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

摘要/Abstract

摘要： 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（500 μg/mL As₂O₃处理）、低剂量天麻多糖组（500 μg/mL As₂O₃以及500 μmol/mL天麻多糖处理）、高剂量天麻多糖组（500 μg/mL As₂O₃以及1 000 μmol/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 vs 0.84±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 mRNA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 mRNA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。

关键词: 天麻多糖, 砷, miR-27a-3p, B7-H3, 结肠癌

Abstract: 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（500 μg/mL As₂O₃ treatment）,low-dose Gastrodia elata polysaccharide group（500 μg/mL As₂O₃ and 500 μmol/mL Gastrodia elata polysaccharide treatment）,and high-dose Gastrodia elata polysaccharide group（500 μg/mL As₂O₃ and 1 000 μmol/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-di-phenytetrazoliumromide（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 OD 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 1 985.36±398.52）,the number of transmembrane（256.68±86.21 vs 2 472.57±186.52）,migration distance[（256.68±86.21）vs（2 472.57±196.52）μm] and B7-H3 mRNA protein expression level（0.64±0.27 vs 2.89±0.23,0.23±0.28 vs 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 OD value（0.95±0.04 vs 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（1 985.36±398.52 vs 1 025.63±325.66,562.96±105.59）,the number of transmembrane（2 472.57±186.52 vs 1 842.36±165.52,635.20±189.36）,migration distance[（2 472.57±196.52） vs（1 842.36±165.52）,（1 235.20±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）% vs（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 OD 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.

Key words: Gastrodia elata polysaccharide, Arsenic, miR-27a-3p, B7-H3, Colon cancer

中图分类号:

R446.7

叶伟, 张曦. 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. [J]职业与健康, 2023, 39(12): 1596-1603.

YE Wei, ZHANG Xi. Effect of Gastrodia elata polysaccharide on proliferation,invasion and apoptosis of colon cancer cells exposed to arsenic via miR-27a-3p-B7-H3 pathway. [J]OCCUPATION AND HEALTH, 2023, 39(12): 1596-1603.

参考文献

[1] TSUJI J S,CHANG E T,GENTRY P R,et al.Dose-response for assessing the cancer risk of inorganic arsenic in drinking water:The scientific basis for use of a threshold approach[J].Crit Rev Toxicol,2019,49(1):36-84.
[2] PULLELLA K,KOTSOPOULOS J.Arsenic exposure and breast cancer risk:A re-evaluation of the literature[J].Nutrients,2020,12(11):3305.
[3] CARDOSO A P F,UDOH K T,STATES J C.Arsenic-induced changes in miRNA expression in cancer and other diseases[J].Toxicol Appl Pharmacol,2020,409(10):115306.
[4] ZHENG F,WANG M,LI Y,et al.CircNR3C1 inhibits proliferation of bladder cancer cells by sponging miR-27a-3p and downregulating cyclin D1 expression[J].Cancer lett,2019,460(3):139-151.
[5] CHEN Q,LIU T,BAO Y I,et al.CircRNA cRAPGEF5 inhibits the growth and metastasis of renal cell carcinoma via the miR-27a-3p/TXNIP pathway[J].Cancer Lett,2020,469(12):68-77.
[6] YING Y,ZHIFANG Y,CENG C A I,et al.The effect of miR-27a-3p on chemotherapy sensitivity in hepatocellular carcinoma cells and its mechanism[J].Oncol Rep,2019,29(10):788-794.
[7] FLEM-KARLSEN K,TEKLE C,ØYJORD T,et al.p38 MAPK activation through B7-H3-mediated DUSP10 repression promotes chemoresistance[J].Sci Rep,2019,9(1):1-9.
[8] KUMAR A,ALI M,KUMAR R,et al.Arsenic exposure in Indo Gangetic plains of Bihar causing increased cancer risk[J].Sci Rep,2021,11(1):1-16.
[9] KANCHAN R K,PERUMAL N,ATRI P,et al.MiR-1253 exerts tumor-suppressive effects in medulloblastoma via inhibition of AsK6 and As276 (B7-H3)[J].Brain Pathol,2020,30(4):732-745.
[10] 张明泽,何春梅,王浩斌,等.兰科药用植物活性多糖研究进展[J].热带亚热带植物学报,2019,27(5):611-622.
[11] ZHU H,LIU C,HOU J,et al.Gastrodia elata blume polysaccharides:a review of their acquisition,analysis,modification,and pharmacological activities[J].Molecules,2019,24(13):2436.
[12] ZHU Z Y,CHEN C J,SUN H Q,et al.Structural characterisation and ACE-inhibitory activities of polysaccharide from Gastrodia elata Blume[J].Nat Prod Res,2019,33(12):1721-1726.
[13] KIDDANE A T,KIM G D.Anticancer and immunomodulatory effects of polysaccharides[J].Nutr Cancer,2021,73(11-12):2219-2231.
[14] KOZONO S,LIN Y M,SEO H S,et al.Arsenic targets Pin1 and cooperates with retinoic acid to inhibit cancer-driving pathways and tumor-initiating cells[J].Nat Commun,2018,9(1):1-17.
[15] SOHRETOGLU D,HUANG S.Ganoderma lucidum polysaccharides as an anti-cancer agent[J].Anti-Cancer Agent Me,2018,18(5):667-674.
[16] WEN-QI H,ZHU Y,DONG-XIN T.Data mining analysis of Professor Liu Shangyi’s prescription characteristics in clinical medicine for the treatment of cancer patients with stomachache[J].Tradit Med Res,2018,3(1):52-54.
[17] GAN Q,WANG J,HU J,et al.Modulation of apoptosis by plant polysaccharides for exerting anti-cancer effects:A review[J].Front Pharmacol,2020,9(2):792-793.
[18] SI S,WANG L,CAO H,et al.Co-deficiency of B7-H3 and B7-H4 identifies high As8+ T cell infiltration and better prognosis in pancreatic cancer[J].BMC cancer,2022,22(1):1-12.
[19] HARLAND N,MAURER F B,ABRUZZESE T,et al.Elevated expression of the immune checkpoint ligand as276(b7-h3)in urothelial carcinoma cell lines correlates negatively with the cell proliferation[J].Int J Mol Sci,2022,23(9):4969.
[20] POONIA T,SINGH N,GARG M C.Contamination of arsenic,chromium and fluoride in the Indian groundwater:A review,meta-analysis and cancer risk assessment[J].Int J Environ Sci Te,2021,18(9):2891-2902.
[21] AGGARWAL C,PRAWIRA A,ANTONIA S,et al.Dual checkpoint targeting of B7-H3 and PD-1 with enoblituzumab and pembrolizumab in advanced solid tumors:Interim results from a multicenter phase I/II trial[J].J Immunother Cancer,2022,10(4):52-54.
[22] LIAO H,DING M,ZHOU N,et al.B7-H3 promotes the epithelial-mesenchymal transition of NSCLC by targeting SIRT1 through the PI3K/AKT pathway[J].Mol Med Rep,2022,25(3):1-9.
[23] CHEN Y,SUN J,ZHAO H,et al.The coexpression and clinical significance of costimulatory molecules B7-H1,B7-H3,and B7-H4 in human pancreatic cancer[Retraction][J].Onco Targets Ther,2022,15(5):699-700.
[24] AMIR TAGHAVI B,ALIZADEH N,SAEEDI H,et al.Targeted therapy of B7 family checkpoints as an innovative approach to overcome cancer therapy resistance:A review from chemotherapy to immunotherapy[J].Molecules,2022,27(11):3545.