The RT-qPCR results showed that the transcriptional level IGF2 gene in non-CSCs group and CSCs group were (1.06±0.24) and (2.17±0.51) respectively, with significant difference (P<0.05). The western blot results showed that the protein expression of IGF2 in CSCs group and non-CSCs group were (1.10±0.55) and (2.14±0.23) respectively, with significant difference (P<0.05). Knockdown of IGF2 significantly decreased the percentage of CD133⁺ cells in CSCs and cell proliferation (P<0.01). Knockdown of IGF2 increased the percentage of G₂/M phase (23.46% of siNC group vs 60.14% of siIGF2 group) and cell apoptosis (2.80% of siNC group vs 40.70% of siIGF2 group), while decreased the percentage of G₀/G₁ phase (40.77% of siNC group vs 17.73% of siIGF2 group). The invasion results showed that the number of cells penetrating into the basement surface in siNC group and siIGF2 group was (109.00±16.37) and (54.00±8.19) respectively, with significant difference (P<0.01). The rate of sphere-forming of colon CSCs in siNC group and siIGF2 group were (51.70±7.42)% and (21.27±2.35)% respectively, with significant difference (P<0.01). The clone formation rate of siNC group and siIGF2 group were (37.20±3.87)% and (18.23±2.25)% respectively, with significant difference (P<0.01). Conclusion IGF2 gene plays an important role in maintaining the biological characteristics of colon cancer stem cells and promoting self-renewal and stemness of colon CSCs.