天麻多糖经miR-27a-3p/B7-H3通路对砷暴露下结肠癌细胞增殖、侵袭、凋亡的影响

Effect of Gastrodia elata polysaccharide on proliferation,invasion and apoptosis of colon cancer cells exposed to arsenic via miR-27a-3p-B7-H3 pathway

目的 探讨天麻多糖经微小核糖核酸-27a-3p(microRNA-27a-3p,miR-27a-3p)/B7同源物3(b7 homolog 3,B7-H3)通路对砷暴露下结肠癌细胞增殖、侵袭、凋亡的影响。方法 结肠癌细胞分为SW480细胞组、砷(arsenic,As)暴露组(500μg/mL As2O3处理)、低剂量天麻多糖组(500μg/mL As2O3以及500μmol/mL天麻多糖处理)、高剂量天麻多糖组(500μg/mL As2O3以及1 000μmol/mL天麻多糖处理),以上各组细胞设6个复孔,培养时间为72 h。试验结束后,3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide,MTT)法测定各组癌细胞增殖情况;细胞迁移实验(transwell,transwell)法、伤口愈合试验测定各组癌细胞侵袭迁移情况;流式细胞仪测定各组癌细胞凋亡水平;逆转录定量聚合酶链反应(reverse transcription quantitative polymerase chain reaction,RT-qPCR)法及蛋白印迹法测定各组癌细胞miR-27a-3p、B7-H3水平。结果 与SW480细胞组比较,As暴露组OD值(0.66±0.03 vs 0.95±0.04)、存活率水平[(60.01±5.50)%vs(89.74±6.07)%]、单克隆形成数目[(126.69±39.65)个vs(1 985.36±398.52)个]、穿膜数[(256.68±86.21)个vs(2 472.57±186.52)个]、迁移距离[(256.68±86.21)μm vs(2 472.57±196.52)μm]、B7-H3 mRNA蛋白表达水平(0.64±0.27 vs 2.89±0.23、0.23±0.28 vs 1.27±0.23)显著升高,凋亡率[(20.30±5.89)%vs(3.40±0.97)%]、miR-27a-3p mRNA表达水平(3.39±0.41 vs 0.66±0.43)显著降低(均P<0.01)。与As暴露组比较,低高剂量天麻多糖组OD值(0.95±0.04 vs0.84±0.03、0.76±0.03)、存活率水平[(89.74±3.07)%vs(81.03±3.04)(63.56±5.05)%]、单克隆形成数目[(1 985.36±398.52)个vs(1 025.63±325.66)(562.96±105.59)个]、穿膜数[(2 472.57±186.52)个vs(1 842.36±165.52)(635.20±189.36)个]、迁移距离[(259.96±15.32)μm vs(1 85.22±9.88)(98.99±8.25)μm]、B7-H3 mRNA蛋白表达水平(2.89±0.23 vs 2.01±0.26、1.53±0.24,1.27±0.23 vs 0.89±0.25、0.53±0.24)显著降低,凋亡率[(3.40±0.97)%vs(12.30±1.58)%、(17.70±0.98)%]、miR-27a-3p m RNA表达水平(0.66±0.43 vs 1.73±0.36、2.65±0.39)显著升高;且高剂量天麻多糖组OD值、存活率水平、单克隆形成数目、穿膜数、迁移距离、B7-H3 mRNA蛋白表达水平明显低于低剂量天麻多糖组,凋亡率、miR-27a-3p m RNA表达水平明显高于低剂量天麻多糖组(均P<0.05)。结论 天麻多糖能抑制砷暴露下结肠癌细胞增殖、侵袭,促进其凋亡;其机制可能与天麻多糖促进miR-27a-3p表达进而抑制了B7-H3的表达有关。

Objective To investigate the effect of Gastrodia elata polysaccharide via microRNA-27a-3p(miR-27a-3p)/homolog 3(B7-H3)on the proliferation,invasion and apoptosis of colon cancer cells exposed to arsenic.Methods The colon cancer cells were divided into SW480 cell group,arsenic(As)exposure group(500μg/mL As20,treatment),low-dose Gastrodia elata polysaccharide group(500μg/mL As203 and 500μmol/mL Gastrodia elata polysaccharide treatment),and high-dose Gastrodia elata polysaccharide group(500μg/mL As20;and 1000μmol/mL Gastrodia elata polysaccharide treatment),the cells of the above groups were set up in six duplicate wells,and the culture time was 72 hours.After the experiment,3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide(MTT)method was used to measure the proliferation of cancer cells in each group,the transwell method and wound healing test were used to measure the invasion and migration of cancer cells in each group,the flow cytometry was used to measure the apoptosis level of cancer cells in each group,the reverse transcription quantitative polymerase chain reaction(RT-qPCR)and Western blotting method were used to determine the levels of miR-27a-3p and B7-H3 in cancer cells of each group.Results Compared with the SW480 cell group,the 0D value(0.66±0.03 vs 0.95±0.04),the survival rate level[(60.01±5.50)%vs(89.74±6.07)%],the number of monoclonal formation(126.69±39.65 vs 1985.36±398.52),the number of transmembrane(256.68±86.21 vs 2472.57±186.52),migration distance[(256.68±86.21)us(2472.57±196.52)μm)and B7-H3 mRNA protein expression level(0.64±0.27 vs 2.89±0.23,0.23±0.28 us 1.27±0.23)in As exposure group significantly increased,while the apoptosis rate[(20.30±5.89)%vs(3.40+0.97)%]and the expression level of miR-27a-3p mRNA(3.39±0.41 vs 0.66±0.43)significantly decreased(all P<0.01).Compared with the As exposure group,the 0D value(0.95±0.04 us 0.84+0.03,0.76±0.03),the survival rate level[(89.74±3.07)%vs(81.03±3.04)%,(63.56±5.05)%],the number of monoclonal formation(1985.36±398.52 vs 1025.63±325.66,562.96±105.59),the number of transmembrane(2472.57±186.52 vs 1842.36±165.52,635.20±189.36),migration distance[(2472.57±196.52)vs(1842.36±165.52),(1235.20±189.36)μm)and B7-H3 mRNA protein expression level(2.89±0.23 vs 2.01±0.26,1.53±0.24;1.27±0.23 vs 0.89±0.25,0.53±0.24)in low-and high-dose Gastrodia elata polysaccharide group significantly decreased,while the apoptosis rate[(3.40+0.97)%us(12.30+1.58)(17.70+0.98)%]and the expression level of miR-27a-3p mRNA(0.66+0.43 vs 1.73±0.36,2.65±0.39)significantly increased(all P<0.01).And 0D value,the survival rate level,the number of monoclonal formation,the number of transmembrane,migration distance and B7-H3 mRNA protein expression level in the high-dose Gastrodia elata polysaccharide group were significantly lower than those in the low-dose Gastrodia elata polysaccharide group,while the apoptosis rate and miR-27a-3p mRNA expression level were significantly higher than those in the low-dose Gastrodia elata polysaccharide group(all P<0.05).Conclusion The Gastrodia elata polysaccharide can inhibit the proliferation and invasion of colon cancer cells colon cancer cells exposed to arsenic and promote their apoptosis,and the mechanism may be related to the fact that Gastrodia elata polysaccharide promotes the expression of miR-27a-3p and then inhibits the expression of B7-H3.