circTHBS1调节miR-135a-5p/RAB1A轴对乳腺癌细胞恶性生物学行为的影响  

Effect of circTHBS1 on Malignant Biological Behaviors of Breast Cancer Cells by Regulating miR-135a-5p/RAB1A Axis

作　　者：张宏旭[1] 刘海旺[2] 王明辉[1] 刘汉成[1] 赵贺宇 马立辉[1] ZHANG Hongxu;LIU Haiwang;WANG Minghui;LIU Hancheng;ZHAO Heyu;MA Lihui(Department of Breast Surgery,Affiliated Hospital of Chengde Medical College,Chengde 067000,China;Department of Pathology,Affiliated Hospital of Chengde Medical College,Chengde 067000,China)

机构地区：[1]承德医学院附属医院乳腺外科,承德067000 [2]承德医学院附属医院病理科,承德067000

出　　处：《中国细胞生物学学报》2024年第10期1791-1800,共10页Chinese Journal of Cell Biology

基　　金：承德市科技计划自筹经费项目(批准号:202204A050)资助的课题。

摘　　要：该文旨在探究环状RNA血栓反应蛋白-1(circTHBS1)调节微小RNA-135a-5p(miR-135a-5p)/Rab家族蛋白1A(RAB1A)轴对乳腺癌(BC)细胞恶性生物学行为的影响。采用实时荧光定量聚合酶链反应(qRT-PCR)检测BC细胞系中circTHBS1、miR-135a-5p和RAB1A mRNA的表达情况,筛选最佳细胞系进行后续实验;通过RNase R处理验证circTHBS1的环状结构。将MCF-7细胞分为Control组、sh-NC组、sh-THBS1组、sh-THBS1+inhibitor-NC组和sh-THBS1+miR-135a-5p inhibitor组。采用qRT-PCR检测转染效率;采用双荧光素酶报告基因法确认circTHBS1和miR-135a-5p、miR-135a-5p和RAB1A的相互作用;Western blot法检测RAB1A在MCF-7细胞中的表达情况;采用平板克隆法检测MCF-7细胞增殖情况;使用流式细胞仪检测MCF-7细胞凋亡情况;划痕实验检测MCF-7细胞迁移情况;Transwell法检测MCF-7细胞侵袭情况;通过裸鼠移植瘤实验检测circTHBS1对瘤组织内BC细胞生长的影响。双荧光素酶报告基因实验结果显示,circTHBS1与miR-135a-5p、RAB1A与miR-135a-5p均具有靶向关系。与正常人乳腺上皮细胞MCF-10A相比,circTHBS1和RAB1A在BC细胞系中表达水平显著增加,miR-135a-5p表达水平显著下降(P<0.05);敲低circTHBS1表达可以抑制MCF-7细胞的增殖、迁移和侵袭,诱导MCF-7细胞凋亡(P<0.05);抑制miR-135a-5p表达可逆转敲低circTHBS1表达对MCF-7细胞的增殖、迁移和侵袭的抑制作用,并抑制MCF-7细胞凋亡(P<0.05);通过裸鼠移植瘤实验发现,敲低circTHBS1表达后,裸鼠BC瘤组织质量和体积下降,同时RAB1A表达量下降,miR-135a-5p表达量升高(P<0.05)。circTHBS1在BC细胞中高表达,敲低circTHBS1可以通过调控miR-135a-5p/RAB1A轴抑制BC细胞的恶性生物学行为。This study aims to investigate the effect of circTHBS1(circular RNA thrombospondin-1)on the malignant biological behaviors of BC(breast cancer)cells by regulating the miR-135a-5p(microRNA-135a-5p)/RAB1A(Rab family protein 1A)axis.qRT-PCR(real-time fluorescence polymerase chain reaction)was used to detect the expression of circTHBS1,miR-135a-5p,and RAB1A mRNA in BC cell lines,and the optimal cell line was screened for subsequent experiments.MCF-7 cells were grouped into Control group,sh-NC group,sh-THBS1 group,sh-THBS1+inhibitor NC group,and sh-THBS1+miR-135a-5p inhibitor group.qRT-PCR was used to detect transfection efficiency.Dual luciferase reporter gene method was used to confirm the interaction between circTHBS1 and miR-135a-5p,miR-135a-5p and RAB1A.Western blot assay was used to detect the expression of RAB1A in MCF-7 cells.Plate cloning method was used to detect the proliferation of MCF-7 cells.Flow cytometry was used to detect the apoptosis of MCF-7 cells.Scratch assay was used to detect the migration of MCF-7 cells.Transwell method was used to detect the invasion of MCF-7 cells.The nude mouse transplantation tumor experiment was used to detect the effect of circTHBS1 on the growth of BC cells in tumor tissue.The dual luciferase reporter gene assay found a targeted relationship between circTHBS1 and miR-135a-5p,RAB1A and miR-135a-5p.Compared with normal breast epithelial cells MCF-10A,the expression of circTHBS1 and RAB1A obviously increased in BC cell lines,while the expression of miR-135a-5p obviously decreased(P<0.05).Knocking down the expression of circTHBS1 was able to inhibit the proliferation,migration,and invasion of MCF-7 cells,and induce MCF-7 cell apoptosis(P<0.05).Inhibiting the expression of miR-135a-5p was able to reverse the inhibitory effect of knocking down circTHBS1 expression on the proliferation,migration,and invasion of MCF-7 cells,and inhibit MCF-7 cell apoptosis(P<0.05).Through nude mouse transplantation experiments,it was found that knocking down the expression of circTHBS1 resulted

关 键 词：环状RNA血栓反应蛋白-1 微小RNA-135a-5p Rab家族蛋白1A 乳腺癌 增殖 凋亡 迁移 侵袭 

分 类 号：R737.9[医药卫生—肿瘤]