摘要
目的分析ZWINT基因沉默对三阴性乳腺癌细胞生长抑制和凋亡的影响,为临床治疗提供参考。方法根据ZWINT基因的互补DNA(cDNA)序列设计合成短发卡RNA(shRNA),以分子克隆方法构建含ZWINT基因shRNA的表达载体,采用酶切分析与脱氧核糖核酸测序确认载体构建。选用G418进行细胞筛选,制备稳定表达ZWINT shRNA的细胞株。采用蛋白印迹技术(Western blotting)分析ZWINT基因表达抑制后蛋白表达水平的变化,利用甲基噻唑基四唑(MTT)法分析细胞增殖抑制能力,设定空白对照组与ZWINT基因沉默组,通过MTT法与流式细胞术测定细胞生长抑制率及凋亡率。结果成功构建ZWINT基因的RNA干扰(RNAi)再组合表达载体,经限制性内切酶分析、DNA序列测定验证所构建载体中序列的精准无误。依托G418抗性筛选药物,构建了ZWINT基因稳定转染细胞。Western blotting显示,ZWINT基因沉默后,相应蛋白的表达量较对照组明显降低,差异有统计学意义(P<0.05)。进一步研究揭示,ZWINT基因沉默组在干预后48、72 h,细胞增殖抑制率分别达41.23%、62.89%,与对照组比较,差异均有统计学意义(t=10.028、9.147,P均<0.05);流式细胞检测细胞凋亡率也显著增加,与对照组比较,差异均有统计学意义(t=12.941、13.019,P均<0.05)。结论ZWINT基因可能在三阴性乳腺癌细胞的生物行为中起着关键作用,通过沉默其基因表达可有效抑制细胞增殖和促使细胞凋亡。
Objective To analyze the impact of ZWINT gene silencing on growth inhibition and apoptosis of triple-negative breast cancer cells.Methods Short hairpin RNA(shRNA)was designed and synthesized based on the complementary DNA(cDNA)sequence of the ZWINT gene.An expression vector containing ZWINT gene specific shRNA was constructed by molecular cloning method.Enzyme digestion analysis and deoxyribonucleic acid sequencing were used to confirm the correctness of vector.G418 was selected for cell screening to prepare cell lines stably expressing ZWINT shRNA.Western blotting was used to analyze the changes in protein expression levels after silencing of ZWINT gene expression.The growth rate inhibition and apoptosis between two groups of cells were determined by MTT method and flow cytometry,respectively.ResultsRNA interference(RNAi)recombined expression vector targeting the ZWINT gene was successfully constructed,and the accuracy and correctness of the sequences in the constructed vector was verified through restriction enzyme analysis and DNA sequencing.Relying on G418 resistance screening drugs,the stable silencing of the ZWINT gene were successfully cultivated.Western blotting showed that after silencing the ZWINT gene,the expression level of the corresponding protein was significantly reduced compared to the control group(P<0.05).At 48 and 72 hours after intervention,the proliferation inhibition rates of ZWINT gene silenced cells reached 41.23% and 62.89%,respectively(t=10.028,9.147;all P<0.05).At the same time,the apoptosis rates also significantly increased to 26.86% and 42.48%,respectively,compared with the control group;the differences were statistically significant(t=12.941,13.019;all P<0.05).Conclusions The ZWINT gene might play a critical role in the biological behavior of triple-negative breast cancer cells.Silencing the expression of ZWINT could effectively inhibit cell proliferation and induce apoptosis.
作者
董小林
李尚日
DONG Xiaolin;LI Shangri(The Third Clinical College of Jinzhou Medical University,Jinzhou,Liaoning 121001,China;Department of Health Management Center,Shiyan People's Hospital,Shiyan,Hubei 442003,China;Department of Oncology,the Second Hospital of Huangshi,Huangshi,Hubei 435000,China)
出处
《热带医学杂志》
2025年第7期894-898,F0002,共6页
Journal of Tropical Medicine
基金
湖北省教育厅科技项目(B2023108)。
关键词
ZWINT基因
沉默
三阴性乳腺癌
生长抑制
凋亡
ZWINT gene
Silencing
Triple-negative breast cancer
Growth inhibition
Apoptosis
