原苏木素A调控结直肠癌SW480细胞生物学行为及其机制研究

Protosapanin A regulates microRNA-431 to reduce the growth and metastasis of colorectal cancer SW480 cells

目的探讨原苏木素A调控结直肠癌细胞生物学行为及机制。方法结直肠癌SW480细胞分别用不同浓度(0、80、160、320、640和1 280 μmol/L)原苏木素A处理筛选有效抑制浓度。SW480细胞根据转染载体分成原苏木素A+Anti-miR-NC组(转染inhibitor control后1 280 μmol/L的原苏木素A处理)、原苏木素A+Anti-miR-431组(转染miR-431 inhibitor后1 280 μmol/L的原苏木素A处理),细胞计数试剂-8法检测细胞增殖,克隆形成实验检测细胞克隆形成能力,Transwell小室检测细胞迁移和侵袭,流式细胞术检测细胞凋亡差异,免疫印迹法检测细胞中E-钙粘蛋白(E-cadherin)、波形蛋白(Vimentin)、B细胞淋巴瘤/白血病-2(bcl-2)、bcl-2相关X蛋白(bax)蛋白表达,实时荧光定量聚合酶链反应(RT-qPCR)检测微小核糖核酸-431(miR-431)表达。两组间比较行t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果 320、640和1 280 μmol/L原苏木素A处理后SW480细胞吸光度(A)值显著低于0、80、160 μmol/L(0.50±0.04、0.37±0.03、0.28±0.02、0.65±0.06、0.63±0.04和0.62±0.05,F=120.991,P<0.01)。320、640和1 280 μmol/L原苏木素A处理后SW480细胞A值显著降低。320、640、1 280 μmol/L原苏木素A处理后的SW480细胞克隆形成数目(65.01±5.17、49.18±4.69、33.05±4.56比78.62±4.11,F=161.813,P<0.01)及bcl-2蛋白表达(0.41±0.04、0.29±0.02、0.20±0.03比0.59±0.05,F=189.500,P<0.01)显著低于0 μmol/L原苏木素A处理组,细胞凋亡率[(8.62±0.52)%、(14.78±1.25)%、(21.08±1.73)%比(4.15±0.16)%,F=403.486,P<0.01]和bax蛋白表达(0.28±0.03、0.37±0.02、0.48±0.05比0.18±0.03,F=125.298,P<0.01)显著高于0 μmol/L原苏木素A处理组。320、640、1 280 μmol/L原苏木素A处理后的SW480细胞迁移数目(172.42±14.97、130.89±12.08、105.50±10.82比224.56±31.58,F=65.715,P<0.01)、侵袭数目(150.43±12.94、114.98±11.33、82.02±9.98比182.34±15.76,F=105.513,P<0.01)和Vimentin蛋白表达(0.42±0.04、0.30±0.03、0.21±0.03比0.65±0.05,F=221.479,P<0.01)显著低于0 μmol/L原苏木素A处理组。320、640、1280 μmol/L原苏木素A处理后的SW480细胞miR-431水平高于0 μmol/L原苏木素A处理组(1.56±0.10、1.92±0.12、2.45±0.18、1.00±0.12,F=188.136,P<0.01)。原苏木素A+Anti-miR-431组SW480细胞A值(0.45±0.04比0.29±0.03)、克隆形成数目(47.04±4.11比34.25±2.78)、bcl-2蛋白表达(0.45±0.05比0.19±0.02)、细胞迁移(165.24±13.06比104.23±9.84)、侵袭数目(147.20±13.94比81.96±8.54)和Vimentin表达(0.38±0.03比0.20±0.02)高于原苏木素A+Anti-miR-NC组,细胞凋亡率[(12.05±1.23)%比(21.84±1.66)%]、细胞中E-cadherin(0.32±0.04比0.56±0.05)和bax蛋白表达(0.24±0.05比0.46±0.04)低于原苏木素A+Anti-miR-NC组,差异有统计学意义(t=9.600、7.733、14.216、14.484、10.307、11.193、11.972、11.245、14.977,P<0.01)。结论原苏木素A可能通过上调miR-431调控SW480细胞生物学行为。

Objective To study the effect and its mechanism of protosapanin A on the biological behavior of colorectal cancer SW480 cells.Methods Colorectal cancer SW480 cells were treated with 0,80,160,320,640,1280μmol/L protosapanin A,and colorectal cancer SW480 cells were divided into neutral protosapanin A+Anti-miR-NC(transfected with inhibitor control,1280μmol/L protosapanin A treatment),protosapanin A+Anti-miR-431 group(transfected with miR-431 inhibitor,1280μmol/L protosapanin A treatment),the cell proliferation was detected by the cell counting kit-8 method,and the cell clone formation ability was determined by the clone formation experiment,Transwell chamber was used to detect cell migration and invasion,flow cytometry was used to detect differences in apoptosis,Western blotting was used to determine the expression of E-cadherin,Vimentin,B-cell lymphoma 2(bcl-2),and bcl-2-associated X(bax)proteins in cells,and microRNA-431(miR-431)was analyzed by quantitative real time polymerase chain reaction(PCR)method.T test was conducted between the two groups,using single-factor variance analysis,and LSD-t test between the two groups.Results 320,640 and 1280μmol/L protosapanin A treatment SW480 cell′s absorbance(A)value were significantly lower than 0,80 and 160μmol/L protosapanin A treatment(0.50±0.04,0.37±0.03,0.28±0.02,0.65±0.06,0.63±0.04,0.62±0.05,F=120.991,P<0.01).Number of SW480 cell clones formed after 320,640,1280μmol/L A treatment(65.01±5.17,49.18±4.69,33.05±4.56,78.62±4.11,F=161.813,P<0.01)and bcl-2 protein expression(0.41±0.04,0.29±0.02,0.20±0.03,0.59±0.05,F=189.500,P<0.01)were significantly lower than 0μmol/L group,while apoptosis rate[(8.62±0.52)%,(14.78±1.25)%,(21.08±1.73)%,(4.15±0.16)%,F=403.486,P<0.01]and bax protein expression(0.28±0.03,0.37±0.02,0.48±0.05,0.18±0.03,F=125.298,P<0.01)were significantly higher.A+Anti-miR-431 group SW480 cell A value(0.45±0.04 vs,0.29±0.03),number of clones(47.04±4.11 vs.34.25±2.78),bcl-2 protein expression(0.45±0.050 vs.0.19±0.02),cell migration(165.24±13.06 vs.104.23±9.84),invasion number(147.20±13.94 vs.81.96±8.54)and vimentin expression(0.38±0.03 vs.0.20±0.02)were higher than A+Anti-miR-NC group,while apoptosis rate[(12.05±1.23)%vs.(21.84±1.66)%],E-cadherin(0.32±0.04 vs.0.56±0.05 in cells)and bax protein expression(0.24±0.05 vs.0.46±0.04)were lower with statistically significant differences(t=9.600,7.733,14.216,14.484,10.307,11.193,11.972,11.245,14.977,P<0.01).Conclusion Protosapanin A may regulate SW480 cell biological behavior by upregulating miR-431.