摘要
目的研究微囊藻毒素-LR(microcystin-LR,MC-LR)致人肝细胞株WRL68细胞恶性增殖过程中长链非编码RNA H19的表达改变及白藜芦醇(resveratrol,Res)的干预作用。方法将WRL68细胞随机分为溶剂对照(0.1%二甲基亚砜)组和25、100 nmol/L白藜芦醇组及MC-LR染毒组(10 nmol/L MC-LR染毒至25代)以及25、100 nmol/L白藜芦醇干预(白藜芦醇+10 nmol/L MC-LR)组,培养至25代。采用MTT法、平板克隆形成实验检测细胞的增殖能力,采用实时荧光定量PCR测定H19的表达水平。结果与溶剂对照组相比,MC-LR染毒不同时间WRL68细胞的存活率均上升,除第15代染毒24 h外,差异均有统计学意义(P<0.05);且MC-LR染毒72、96 h时第25代WRL68细胞的存活率高于15代细胞,差异均有统计学意义(P<0.05)。MC-LR染毒第25代WRL68细胞的克隆形成率为114.6%,高于溶剂对照组(22.87%),差异有统计学意义(P<0.05)。与溶剂对照组相比,100 nmol/L白藜芦醇处理24~96 h的第25代WRL68细胞的存活率下降,差异均有统计学意义(P<0.05);100 nmol/L白藜芦醇干预组处理24~96 h的第25代WRL68细胞的存活率低于MC-LR染毒组和25 nmol/L白藜芦醇干预组,差异均有统计学意义(P<0.05)。而MC-LR染毒组和25 nmol/L白藜芦醇干预组染毒24~96 h的第25代WRL68细胞的存活率间比较,差异均无统计学意义(P>0.05)。与溶剂对照组比较,MC-LR染毒组第10、15、20、25代WRL68细胞中H19的表达水平均较高,而100 nmol/L白藜芦醇组表达水平均较低,差异均有统计学意义(P<0.05);与MC-LR染毒组比较,100 nmol/L白藜芦醇干预组第10、15、20、25代WRL68细胞中H19的表达水平均较低,差异均有统计学意义(P<0.05)。随着处理时间的延长,MC-LR染毒组WRL68细胞中H19的表达水平呈上升趋势,而100 nmol/L白藜芦醇组和100 nmol/L白藜芦醇干预组WRL68细胞中H19的表达水平均呈下降趋势。第5代不同处理组的细胞中H19的表达水平无明显变化。结论白藜芦醇可下调MC-LR染毒细胞中H19的表达水平并抑制MC-LR诱导的肝细胞恶性增殖。
Objective To understand the changes of long non-coding RNA H19 expression in human liver cell line(WRL68)during malignant proliferation induced by microcystin-LR(MC-LR)as well as the intervention effect of resveratrol.Methods WRL68 cells were randomly divided into the solvent control(0.1%dimethyl sulfoxide)group,25 and 100 nmol/L resveratroltreated groups,MC-LR exposure group(10 nmol/L MC-LR treated until to the 25 th passage),and 25,100 nmol/L resveratrol intervention groups(combined with 10 nmol/L MC-LR)respectively,which were cultured for 25 passages.The proliferation capacity of cell was determined by MTT assay and plate clone formation assay.The expression of H19 was detected by realtime PCR.Results Compared with the solvent control group,the viabilities of WRL68 cells exposed to MC-LR for different times were increased except the 15 th passage MC-LR exposure for 24 h(P<0.05);The viabilities of the 25 th passage WRL68 cells treated with MC-LR for 72 and 96 h was significantly higher than those of the 15 th passage MC-LR-treated cells(P<0.05).The colony formation rate of the 25 th passage cells treated with MC-LR was 114.6%,which were significantly more than solvent control(22.87%)(P<0.05).Compared with solvent control group,the survival rate of the 25 th passage WRL68 cells exposed to 100 nmol/L resveratrol for 24-96 h decreased significantly(P<0.05).The viabilities of the 25 th passage WRL68 cells in 100 nmol/L resveratrol intervention group with exposure time of 24-96 h were less than MC-LR exposure group as well as25 nmol/L resveratrol intervention group respectively(P<0.05).No significant difference was seen in the survival rate of the25 th passage WRL68 cells with exposure time of 24-96 h between the MC-LR exposure group and 25 nmol/L resveratrol intervention group(P>0.05).Compared with the solvent control group,the expressions of H19 of the 10 th,15 th,20 th and 25 th passage WRL68 cells in MC-LR exposure group were up-regulated,while the expression of H19 in 100 nmol/L resveratrol exposure group was down-regulated(P<0.05).Compared with MC-LR exposure group,the expressions of H19 in the 10,15,20 and 25 passage WRL68 cells in 100 nmol/L resveratrol intervention group was decreased(P<0.05).Furthermore,with the prolonged exposure time of MC-LR,the expression of H19 in WRL68 cells exposed to MC-LR showed an uptrend,and that showed the downtrend in the 100 nmol/L resveratrol exposure group and 100 nmol/L resveratrol intervention group.The expression of H19 showed no significant difference in the 5 th passage WRL68 cells among different treatment groups.Conclusion Resveratrol may down-regulate H19 expression in cells exposed to MC-LR and inhibit malignant proliferation of hepatocytes induced by MC-LR.
作者
关斌
农清清
费梦雪
贾雪姣
黄秋月
马智星
王佑新
唐燕梅
GUAN Bin;NONG Qing-qing;FEI Meng-xue;JIA Xue-jiao;HUANG Qiu-yue;MA Zhi-xing;WANG You-xin;TANG Yan-mei(Department of Environmental Health,School of Public Health,Guangxi Medical University,Nanning,Guangxi 530021,China;不详)
出处
《环境与健康杂志》
CAS
北大核心
2020年第7期576-580,共5页
Journal of Environment and Health
基金
国家自然科学基金(81960582,81660529)
