国际肿瘤学杂志

国际肿瘤学杂志 ›› 2021, Vol. 48 ›› Issue (10): 583-590.doi: 10.3760/cma.j.cn371439-20201208-00115

• 论著 • 上一篇    下一篇

麦冬皂苷D联合沉默环氧合酶-2基因对人胰腺癌BxPC-3细胞增殖、迁移和侵袭的影响

钟扬1,2, 何苗1,2, 刘志1,2, 陈建宇1,2, 张光年1,2, 秦龙3, 李婷3, 李建水1,2()   

  1. 1川北医学院附属医院肝胆外二科,南充 637000
    2川北医学院肝胆胰肠疾病研究所,南充 637000
    3四川省南充市中心医院胃肠外科 637000
  • 收稿日期:2020-12-08 修回日期:2021-05-19 出版日期:2021-10-08 发布日期:2021-11-24
  • 通讯作者: 李建水 E-mail:ljs2005doctor@126.com
  • 基金资助:
    南充市市校科技战略合作专项资金项目(18SXHZ0523)

Effects of Ophiopogon D combined with cyclooxygenase-2 silencing on proliferation, migration and invasion of human pancreatic cancer BxPC-3 cells

Zhong Yang1,2, He Miao1,2, Liu Zhi1,2, Chen Jianyu1,2, Zhang Guangnian1,2, Qin Long3, Li Ting3, Li Jianshui1,2()   

  1. 1Second Department of Hepatobiliary Surgery, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China
    2Institute of Hepato-Biliary-Pancreas and Intestinal Disease, North Sichuan Medical College, Nanchong 637000, China
    3Department of Gastrointestinal Surgery, Nanchong Central Hospital of Sichuan Province, Nanchong 637000, China
  • Received:2020-12-08 Revised:2021-05-19 Online:2021-10-08 Published:2021-11-24
  • Contact: Li Jianshui E-mail:ljs2005doctor@126.com
  • Supported by:
    Nanchong City School Science and Technology Strategic Cooperation Special Fund(18SXHZ0523)

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摘要：

目的 探讨麦冬皂苷D联合沉默环氧合酶-2(COX-2)基因对人胰腺癌BxPC-3细胞增殖、迁移、侵袭的影响。方法 将BxPC-3细胞分为空白对照组、麦冬皂苷D高剂量组(40 μmol/L)、中剂量组(20 μmol/L)、低剂量组(10 μmol/L);将沉默COX-2后的细胞分为空白组、COX-2抑制组(50 pmol/ml siRNA-COX-2)、麦冬皂苷D组(20 μmol/L)与联合用药组(麦冬皂苷D 20 μmol/L+50 pmol/ml siRNA-COX-2),采用CCK-8法检测细胞增殖活力,划痕实验检测细胞迁移距离,Transwell法检测细胞侵袭程度,采用实时定量PCR(RT-qPCR)法检测BxPC-3细胞中COX-2基因的相对表达水平,蛋白质印迹法检测BxPC-3细胞中COX-2、缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)蛋白相对表达水平。结果 空白对照组,麦冬皂苷D高、中、低剂量组的细胞增殖率分别为(100.0±4.9)%、(71.8±5.4)%、(80.5±5.8)%和(89.7±5.7)%,细胞迁移距离分别为(279.8±24.0)μm、(141.9±21.2)μm、(168.8±37.1)μm和(224.6±19.9)μm,侵袭数量吸光度(A)值分别为1.107±0.095、0.390±0.030、0.596±0.017和0.826±0.034,差异均具有统计学意义(F=19.770,P<0.001;F=48.270,P<0.001;F=198.400,P<0.001),麦冬皂苷D高、中、低剂量组均明显低于空白对照组(均P<0.05);COX-2基因相对表达水平分别为1.007±0.178、0.387±0.169、0.567±0.142和0.740±0.030,蛋白相对表达水平分别为1.000±0.033、0.654±0.085、0.762±0.110和0.881±0.049,差异均具有统计学意义(F=10.280,P=0.004;F=11.780,P=0.003),麦冬皂苷D高、中剂量组均低于空白对照组(均P<0.05),麦冬皂苷D低剂量组与空白对照组差异均无统计学意义(均P>0.05);选择麦冬皂苷D中剂量(20 μmol/L)作为后续实验浓度。沉默COX-2后,空白组、COX-2抑制组、麦冬皂苷D组和联合用药组的细胞增殖率分别为(100.0±2.8)%、(68.4±6.7)%、(67.7±5.9)%和(57.0±8.5)%,迁移距离分别为(274.4±23.8)μm、(217.0±18.8)μm、(186.2±18.6)μm和(115.7±15.8)μm,侵袭数量A值分别为1.143±0.092、0.791±0.058、0.715±0.026和0.424±0.058,差异均具有统计学意义(F=34.430,P<0.001;F=103.400,P<0.001;F=131.100,P<0.001),各处理组细胞的增殖率、迁移距离、侵袭数量均明显低于空白组(均P<0.001);与COX-2抑制组和麦冬皂苷D组相比,联合用药组细胞增殖、迁移、侵袭能力均被明显抑制(均P<0.05);COX-2抑制组与麦冬皂苷D组相比,仅迁移距离差异有统计学意义(P<0.05)。各组COX-2蛋白相对表达水平分别为0.995±0.037、0.779±0.060、0.806±0.076和0.645±0.079,HIF-1α蛋白相对表达水平分别为1.083±0.104、0.749±0.070、0.736±0.070和0.394±0.016,VEGF蛋白相对表达水平分别为1.016±0.103、0.757±0.090、0.745±0.021和0.603±0.023,差异均具有统计学意义(F=14.650,P=0.001;F=45.220,P<0.001;F=18.180,P<0.001),各处理组3种蛋白表达水平均明显低于空白组(均P<0.05);与COX-2抑制组和麦冬皂苷D组相比,联合用药组COX-2、HIF-1α与VEGF蛋白相对表达水平均显著降低(均P<0.05);COX-2抑制组与麦冬皂苷D组相比,3种蛋白表达差异均无统计学意义(均P>0.05)。结论 麦冬皂苷D联合沉默COX-2基因能抑制胰腺癌细胞增殖、迁移与侵袭,其机制可能与抑制COX-2通路并降低HIF-1α、VEGF蛋白表达水平有关。

关键词: 胰腺肿瘤, 细胞增殖, 肿瘤侵润, 环氧化酶2, 麦冬皂苷D

Abstract:

Objective To explore the effects of Ophiopogon D combined with cyclooxygenase-2 (COX-2) gene silencing on the proliferation, migration and invasion of human pancreatic cancer BxPC-3 cells. Methods BxPC-3 cells were divided into blank control group, Ophiopogonin D high-dose group (40 μmol/L), medium-dose group (20 μmol/L) and low-dose group (10 μmol/L). The COX-2-slienced cells were divided into control group, COX-2 inhibited group (50 pmol/ml siRNA-COX-2), Ophiopogonin D group (20 μmol/L) and combination treatment group (Ophiopogonin D 20 μmol/L+50 pmol/ml siRNA-COX-2). The proliferation activity of BxPC-3 cells was detected by CCK-8, and the migration distance of BxPC-3 cells was detected by scratched assay. The invasion degree of BxPC-3 cells was detected by Transwell, the relative expression level of COX-2 gene in BxPC-3 cells was detected by real-time quantitative PCR (RT-qPCR), and the relative expressions of COX-2,hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) proteins in BxPC-3 cells were detected by Western blotting. Results The cell proliferation rates of blank control group, Ophiopogonin D high-dose, medium-dose and low-dose groups were (100.0±4.9)%, (71.8±5.4)%, (80.5±5.8)% and (89.7±5.7)%, respectively. The migration distances were (279.8±24.0) μm, (141.9±21.2) μm, (168.8±37.1) μm and (224.6±19.9) μm, respectively. The absorbance (A) values of invasion number were 1.107±0.095, 0.390±0.030, 0.596±0.017 and 0.826±0.034, respectively.There were statistically significant differences (F=19.770, P<0.001; F=48.270, P<0.001; F=198.400, P<0.001).The above indexes of the Ophiopogonin D high-, medium- and low-dose groups were significantly lower than those in the blank control group (all P<0.05).The relative expression levels of COX-2 gene were 1.007±0.178, 0.387±0.169, 0.567±0.142 and 0.740±0.030, respectively,and the relative protein expression levels were 1.000±0.033, 0.654±0.085, 0.762±0.110 and 0.881±0.049, respectively, with statistically significant differences (F=10.280, P=0.004;F=11.780, P=0.003). The above indexes of the Ophiopogonin D high- and medium-dose groups were significantly lower than those in the blank control group (all P<0.05), and there was no statistically significant difference between the Ophiopogonin D low-dose group and blank control group (both P>0.05). The medium-dose of Ophiopogonin D (20 μmol/L) was selected as the subsequent concentration.After COX-2 silencing,the proliferation rates of the control group, COX-2 inhibited group, Ophiopogonin D group and combination treatment group were (100.0±2.8)%, (68.4±6.7)%, (67.7±5.9)% and (57.0±8.5)%, respectively,the migration distances were (274.4±23.8) μm, (217.0±18.8) μm, (186.2±18.6) μm and (115.7±15.8) μm, respectively,and the A values of invasion number were 1.143±0.092, 0.791±0.058, 0.715±0.026 and 0.424±0.058, respectively, with statistically significant differences (F=34.430, P<0.001; F=103.400, P<0.001; F=131.100, P<0.001). The proliferation rates, migration distances and invasion numbers in each treatment group were significantly lower than those in the control group (all P<0.001). Compared with the COX-2 inhibited group and Ophiopogonin D group, the cell proliferation, migration and invasion were significantly inhibited in the combination treatment group (all P<0.05). Compared with the Ophiopogonin D group, only the migration distance of the COX-2 inhibited group was significantly different (P<0.05).The relative expression levels of COX-2 protein in the above groups were 0.995±0.037, 0.779±0.060, 0.806±0.076 and 0.645±0.079, respectively,the relative expression levels of HIF-1α were 1.083±0.104, 0.749±0.070, 0.736±0.070 and 0.394±0.016, respectively,and the relative expression levels of VEGF protein were 1.016±0.103, 0.757±0.090, 0.745±0.021 and 0.603±0.023, respectively,with statistically significant differences (F=14.650, P=0.001; F=45.220, P<0.001; F=18.180, P<0.001).The expression levels of the three proteins in each treatment group were significantly lower than those in the control group (all P<0.05). Compared with the COX-2 inhibited group and Ophiopogonin D group, the relative protein expression levels of COX-2, HIF-1α and VEGF in the combination treatment group were significantly decreased (all P<0.05). Compared with the Ophiopogonin D group, there were no significant differences in the expression of the three proteins in the COX-2 inhibited group (all P>0.05). Conclusion Ophiopogon D combined with COX-2 gene silencing can inhibit the proliferation, migration and invasion of pancreatic cancer cells, and the mechanism may be related to the inhibition of COX-2 pathway and the decrease of HIF-1α and VEGF protein expression levels.

Key words: Pancreatic neoplasms, Cell proliferation, Neoplasm invasiveness, Cyclooxygenase 2, Ophiopogon D