黄芪多糖通过调控miR-20a/TGFBR2 分子轴降低结直肠癌HT-29/DDP细胞的顺铂耐药性

摘要] 目的：探讨黄芪多糖（APS）通过调控miR-20a/TGFBR2 分子轴对结直肠癌（CRC）顺铂耐药细胞HT-29/DDP增殖、侵袭、凋亡和耐药性的影响及其机制。方法：以人CRC HT-29 细胞、HT-29/DDP细胞为亲本和耐药细胞模型，将HT-29/DDP细胞随机分为4 组：对照组、APS 处理组、过表达miR-20a + APS 组、沉默TGFBR2 + APS 组。用不同质量浓度的APS（0、0.5、1.0、1.5 和2.0 mg/ml）处理HT-29/DDP细胞后，以qPCR和Wb实验检测细胞中miR-20a 和TGFBR2 的表达水平；用CCK-8、Transwell 和Annexin V-FITC/PI 染色流式细胞术检测对HT-29/DDP细胞增殖、侵袭和凋亡的影响；用双荧光素酶报告基因验证miR-20a 与TGFBR2的靶向作用关系。构建裸鼠皮下CRC HT-29/DDP细胞移植瘤模型，观察APS对移植瘤生长的影响及其机制。结果：APS显著抑制HT-29/DDP 细胞的增殖（P<0.01），且呈剂量依赖性。miR-20a 在经APS 处理后的HT-29/DDP 细胞中低表达（P<0.01）、TGFBR2 的表达水平显著上调（P<0.01）。双荧光素酶报告基因证实miR-20a 靶向作用TGFBR2 并下调其表达水平。体内外实验证实APS通过靶向下调miR-20a 对TGFBR2 的抑制作用，提高HT-29/DDP细胞的药物敏感性，进而抑制该细胞增殖、侵袭和促进凋亡（均P<0.01)；同时发现，该作用与抑制PCNA、Bcl-2 蛋白而促进Bax、Caspase-3 蛋白表达有关联。结论：APS通过下调miR-20a对TGFBR2 表达的抑制作用，从而逆转HT-29/DDP细胞对顺铂的耐药性。

Astragalus polysaccharides suppressed cisplatin-resistance of colorectal cancer TH-29/DDP cells via regulating miR-20a/TGFBR2 axis

Abstract] Objective: To investigate the effect of astragalus polysaccharides (APS) on proliferation, invasion, apoptosis and drugresistance of cisplatin-resistant colorectal cancer (CRC) HT-29/DDP cells through regulating miR-20a/TGFBR2 axis, and to explore the possible mechanism. Methods: Human CRC HT-29 cells and HT-29/DDP cells were used as non-drug resistant and resistant cell models,respectively; HT-29/DDP cells were randomly divided into four groups, including untreated (HT-29/DDP) group, APS treatment group, miR-20a mimics + APS group, and si-TGFBR2 + APS group. qPCR and Western blotting were applied to detect the expressions of miR-20a and TGFBR2 in HT-29/DDP cells treated with different concentrations of APS (0, 0.5, 1.0, 1.5 and 2.0 mg/ml). Subsequently,dual luciferase reporter gene assay was used to verify whether TGFBR2 was a target gene of miR-20a. In addition, CCK-8, Transwell and Annexin V-FITC/PI double staining were applied to examine the effect of APS on proliferation, invasion and apoptosis of HT-29/DDP cells. Furthermore, subcutaneous HT-29/DDP cell xenograft model was established on nude mice, and the effect of APS on the growth of transplanted tumor was observed. Results: APS significantly inhibited the proliferation of HT-29/DDP cells in a dose-dependent manner (P<0.01). Meanwhile, the expression of miR-20a was down-regulated in HT-29/DDP cells treated with APS, while the expression of TGFBR2 was significantly up-regulated (all P<0.01). Additionally, dual luciferase reporter gene assay result showed that TGFBR2 was a direct target of miR-20a in HT-29/DDP cells and its expression was suppressed. Furthermore, APS could enhance the drug sensitivity of HT-29 /DDP cells through downregulating the inhibitory effect of miR-20a on TGFBR2 expression, thereby suppressed proliferation and invasion, and induced apoptosis of HT-29/DDP cells in vitro and in vivo. It was also found that this effect was related with the suppression of PCNA and Bcl-2 proteins and promotion of Bax and Caspase-3 proteins. Conclusion: APS reverses the resistance of HT-29/DDP cells to cisplatin by down-regulating the inhibitory effect of miR-20a on TGFBR2 expression.