摘要
目的:研究circ_0022382对乳腺癌细胞增殖和迁移的影响及可能的作用机制。方法:①筛选乳腺癌中高表达的circRNA,首先分别对乳腺癌circRNA相关GEO数据集(GSE165884)和miRNA相关GEO数据集(GSE45498)进行差异分析,然后通过ENCORI数据库预测差异表达的circRNA所结合的miRNA,最后通过韦恩图对circRNA结合的miRNA和GEO数据库预测的miRNA取交集,得到交集的miRNA及其对应的circRNA。②分别予以收敛引物和发散引物对乳腺癌细胞MDA-MB-231来源的互补DNA(cDNA)和基因组DNA(gDNA)进行PCR,并通过琼脂糖凝胶电泳实验对PCR产物进行分离以验证circ_0022382的环形结构。③实时荧光定量PCR(qRT-PCR)分别检测乳腺癌细胞MDA-MB-231和MCF-7及正常乳腺上皮细胞MCF-10A,乳腺癌组织和癌旁组织中circ_0022382表达水平。④在MDA-MB-231细胞和MCF-7细胞中分别转染si-NC、si-circ_0022382,采用qRT-PCR检测转染效率,分别采用克隆形成实验和划痕愈合实验检测乳腺癌细胞增殖和迁移;qRT-PCR检测let-7a-5p的表达水平;在si-circ_0022382组中分别转染let-7a-5p inhibitor NC、let-7a-5p inhibitor,克隆形成实验和划痕愈合实验检测乳腺癌细胞增殖和迁移。⑤通过京都基因与基因组百科全书(KEGG)富集分析let-7a-5p的下游信号通路;在MDA-MB-231细胞和MCF-7细胞中分别转染let-7a-5p NC、let-7a-5p mimic,蛋白质印迹法和细胞荧光免疫法检测PI3K/AKT/mTOR信号通路相关蛋白表达,蛋白质印迹法检测let-7a-5p inhibitor和si-circ_0022382共转染后乳腺癌细胞p-AKT表达。结果:①GSE165884差异分析得到37个高表达的circRNA,GSE45498差异分析得到6个低表达的miRNA,37个高表达的circRNA中,circ_0022382对应的miRNA和6个低表达的miRNA有交集基因let-7。②乳腺癌细胞MDA-MB-231来源的cDNA既可以通过发散引物又可以通过收敛引物来扩增,而gDNA只能通过收敛引物来扩增。③与MCF-10A细胞相比,circ_0022382在乳腺癌细胞MDA-MB-231和MCF-7中均显著高表达(P均<0.001);乳腺癌组织中circ_0022382表达亦明显高于癌旁组织(P<0.05)。④下调circ_0022382表达后,MDA-MB-231和MCF-7细胞的增殖和迁移能力均显著下降(P均<0.001),let-7a-5p的表达水平均显著升高(P<0.01和P<0.05);在si-circ_0022382组中,共转染let-7a-5p inhibitor能够显著挽救MDA-MB-231和MCF-7细胞增殖和迁移能力的下降(P<0.01和P<0.001)。⑤KEGG信号通路富集分析结果显示,PI3K-AKT和mTOR信号通路显著富集;与let-7a-5p NC组相比,let-7a-5p mimic组的p-AKT、p-PI3K和mTOR的蛋白水平显著下降;在si-circ_0022382组中,共转染let-7a-5p inhibitor能够显著挽救MDA-MB-231和MCF-7细胞中p-AKT表达水平的降低。结论:circ_0022382在乳腺癌中高表达,通过靶向let-7a-5p/PI3K/AKT/mTOR轴促进乳腺癌细胞的增殖和迁移能力,其有可能作为乳腺癌治疗和诊断的潜在靶点。
Objective:To study the effects of circ_0022382 on the proliferation and migration of breast cancer cells and the possible underlying mechanism.Methods:①Screening for circRNAs that are highly expressed in breast cancer.Firstly,the differential analysis of the circRNA-related GEO dataset(GSE165884)and miRNA-related GEO dataset(GSE45498)of breast cancer were carried out,respectively,and then the miRNAs bound by the differentially expressed circRNAs were predicted by the ENCORI database,and finally the intersecting miRNAs and the miRNAs predicted by the GEO database were obtained by the Venn diagram.②The complementary DNA(cDNA)and genomic DNA(gDNA)derived from MDA-MB-231 of breast cancer cells were PCR with convergent primers and divergent primers,respectively,and the PCR products were separated by agarose gel electrophoresis to verify the circular structure of the circ_0022382.③Real-time quantitative PCR(qRT-PCR)was used to detect circ_0022382 expression in breast cancer cells MDA-MB-231 and MCF-7 and normal breast epithelial cell MCF-10A,as well as in breast cancer tissues and adjacent tissues,respectively.④MDA-MB-231 cells and MCF-7 cells were transfected with si-NC and si-circ_0022382,qRT-PCR was used to detect transfection efficiency,clone formation assay and scratch healing assay were used to detect the proliferation and migration of breast cancer cells.qRT-PCR was used to detect the expression level of let-7a-5p.The si-circ_0022382 group was transfected with let-7a-5p inhibitor NC and let-7a-5p inhibitor,respectively,and the proliferation and migration of breast cancer cells were detected by clone formation assay and scratch healing assay.⑤The downstream signaling pathway of let-7a-5p was analyzed by Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment.MDA-MB-231 cells and MCF-7 cells were transfected with let-7a-5p NC and let-7a-5p mimic,respectively,and the expression of PI3K/AKT/mTOR signaling pathway-related proteins was detected by Western blotting and cellular immunofluorescence,and the expression of p-AKT in breast cancer cells co-transfected with let-7a-5p inhibitor and si-circ_0022382 was detected by Western blotting.Results:①GSE165884 differential analysis yielded 37 high-expressing circRNAs,GSE45498 differential analysis yielded 6 low-expressing miRNAs,and among the 37 high-expression circRNAs,circ_0022382 corresponding miRNAs and 6 low-expressing miRNAs had intersecting gene let-7.②cDNA derived from breast cancer cell MDA-MB-231 can be amplified by both divergent primers and convergent primers,whereas gDNA can only be amplified by convergent primers.③Compared with MCF-10A,circ_0022382 was significantly more expressed in MDA-MB-231 and MCF-7(P<0.001).The expression of circ_0022382 in breast cancer tissues was also higher than that in adjacent tissues(P<0.05).④After knocking down circ_0022382,the proliferation and migration of MDA-MB-231 and MCF-7 cells decreased significantly(P<0.001),and the expression level of let-7a-5p increased significantly(P<0.01 and P<0.05).Co-transfection of let-7a-5p inhibitor significantly rescued the decreased proliferation and migration of MDA-MB-231 and MCF-7 cells transfected with si-circ_0022382(P<0.01 and P<0.001).⑤The results of KEGG signaling pathway enrichment analysis showed that PI3K-AKT and mTOR signaling pathways were significantly enriched.Compared with the let-7a-5p NC group,the expression of p-AKT,p-PI3K and mTOR proteins in the let-7a-5p mimic group significantly decreased.Co-transfection of let-7a-5p inhibitor significantly rescued the decrease of p-AKT expression of MDA-MB-231 and MCF-7 cells transfected with si-circ_0022382.Conclusion:Circ_0022382 is highly expressed in breast cancer and promotes the proliferation and migration of breast cancer cells by targeting the let-7a-5p/PI3K/AKT/mTOR axis,which may be a potential target for breast cancer treatment and diagnosis.
作者
刘伟
张俊
张佳雯
叶雨
诸健琴
虞绮雯
李涛
孙晓春
陈华标
LIU Wei;ZHANG Jun;ZHANG Jiawen;YE Yu;ZHU Jianqin;YU Qiwen;LI Tao;SUN Xiaochun;CHEN Huabiao(School of Medicine,Jiangsu University,Zhenjiang Jiangsu 202013;Department of Clinical Laboratory,Affiliated Hospital of Yangzhou University,Yangzhou Jiangsu 225012;School of Medicine,Ningbo University,Ningbo Zhejiang 315211,China)
出处
《江苏大学学报(医学版)》
2025年第2期145-153,共9页
Journal of Jiangsu University:Medicine Edition
