【摘要】 目的探讨分化抑制因子3(Id3)协同Id1对结直肠癌细胞干性的影响,并阐明其可能的机制。方法应用慢病毒感染系统Id1短发夹RNA(shRNA)质粒构建Id1单基因敲减(sh-Id1)、sh-Id3和双基因敲减(sh-Id1Id3)的肠癌HCT116细胞株,荧光显微镜下观察荧光强度,实时荧光定量PCR和Western印迹法检测敲减效果。实验分为4组:HCT116空载质粒对照组(简称对照组)、sh-Id1组、sh-Id3组和Sh-Id1Id3组。实时无标记细胞检测技术(RTCA)检测细胞增殖信号,克隆形成实验检测细胞克隆形成数量和直径,流式细胞术AnnexinⅤ/PI法检测细胞凋亡,体外无血清悬浮培养法检测细胞成球的数量和大小,流式细胞术检测基因敲减的HCT116细胞CD24和CD133表达;Western印迹法检测基因敲减的HCT116细胞及过表达c-myc后CD24、CD133、EpCAM、Oct4、EZH2、Lgr5和β联蛋白表达水平。结果荧光显微镜下可见对照组、sh-Id1组、sh-Id3组和Sh-Id1Id3组细胞绿荧光率均>90%;实时荧光定量PCR和Western印迹结果显示,与对照组相比,sh-Id1组Id1表达降低(均P<0.01),sh-Id3组Id3表达降低(均P<0.01),sh-Id1Id3组的Id1和Id3表达均降低(均P<0.01),说明sh-Id1,sh-Id3和sh-Id1Id3的HCT116细胞株构建成功。RTCA结果显示,与对照组相比,sh-Id1,sh-Id3和sh-Id1Id3组细胞增殖信号比对照组显著降低(P<0.05)。克隆形成实验结果显示,sh-Id1,sh-Id3和sh-Id1Id3组克隆形成的数量及大小均比对照组显著降低(P<0.05)。流式细胞术检测细胞凋亡结果显示,sh-Id1,sh-Id3和sh-Id1Id3组细胞凋亡显著高于对照组(P<0.05);无血清悬浮培养实验结果显示,sh-Id1,sh-Id3和sh-Id1Id3组细胞球性形成率均比对照组显著降低(P<0.05)。流式细胞术结果显示,与对照组相比,sh-Id1,sh-Id3和sh-Id1Id3组中CD133+和CD24+细胞比例均显著降低(P<0.05),且上述结果中,sh-Id1Id3组与sh-Id1和Sh-Id3组相比显著降低(P<0.05)。Western印迹结果显示,与对照组相比,sh-Id1,sh-Id3和sh-Id1Id3组CD24、CD133(CD133/2亚型)、EpCAM、Oct4、EZH2、β联蛋白、Lgr5蛋白表达均显著性下调(P<0.05)。sh-Id1、sh-Id3和sh-Id1Id3组中Wnt通路的下游分子c-myc、周期蛋白D1和凋亡抑制因子蛋白水平均明显下降(P<0.05),同时Shh通路关键分子GLI1,GLI2和Shh蛋白水平明显下调(P<0.05),兔抗人Hedgehes(PTCH)2、融合抑制剂(SUFU)显著升高(P<0.05)。实验过表达c-myc后,sh-Id1,sh-Id3和sh-Id1Id3降低的干性标志物CD133,Oct4,EZH2,EpCAM和Lgr5蛋白表达均重新上调(P<0.05)。结论 Id3和Id1通过Wnt/β联蛋白、Shh通路共同调控直肠癌细胞的干性相关蛋白的表达。更多还原

【Abstract】 OBJECTIVE To explore the effect of inhibitor of differentiation 3(Id3) and Id1 on the stemness of colonrectal cancer cells, and clarify its possible mechanism. METHODS The lentiviral infection system was used to construct the Id1 and Id3 single gene knockdown(sh-Id1, sh-Id3) and double gene knockdown HCT116 cell lines(sh-Id1 Id3). The fluorescence intensity was observed under a fluorescence microscope, and detected by real-time quantitative PCR(qRT-PCR) and Western blotting. The experiments were divided into four groups: HCT116 no-load plasmid control(control), sh-Id1,sh-Id3, and sh-Id1 Id3 groups. Real time cel I ana Iysis(RTCA) was used to detect cell proliferation signals,while clone formation experiments were conducted to detect the amount and size of cell cloning. AnnexinⅤ/PI method was employed to detect apoptosis, in vitro serum-free suspension culture method was used to detect the amount and size of cell formation, and flow cytometry was adopted to detect CD24 and CD133 expressions. The expressions of CD24, CD133, EpCAM, Oct4, EZH2, Lgr5 and β-catenin were detected by Western blotting, and the expressions of these proteins were also detected after overexpression of c-myc. RESULTS The green fluorescence rates of control, sh-Id1, sh-Id3 and sh-Id1 Id3 groups were all over 90% under a fluorescence microscope. The results of q RT-PCR and Western blotting showed that the expression of Id1 in sh-Id1 group was decreased, so did the expression of Id3 in sh-Id3 group, and the expressions of Id1 and Id3 in sh-Id1 Id3 group, indicating that the HCT116 cell lines of sh-Id1, sh-Id3 and sh-Id1 Id3 were constructed. RTCA results showed that the proliferation signals of sh-Id1, sh-Id3 and sh-Id1 Id3 groups decreased significantly(P<0.05). The number and size of clones in sh-Id1, sh-Id3 and sh-Id1 Id3 groups were significantly smaller than those in the control group(P<0.05). The results of flow cytometry showed that the apoptosis of sh-Id1, sh-Id3 and sh-Id1 Id3 groups was significantly higher than that of the control group(P<0.05). The results of serum-free suspension culture showed that the spheroid formation rate of sh-Id1, sh-Id3 and sh-Id1 Id3 groups was significantly lower than that of the control group(P<0.05). The results of flow cytometry showed that the proportions of CD133+and CD24+cells in the sh-Id1, sh-Id3 and sh-Id1 Id3 groups were significantly different from those in the control group(P<0.05), and that there was also significant difference between sh-Id1 Id3 group and sh-Id1, sh-Id3 groups(P<0.05). Western blot results showed that the protein expressions of CD24, CD133(CD133/2 subtype), EpCAM, Oct4, EZH2, β-catenin and Lgr5 were significantly down regulated in the sh-Id1, sh-Id3 and sh-Id1 Id3 groups compared with the control group(P<0.05). The study on Wnt signaling pathway indicated that the protein levels of c-myc, cyclin D1 and inhibitor of apoptosis of Wnt pathway in the sh-Id1, sh-Id3 and sh-Id1 Id3 groups were significantly decreased(P<0.05), so did the protein levels of GLI1, GLI2 and Shh, the key molecules of Shh pathway(P<0.05), while PTCH2 and SUFU were significantly increased(P<0.05). At the same time, after overexpression of c-myc, the expressions of CD133, Oct4, EZH2, EpCAM and Lgr5 were up-regulated a second time(P<0.05). CONCLUSION Id3 and Id1 work together to regulate the expressions of stemness related proteins of CRC through Wnt/β-catenin and Shh pathways.更多还原