过表达miR-613通过靶向下调Wee1提高结直肠癌细胞放疗敏感性的机制研究

Overexpression of miR-613 enhanced radiosensitivity of colorectal cancer cells via targeting downregulation of Wee1

目的研究微小RNA-613(miR-613)/Wee1分子轴影响结直肠癌细胞放疗敏感性的分子机制。方法选取2016年11月至2017年5月昆明医科大学附属延安医院收治的放疗敏感结直肠癌患者20例、放疗抵抗患者20例,另选取人结直肠癌细胞株LoVo、HCT116,并建立放疗抵抗细胞株LoVo/R、HCT116/R,采用实时荧光定量PCR(qRT-PCR)检测miR-613和Wee1在结直肠癌组织和细胞株中的表达情况。在结直肠癌放疗抵抗细胞中转染miR-613 mimic,未转染细胞为对照组,采用CCK-8、Transwell和Western blotting方法检测在不同放疗剂量下,过表达miR-613对细胞增殖活力、侵袭能力和细胞周期的影响。进一步实验,采用双荧光素酶报告基因验证miR-613与Wee1的靶向关系。在结直肠癌放疗抵抗细胞中转染si-Wee1或同时转染si-Wee1和miR-613 inhibitor,未转染细胞为对照组,采用CCK-8、Transwell和Western blotting方法检测在不同放疗剂量下,miR-613/Wee1分子轴对细胞增殖活力、侵袭能力和细胞周期的影响。结果miR-613在放疗抵抗结直肠癌患者组织中的表达量低于放疗敏感患者(1.54±0.25∶2.64±0.45;t=3.140,P=0.009),其在放疗抵抗结直肠癌细胞株中低表达(LoVo/R∶LoVo为1.03±0.12∶3.05±0.15,t=8.944,P=0.006;HCT116/R∶HCT116为1.01±0.11∶2.85±0.16,t=8.050,P=0.008)。同时,与对照组相比,过表达miR-613显著抑制结直肠癌放疗抵抗LoVo/R和HCT116/R细胞增殖(LoVo/R细胞:t6 Gy=6.018,P=0.013;HCT116/R细胞:t6 Gy=5.634,P=0.015),抑制细胞侵袭(LoVo/R细胞:45.00±8.95∶180.15±6.95,t6 Gy=11.93,P=0.003;HCT116/R细胞:49.97±6.21∶170.20±7.03,t6 Gy=12.82,P=0.006),并下调G2-M期相关蛋白细胞周期蛋白依懒性激酶1(CDK1)和cyclin B的表达水平。此外,双荧光素酶报告基因证实miR-613靶向作用于Wee1。进一步实验发现,miR-613通过靶向下调Wee1显著抑制LoVo/R和HCT116/R细胞增殖(转染si-Wee1、同时转染si-Wee1和miR-613 inhibitor以及对照组3组比较,LoVo/R细胞:F8 Gy=40.742,P=0.007;HCT116/R细胞:F8 Gy=28.958,P=0.011),抑制细胞侵袭(LoVo/R细胞:F8 Gy=55.413,P=0.004;HCT116/R细胞:F8 Gy=65.634,P=0.003),并将细胞阻滞在G2-M期,进而上调LoVo/R和HCT116/R细胞对放疗的敏感性。结论miR-613/Wee1分子轴与结直肠癌细胞放疗抵抗性存在调控关系,且过表达miR-613可逆转结直肠癌细胞的放疗抵抗性。

Objective To explore the effect of microRNA-613 (miR-613)/Wee1 axis on the radiosensitivity of colorectal cancer cells. Methods A total of 20 patients with radiosensitive colorectal cancer and 20 patients with radioresistance were selected from Yan′an Hospital Affiliated to Kunming Medical University from November 2016 to May 2017. Human colorectal cancer cell lines LoVo and HCT116 were selected and the radioresistant cell lines LoVo/R and HCT116/R were established for subsequent experiments. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of miR-613 and Wee1 in colorectal can-cer tissues and cell lines. The radioresistant cells were transfected by miR-613 mimic, and non-transfected cells were used as control group. The effects of miR-613 overexpression on the proliferation, invasion and cell cycle of radiation resistance of colorectal cancer cells at different radiation doses were evaluated by CCK-8 assay, Transwell assay and Western blotting, respectively. Furthermore, dual-luciferase reporter gene assay was used to verify whether Wee1 was a target gene of miR-613. si-Wee1 was transfected into radioresistant cells of colo-rectal cancer, or co-transfected with si-Wee1 and miR-613 inhibitor, and non-transfected cells were used as control group. The effects of miR-613/Wee1 axis on cell proliferation, invasion and cell cycle were detected by CCK-8, Transwell and Western blotting at different radiation doses. Results The expression of miR-613 was downregulated in the radiation resistance group of patients (1.54±0.25 vs. 2.64±0.45;t=3.140, P=0.009) and radiation resistance cell lines (LoVo/R vs. LoVo: 1.03±0.12 vs. 3.05±0.15;t=8.944, P=0.006;HCT116/R vs. HCT116: 1.01±0.11 vs. 2.85±0.16;t=8.050, P=0.008). Overexpression of miR-613 was significantly inhibited the proliferation (LoVo/R: t6 Gy=6.018, P=0.013;HCT116/R: t6 Gy=5.634, P=0.015) and invasion (LoVo/R: 45.00±8.95 vs. 180.15±6.95, t6 Gy=11.93, P=0.003;HCT116/R: 49.97±6.21 vs. 170.20±7.03, t6 Gy=12.82, P=0.006) of LoVo/R and HCT116/R cells and decreased the expression levels of G2-M phase cell cycle correlated proteins (CDK1 and cyclin B). Moreover, dual-luciferase reporter gene assay confirmed that Wee1 was a target of miR-613. Mechanistically, overexpression of miR-613 promoted the radiosensitivity of LoVo/R and HCT116/R cells through inhibiting cell proliferation (compared with si-Wee1 group, co-transfected with si-Wee1 and miR-613 inhibitor, and control group, LoVo/R: F8 Gy=40.742, P=0.007;HCT116/R: F8 Gy=28.958, P=0.011), invasion (LoVo/R: F8 Gy=55.413, P=0.004;HCT116/R: F8 Gy=65.634, P=0.003) and arresting cell at G2-M phase via downregulating Wee1. Conclusion miR-613/Wee1 axis plays a certain role in regulating the radiation resistance of colo-rectal cancer cells, overexpression of miR-613 may reverse the radiation resistance of colorectal cancer cells.