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目的结直肠癌(colorectal cancer,CRC)是世界范围内最常见的恶性肿瘤之一,本研究探讨应用小分子干扰RNA(siRNA)沉默Twist信号通路对结直肠癌顺铂化疗敏感性的影响及其可能机制。方法 0、0.0625、0.125、0.25、0.5和1μg/mL的顺铂作用于SW480和LoVo这种结直肠癌细胞株48h。Twist-siRNA转染这2种结直肠癌细胞株。CCK-8法计算转染前后顺铂对于两者的抑制率。EdU法检测癌细胞增殖能力。Hoechest 3344荧光染色检测细胞形态。蛋白印迹法检测不同药物作用下的Twist及上皮间质化相关标志物的表达变化。结果 LoVo和SW480细胞组中Twist表达量分别为0.824±0.124和0.191±0.039 9;Twist在LoVo中高表达,在SW480中低表达,差异有统计学意义,P<0.05。SW480和LoVo对于顺铂的IC50值分别为(3.394±0.083)和(6.737±0.796)μg/mL,P<0.05。wist siRNA干扰后SW480和LoVo对于顺铂的IC50值分别为(0.113 7±0.008 8)和(0.459 7±0.025 0)μg/mL,与单用顺铂组相比,差异均有统计学意义,均P<0.05。Hoechest3344荧光染色结果表明,Twist转染组比未转染组出现了更加明显的细胞凋亡形态变化。CCK-8检测结果显示,采用Twist siRNA干扰后SW480和LoVo对于顺铂的IC50值分别为(0.113 7±0.008 8)和(0.459 7±0.025 0)μg/mL,与单用顺铂组相比,差异有统计学意义,P<0.05。EdU法检测结果显示,Twist siRNA+顺铂组、顺铂组和正常组SW480细胞增殖率分别为(14.63±1.33)%、(25.55±1.73)%和(51.23±3.31)%;LoVo细胞增殖率分别为(22.35±2.39)%、(29.27±1.85)%和(64.62±3.61)%;与正常组和单用顺铂组相比,Twist siRNA+顺铂组的细胞增殖率明显降低,差异均有统计学意义,均P<0.05。蛋白印迹法结果表明,Twist siRNA作用的同时伴随E-cadherin表达升高,而Vimentin表达降低,差异有统计学意义,P<0.05。结论应用siRNA降低Twist信号通路活性可通过抑制上皮间质化过程而增加结直肠癌对于顺铂的化疗敏感性。
OBJECTIVE: Colorectal cancer (CRC) is one of the most common malignant tumors in the world. This study investigated the effect of silencing Twist signaling pathway on the chemosensitivity of cisplatin to colorectal cancer using small interfering RNA (siRNA) Possible mechanism. Methods 0, 0.0625, 0.125, 0.25, 0.5 and 1 μg / mL of cisplatin were applied to SW480 and LoVo colorectal cancer cell lines 48h. Twist-siRNA transfected these two colorectal cancer cell lines. The CCK-8 method was used to calculate the inhibition rate of cisplatin before and after transfection. EdU assay of cancer cell proliferation. Hoechest 3344 fluorescence staining for cell morphology. Western blotting was used to detect the expression changes of Twist and epithelial-mesenchymal-related markers under the influence of different drugs. Results The expression of Twist in LoVo and SW480 cells was 0.824 ± 0.124 and 0.191 ± 0.0399, respectively. Twist was highly expressed in LoVo and low in SW480, with statistical significance (P <0.05). The IC50 values of SW480 and LoVo for cisplatin were (3.394 ± 0.083) and (6.737 ± 0.796) μg / mL, respectively, P <0.05. The IC50 values of SW480 and LoVo against cisplatin were (0.113 7 ± 0.008 8) and (0.459 7 ± 0.025 0) μg / mL respectively after wist siRNA interference, which were significantly different from that of cisplatin alone. All P <0.05. Hoechest3344 fluorescence staining results showed that Twist transfection group than the untransfected group appeared more obvious morphological changes of apoptosis. The results of CCK-8 showed that the IC50 values of SW480 and LoVo for cisplatin were (0.113 7 ± 0.008 8) and (0.459 7 ± 0.025 0) μg / mL, respectively, compared with those treated with cisplatin alone , The difference was statistically significant, P <0.05. The results of EdU assay showed that the proliferation rates of SW480 cells in Twist siRNA group, cisplatin group and normal group were (14.63 ± 1.33)%, (25.55 ± 1.73)% and (51.23 ± 3.31)%, respectively. The proliferation rates of LoVo cells (22.35 ± 2.39)%, (29.27 ± 1.85)% and (64.62 ± 3.61)%, respectively. Compared with normal group and cisplatin alone, the cell proliferation rate of Twist siRNA + cisplatin group was significantly lower Significance, both P <0.05. The results of Western blotting showed that the expression of E-cadherin increased while the expression of Vimentin decreased in Twist siRNA group. The difference was statistically significant (P <0.05). Conclusions The application of siRNA to decrease the activity of Twist signaling pathway can increase the chemosensitivity of cisplatin to colorectal cancer by inhibiting the process of epithelialization.