尿刊酸修饰壳聚糖载体负载中性粒细胞明胶酶相关载脂蛋白干扰RNA对结肠癌细胞增殖和迁移及凋亡的实验研究

Experimental research of neutrophil gelatinase-associated lipocalin siRNA encapsulated by urocanic acid-coupled chitosan on colon cancer cells

目的 探讨尿刊酸修饰壳聚糖（UAC）负载siRNA对中性粒细胞明胶酶相关载脂蛋白（NGAL）的基因沉默作用及对结肠癌细胞增殖、迁移和凋亡的影响。方法 采用UAC和壳聚糖（CTS）分别包裹NGAL siRNA转染结肠癌细胞株HT-29，采用实时荧光定量PCR检测NGAL的表达量，并分析NGAL基因沉默与细胞增殖、迁移及凋亡的关系。结果 荧光显微镜下显示，UAC-siRNA的转染效率为（37.52 ± 7.17）%，高于CTS-siRNA的（11.32 ± 3.39）%，差异有统计学意义（t= 6.102，P= 0.005）。siRNA转染48 h后，以GAPDH为内参，测得UAC组NGAL基因的相对表达量为0.350，CTS组NGAL基因的相对表达量为0.529，差异有统计学意义（t=-3.743，P= 0.02）。增殖实验显示，UAC和CTS两组与对照组相比，细胞增殖率稍有下降，但差异无统计学意义（F=9.520，P= 0.438）。而迁移实验显示，UAC组和CTS组HT29细胞的24 h迁移率均低于对照组（F= 6.756，P= 0.029）；其中UAC组HT29细胞的24 h迁移率为（77.90 ± 7.14）%，低于CTS组的（87.67 ± 3.98）%（t=-1.704，P= 0.164）。凋亡检测显示，转染后2 d，UAC组细胞凋亡率为（15.800 ± 1.054）%，高于CTS组的（12.900 ± 0.656）%和对照组的（11.933 ± 1.914）%，差异有统计学意义（F= 7.004，P= 0.027）。结论 CTS经尿刊酸修饰为UAC后，包裹能力及转染效率显著增加；以UAC为载体的siRNA干扰NGAL基因后，可沉默HT29结肠癌细胞株NGAL基因的表达，抑制HT29结肠癌细胞迁移，促进细胞凋亡。

Objective To explore the impact of neutrophil gelatinase-associated lipocalin （NGAL） knockdown by NGAL siRNA encapsulated with urocanic acid-modified chitosan nanoparticles （UAC） on the proliferation, migration and apoptosis of human colon cancer cells.Methods NGAL siRNA was encapsulated by UAC and chitosan （CTS） respectively, and then was transfected into human colon cancer cell lines HT29. The NGAL mRNA was detected by real-time quantitative PCR （RT-QPCR） . Relationships of NGAL gene silencing with the proliferation, migration and apoptosis of HT29 cell were analyzed.Results Under the fluorescence microscope, the transfection efficiency of siRNA in UAC group was （37.52 ± 7.17） %, which was significantly higher than （11.32 ± 3.39） % in CTS group （t= 6.102, P= 0.005） . Forty-eight hours after transfection, RT-QPCR examination showed that the level of NGAL mRNA expression was 0.350 in UAC group and 0.529 in CTS group with significant difference （t=-3.743, P= 0.02） , meanwhile both levels were significantly lower as compared to control group （F= 163.538, P 〈 0.001） . Proliferation analysis revealed that after silencing NGAL gene, proliferation rate of UAC group and CTS group was slightly lower than control group, and no significant differences were found （F= 9.520, P= 0.438） . However, migration assay demonstrated that the 24-hour migration rate of UAC group and CTS group was significantly lower than that of control group （F= 6.756, P= 0.029） , meanwhile the migration rate of UAC group was slightly lower than that of CTS group[ （77.90 ± 7.14） % vs. （87.67 ± 3.98） %, t=-1.704, P= 0.164]. Apoptosis detection revealed that the apoptosis rate in UAC group was significantly higher than that in CTS group and the control group 2 days after transfection[ （15.800 ± 1.054） % vs. （12.900 ± 0.656） %, （11.933 ± 1.914） %, F=7.004, P= 0.027].Conclusions The encapsulated ability and transfection efficiency of chitosan modified by urocanic acid elevate significantly. Silencing NGAL gene by UAC carrier can down-regulate the expression of NGAL mRNA in HT29 colon cell line, inhibit their migration and facilitate their apoptosis.