草苁蓉多糖对人肝癌细胞侵袭迁移的影响及机制

Effect of Boschniakia Rossica polysaccharides on the invasion and migration of human hepatocarcinoma cells and its mechanism

目的探讨草苁蓉多糖(BRPS)对人肝癌细胞侵袭迁移的影响及其可能机制。方法使用不同浓度的BRPS(12.5、25、50、100、200μg/L)干预肝癌HepG2细胞,筛选合适作用浓度。取对数生长期细胞,随机分为对照组(不作处理)、缺氧诱导因子-1α(HIF-1α)组(转染LV-HIF-1α)、HIF-1α-NC组(转染LV-HIF-1α-NC)、BRPS组(加入50μg/L BRPS)、BRPS+HIF-1α组(转染LV-HIF-1α,加入50μg/L BRPS),采用Hoechst33258染色观察肝癌细胞凋亡情况,流式细胞术检测肝癌细胞凋亡率,划痕实验检测肝癌细胞迁移能力,Transwell实验检测肝癌细胞侵袭能力,Western blotting检测肝癌细胞中HIF-1α以及上皮-间质转化(EMT)标志物E-钙黏蛋白、波形蛋白的表达水平。结果选取BRPS浓度50μg/L进行实验。Hoechst33258染色结果显示,对照组和HIF-1α-NC组蓝色荧光微弱,HIF-1α组蓝色荧光较弱,BRPS组蓝色荧光较强,BRPS+HIF-1α组蓝色荧光较BRPS组减弱。与对照组[细胞凋亡率:3.89%±1.25%,划痕愈合率:63.35%±3.35%,穿膜细胞数:(122.60±3.29)个]比较,HIF-1α组细胞凋亡率(1.67%±0.86%)降低,划痕愈合率(87.48%±3.92%)增高,穿膜细胞数[(208.60±6.17)个]增多,BRPS组细胞凋亡率(26.58%±1.63%)增高,划痕愈合率(14.82%±3.81%)降低,穿膜细胞数[(68.80±4.25)个]减少,差异有统计学意义(P<0.05);BRPS+HIF-1α组细胞凋亡率(12.14%±1.05%)高于HIF-1α组,低于BRPS组,划痕愈合率(32.59%±3.76%)、穿膜细胞数[(123.40±4.94)个]低于HIF-1α组,高于BRPS组,差异有统计学意义(P<0.05)。与对照组比较,HIF-1α组HIF-1α、波形蛋白表达水平升高,E-钙黏蛋白表达水平降低,BRPS组HIF-1α、波形蛋白表达水平降低,E-钙黏蛋白表达水平升高,差异有统计学意义(P<0.05);BRPS+HIF-1α组HIF-1α、波形蛋白表达水平低于HIF-1α组,高于BRPS组,E-钙黏蛋白表达水平高于HIF-1α组,低于BRPS组,差异有统计学意义(P<0.05)。结论BRPS可促进肝癌HepG2细胞凋亡,抑制其迁移和侵袭,其机制可能为通过调控HIF-1α而抑制EMT的发生。

Objective To investigate the effect and potential mechanism of Boschniakia rossica polysaccharides(BRPS)on the invasion and migration of human liver cancer cells.Methods Different concentrations of BRPS(12.5,25,50,100 and 200μg/L)were screened on liver cancer HepG2 cells to identify the appropriate concentration.Based on culture conditions,the HepG2 cells were named control group(no treatment),hypoxia-inducible factor-1α(HIF-1α)group(transfected with LV-HIF-1α),HIF-1α-NC group(transfected with LV-HIF-1α-NC),BRPS group(add 50μg/L BRPS),and BRPS+HIF-1αgroup(transfected with LV-HIF-1α,and add 50μg/L BRPS).Hoechst33258 staining was used to evaluate cell apoptosis;flow cytometry was used to detect cell apoptosis rate;the scratch test was used to detect cells migration ability;Transwell test was used to detect cell invasion ability;Western blotting was used to detect HIF-1αand epithelial-mesenchymal transition(EMT)related factors E-cadherin and vimentin protein expressions in cells.Results The ideal BRPS concentration was identified as 50μg/L.The results of Hoechst33258 staining showed that the blue fluorescence of hepatocarcinoma cells was weak in control group and HIF-1α-NC group,the blue fluorescence of HIF-1αgroup was dimer than control group;the blue fluorescence of BRPS group was brighter than control group;the blue fluorescence of BRPS+HIF-1αgroup was lower than that in BRPS group.In the control group,the apoptosis rate was 3.89%±1.25%;the cell healing rate was 63.35%±3.35%;the number of transwell cells was 122.60±3.29.Compared with control group,HIF-1αgroup showed a lower apoptosis rate(1.67%±0.86%,P<0.05),higher cell healing rate(87.48%±3.92%,P<0.05),and higher number of transwell cells(208.60±6.17,P<0.05).On the contrary,BRPS group showed a higher apoptotic rate(26.58%±1.63%,P<0.05),lower cell healing rate(14.82%±3.81%,P<0.05),and decreased number of transwell cells(68.80±4.25,P<0.05).Interestingly,in BRPS+HIF-1αgroup,the apoptosis rate was 12.14%±1.05%,higher than HIF-1αgroup but lower than BRPS group;the cell healing rate and the number of transmembrane cells were 32.59%±3.76%and(123.40±4.94)cells,respectively,both of which were lower than HIF-1αgroup but higher than that in BRPS group(P<0.05).Compared with control group,in HIF-1αgroup,the expression levels of HIF-1αand vimentin increased while E-cadherin decreased;however,in BRPS group,the expression levels of HIF-1αand vimentin decreased while E-cadherin increased(P<0.05).As expected,the expression levels of HIF-1αand vimentin in BRPS+HIF-1αgroup were lower than those in HIF-1αgroup but higher than those in BRPS group;the expression levels of E-cadherin was higher than that in HIF-1αgroup but lower than that in BRPS group(P<0.05).Conclusion BRPS can promote HepG2 cell apoptosis,inhibit cell migration and invasion.Its mechanism may be that it can suppress EMT by regulating HIF-1α.