摘要
目的:探讨长链非编码RNA(lncRNA)前列腺癌相关ncRNA转录本1(PCAT1)对乳腺癌细胞增殖、凋亡和放射敏感性的影响,并分析其分子机制。方法:实时定量PCR分析lncRNA PCAT1和miR-329-3p在乳腺癌细胞系(MCF-7、T47D、Bcap-37)以及正常乳腺上皮细胞(MCF-10A)中表达水平。转染lncRNA PCAT1小干扰RNA、miR-329-3p模拟物至T47D细胞,采用噻唑蓝(MTT)实验、流式细胞术、细胞克隆实验分析干扰lncRNA PCAT1表达或过表达miR-329-3p对T47D细胞增殖活力、凋亡和放射敏感性的影响。双荧光素酶实验验证lncRNA PCAT1与miR-329-3p之间的关系。免疫印迹法检测细胞周期素D1(CyclinD1)、磷酸化组蛋白(γ-H2AX)、切割的半胱氨酸蛋白酶3(cleaved-caspase-3)以及β-连环蛋白(β-Catenin)表达。结果:与MCF-10A细胞比较,MCF-7、T47D、Bcap-37细胞中lncRNA PCAT1表达量显著升高(P<0.05),miR-329-3p表达量显著降低(P<0.05)。干扰lncRNA PCAT1表达或过表达miR-329-3p显著下调CyclinD1表达(P<0.05),上调Cleaved-caspase-3表达(P<0.05),降低T47D细胞活力(P<0.05),促进细胞凋亡(P<0.05),并增加细胞的放射敏感性(P<0.05)。miR-329-3p与lncRNA PCAT1序列直接结合,干扰lncRNA PCAT1后miR-329-3p的表达显著升高(P<0.05),过表达lncRNA PCAT1后miR-329-3p的表达显著降低(P<0.05)。干扰lncRNA PCAT1表达显著抑制β-Catenin蛋白表达(P<0.05)。抑制miR-329-3p表达可减弱干扰lncRNA PCAT1对T47D细胞增殖活力、凋亡、放射敏感性以及β-Catenin蛋白表达的影响(P<0.05)。结论:干扰lncRNA PCAT1可抑制乳腺癌细胞增殖,诱导细胞凋亡,增加细胞放射敏感性,其机制可能与靶向miR-329-3p调控Wnt/β-Catenin信号通路相关。
Objective:To investigate the effect of long non-coding RNA(lncRNA)prostate cancerassociated transcript 1(PCAT1)ncRNA on proliferation,apoptosis and radiosensitivity of breast cancer cells,and the underlying molecular mechanism.Methods:Real-time quantitative PCR was used to determine the expression levels of lncRNA PCAT1 and miR-329-3p in breast cancer cell lines(MCF-7,T47D,Bcap-37)and normal breast epithelial cells(MCF-10A).Then the T47D cells were transfected with lncRNA PCAT1 small interfering RNA and miR-329-3p mimics.Thiazolyl blue(MTT)assay,flow cytometry,and cell clonogenci assay were used to determine the effect of interferencing lncRNA PCAT1 expression or overexpressing miR-329-3p on proliferation,viability,apoptosis and radiosensitivity of T47D cells.Dual luciferase reporter assay was used to verify the relationship between lncRNA PCAT1 and miR-329-3p.Western blotting was used to detect the expression of Cyclin D1,phosphorylated histone(γ-H2AX),cleaved-caspase-3 andβ-catenin.Results:The MCF-7,T47D and Bcap-37 cells showed significantly upregulated lncRNA PCAT1 expression and down-regulated miR-329-3p expression,compared with the MCF-10A cells(allP<0.05).Interferencing lncRNA PCAT1 expression or overexpression of miR-329-3p led to significant down-regulation of CyclinD1(P<0.05),up-regulation of Cleavedcaspase-3(P<0.05),as well as lowered viability(P<0.05),promoted apoptosis(P<0.05),and higher radiosensitivity of T47D cells(P<0.05).miR-329-3p was found to bind directly to the lncRNA PCAT1 sequence.The expression of miR-329-3p was significantly increased after interferencing lncRNA PCAT1(P<0.05),and suppressed after overexpressing lncRNA PCAT1(P<0.05).Interferencing the expression of lncRNA PCAT1significantly suppressed the expression ofβ-Catenin protein(P<0.05).Inhibiting the expression of miR-329-3p attenuated the effect of interferencing lncRNA PCAT1 on T47D proliferation,apoptosis and radiosensitivity,andβ-Catenin protein expression(P<0.05).Conclusion:Interferencing the lncRNA PCAT1 inhibits the proliferation,induces apoptosis,and increases the radiosensitivity of breast cancer cells.The mechanism may be related to regulation of Wnt/β-Catenin signaling pathway via targeting miR-329-3p.
作者
李东原
王艳梅
吴博
闫美宏
毕华
Li Dongyuan;Wang Yanmei;Wu Bo;Yan Meihong;Bi Hua(Department of Histology and Embryology,Yan'an University School of Medicine,Yan'an 716000,Shaanxi,China)
出处
《广州医科大学学报》
2022年第1期1-9,共9页
Academic Journal of Guangzhou Medical University
基金
陕西省教育厅科研专项计划项目(19JK0970)
陕西省大学生创新项目(S202010719087)
延安大学校级科研项目(YDY2020-20)。
作者简介
通讯作者:王艳梅,Email:421858686@qq.com。
