下调胰岛素样生长因子2基因对HCT116结肠癌干细胞生物学特性的影响

Effects of down-regulation of IGF2 gene on the biological characteristics of HCT116 colon cancer stem cells

目的探讨下调胰岛素样生长因子2(IGF2)基因对HCT116结肠癌干细胞生物学特性的影响。方法采用流式细胞分选技术利用CD133抗体从结肠癌HCT116细胞株分离结肠癌干细胞。采用流式细胞术检测分离前后CD133+细胞的比例,通过成球实验、免疫荧光染色、流式细胞术测定肿瘤干细胞标志蛋白的表达和软琼脂克隆形成实验等方法鉴定结肠癌干细胞。采用实时荧光定量聚合酶链反应(RT-qPCR)和Western blot检测结肠癌干细胞中IGF2的表达,应用小干扰RNA建立IGF2瞬时干涉细胞模型,通过细胞增殖实验、细胞周期和凋亡测定、细胞侵袭实验、克隆形成实验等观察下调IGF2对结肠癌干细胞生物学特性的影响。结果从结肠癌HCT116细胞中成功分离出干细胞,并可在无血清干细胞培养基中培养形成细胞球,连续消化传代后仍能保持与原代相同的形态特征。免疫荧光染色分析显示,干细胞标志物CD133和乙醛脱氢酶(ALDH)在结肠癌干细胞表面持续阳性表达。在软琼脂培养基上培养13 d后,非肿瘤干细胞组的克隆形成率为(28.10±2.66)%,肿瘤干细胞组的克隆形成率为(43.73±2.30)%,差异有统计学意义(P<0.01)。RT-qPCR结果显示,非肿瘤干细胞组和肿瘤干细胞组中IGF2的基因表达水平分别为1.06±0.24和2.17±0.51,差异有统计学意义(P<0.05)。Western blot检测结果显示,非肿瘤干细胞组和肿瘤干细胞组中IGF2的蛋白表达水平分别为1.10±0.55和2.14±0.23,差异有统计学意义(P<0.05)。下调IGF2基因后,结肠癌干细胞的增殖受到抑制(P<0.01);结肠癌干细胞中CD133+细胞的比例明显降低;细胞周期中G2/M期细胞比例增高(siNC组为23.46%,siIGF2组为60.14%),同时G0/G1期细胞比例降低(siNC组为40.77%,siIGF2组为17.73%);细胞凋亡率显著增高(siNC组为2.80%,siIGF2组为40.70%);siNC组和siIGF2组穿透至基底膜下室面的细胞数分别为109.00±16.37和54.00±8.19,差异有统计学意义(P<0.01);siNC组和siIGF2组的干细胞成球率分别为(51.70±7.42)%和(21.27±2.35)%,差异有统计学意义(P<0.01);siNC组和siIGF2组的克隆形成率分别为(37.20±3.87)%和(18.23±2.25)%,差异有统计学意义(P<0.01)。结论IGF2基因在维持结肠癌干细胞生物学特性中发挥重要作用,并促进了干细胞的自我更新和干性维持。

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.