以KRAS启动子G-四链体DNA为靶点的小檗碱对人结肠癌细胞SW620的作用及机制

Function and Mechanism of Berberine on Colon Cancer Cell SW620 Based on KRAS Promoter G-quadruplex DNA Target

目的：通过研究小檗碱对结肠癌细胞增殖的影响以及小檗碱是否通过调控Kirsten rat sarcoma viral oncogene homolog（KRAS）启动子G-四链体结构抑制结肠癌细胞Kirsten rat sarcoma viral oncogene homolog（KRAS）mRNA的表达,探究以KRAS启动子G-四链体DNA为靶点的小檗碱对人结肠癌细胞系SW620的作用及可能机制。方法：采用噻唑蓝（MTT）比色法检测小檗碱对SW620细胞的增殖抑制率。采用荧光光谱研究小檗碱和KRAS启动子G-四链体DNA的结合能力,采用阴离子淬灭实验和荧光各向异性及荧光偏振实验研究二者结合的结合模式,采用圆二色光谱方法研究小檗碱对KRAS启动子G-四链体DNA构型的影响,采用圆二色光谱变温实验研究小檗碱对KRAS启动子G-四链体DNA构型热稳定性的影响,采用实时荧光定量聚合酶链式反应（Real-time PCR）检测小檗碱对SW620细胞KRAS基因转录的影响。结果：小檗碱可以抑制SW620细胞的增殖,且呈剂量依赖性。KRAS启动子G-四链体与小檗碱以近1∶1计量比结合,二者的结合常数为（0.93±0.21）×10^6L·mol^-1,结合能力较强,结合模式为末端堆积和沟槽结合的复合模式。小檗碱可维持KRAS启动子的平行G-四链体构型及热稳定性。100μmol·L^-1小檗碱能抑制SW620细胞KRAS Mrna的表达。结论：小檗碱能够与KRAS启动子G-四链体DNA结合并维持其平行构型的稳定性,小檗碱可能通过调控KRAS启动子G-四链体结构下调SW620细胞KRAS基因转录,这可能是小檗碱抑制其细胞增殖的机制之一。KRAS启动子区域G-四链体结构可能成为结肠癌治疗的新靶点。

Objective： To explore the function and possible mechanism of berberine on colon cancer cell SW620 based on Kirsten rat sarcoma viral oncogene homolog（ KRAS） promoter G-quadruplex target, by investigating its effect on colon cancer cells proliferation and whether berberine could inhibit mRNA expression of KRAS in colon cancer cells through regulating its promoter G-quadruplex. Method： The inhibition rate of berberine on SW620 cell lines proliferation was determined by using 3-（ 4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-Htetrazolium bromide（ MTT） assay. The binding capacity of KRAS G-quadruplex DNA with berberine was detected by methods of fluorescence spectra,and the anionic quenching experiment,and fluorescence anisotropy and fluorescence polarization experiment were used to investigate their binding mode. The effect of berberine on conformation of KRAS G-quadruplex was studied by using circular dichroism spectra; the effect of berberine on thermal stability of KRAS G-quadruplex was studied by using circular dichroism spectra variable-temperature experiment,and the effect of berberine on KRAS genetic transcription of SW620 cells was detected by real-time quantitative polymerase chain reaction（ Real-time PCR）. Result： Berberine can inhibit the proliferation of SW620 cells in a dose dependent manner. KRAS G-quadruplex DNA had a stronger binding capacity with berberine at a stoichiometric ratio of about 1 ∶ 1,with a binding constant of（ 0. 93 ± 0. 21） × 10^6 L·mol^-1,under a binding mode of complex patterns of terminal stacking and groove binding. Berberine can keep the parallel conformation and thermostability of KRAS G-quadruplex,and 100 μmol·L^-1 berberine can inhibit the Mrna expression of KRAS in SW620 cells. Conclusion： Berberine can bind with KRAS promoter G-quadruplex DNA and keep the thermostability of its parallel conformation,and may down-regulate KRAS genetic transcription of SW620 cells by affecting its G-quadruplex structure,which may be one of the mechanisms for inhibiting the proliferation of cells.KRAS promoter G-quadruplex structure may become a new target in colon cancer therapy.