下调lncRNA LOXL1-AS1表达对乳腺癌细胞增殖和侵袭的影响

Effect of downregulating lncRNA LOXL1-AS1 expression on proliferation and invasion of breast cancer cells

目的探讨长链非编码RNA(lncRNA)类赖氨酰氧化酶1反义RNA1(LOXL1-AS1)对乳腺癌MCF-7细胞增殖、侵袭和凋亡的影响及其作用机制。方法实时荧光定量PCR检测lncRNA LOXL1-AS1、miR-28-5p和IGF-1在乳腺癌细胞(MCF-7细胞)和人正常乳腺上皮细胞(MCF-10A细胞)及收集的55例乳腺癌组织和癌旁正常组织中的表达量。采用lncRNA LOXL1-AS1 siRNA、miR-28-5p inhibitor、IGF-1 siRNA分别转染MCF-7细胞,MTT法检测MCF-7细胞的增殖活力,Transwell分析MCF-7细胞的侵袭情况,流式细胞仪检测MCF-7细胞凋亡情况。生物信息学软件(miRanda)和双荧光素酶报告基因实验分析lncRNA LOXL1-AS1和miR-28-5p之间的作用靶点及相关性,TargetScan和双荧光素酶报告基因实验分析miR-28-5p与IGF-1之间的作用靶点及相关性;pcDNA-LOXL1-AS1与miR-28-5p mimics共同转染MCF-7细胞后,MTT法检测MCF-7细胞的增殖活力,Transwell分析MCF-7细胞的侵袭情况,流式细胞仪检测MCF-7细胞凋亡情况。结果MCF-7细胞内lncRNA LOXL1-AS1和IGF-1表达高于MCF-10A细胞(P<0.01),miR-28-5p表达低于MCF-10A细胞(P<0.01)。与癌旁组织相比,乳腺癌组织中lncRNA LOXL1-AS1和IGF-1表达明显升高(P<0.01),miR-28-5p表达明显降低(P<0.01)。转染lncRNA LOXL1-AS1 siRNA或IGF-1 siRNA后,MCF-7细胞增殖活力降低,细胞侵袭数目减少,细胞凋亡率增加(均P<0.01)。lncRNA LOXL1-AS1靶向miR-28-5p,miR-28-5p靶向IGF-1;miR-28-5p表达下调后,MCF-7细胞增殖活力提高,细胞侵袭数目增加,细胞凋亡率减少(P<0.05);pcDNA-LOXL1-AS1与miR-28-5p mimics共同转染MCF-7细胞后,MCF-7细胞增殖活力提高,侵袭数目增加,细胞凋亡率减少(均P<0.05)。结论lncRNA LOXL1-AS1在MCF-7细胞中表达上调,下调lncRNA LOXL1-AS1表达通过影响miR-28-5p/IGF-1轴抑制了乳腺癌增殖和侵袭。

Objective To investigate the effects of lncRNA LOXL1-AS1 on the proliferation,invasion abilities and the apoptosis of breast cancer MCF-7 cells and its potential mechanism.Methods The expression levels of lncRNA LOXL1-AS1,miR-28-5p and IGF-1 were detected in MCF-7 and MCF-10A cells,and in 55 samples of breast cancer tissues and adjacent normal tissues by real-time quantitative PCR.LncRNA LOXL1-AS1 siRNA,miR-28-5p inhibitor and IGF-1 siRNA were respectively transfected into MCF-7 cells,and then MTT assay was used to detect the proliferation activity of MCF-7 cells,Transwell assay was used to analyze the invasion of MCF-7 cells,and flow cytometry was used to detect the apoptosis of MCF-7 cells.The target and correlation between lncRNA LOXL1-AS1 and miR-28-5p were analyzed by miRanda and dual luciferase reporter gene assay.The target and correlation between miR-28-5p and IGF-1 were analyzed by TargetScan and dual luciferase reporter gene assay.After pcDNA-LOXL1-AS1 and miR-28-5p mimics were co-transfected into MCF-7 cells,the proliferation activity of MCF-7 cells was detected by MTT assay,the invasion of MCF-7 cells was analyzed by Transwell,and the apoptosis of MCF-7 cells was detected by flow cytometry.Results The expression levels of lncRNA LOXL1-AS1 and IGF-1 in MCF-7 cells were higher than those in MCF-10A cells(P<0.01),and the expression of miR-28-5p was lower than that of MCF-10A cells(P<0.01).Compared to paracancer tissue,the expression of lncRNA LOXL1-AS1 and IGF-1 was significantly increased(P<0.01),and the expression of miR-28-5p was significantly decreased in breast cancer tissues(P<0.01).After transfection of lncRNA LOXL1-AS1 siRNA and IGF-1 siRNA,the proliferation activity of MCF-7 cells was decreased,the number of cell invasion was decreased,and the apoptosis rate was increased(all P<0.01).LncRNA LOXL1-AS1 targeted miR-28-5p and miR-28-5p targeted IGF-1.After the expression of miR-28-5p was down-regulated,the proliferation activity of MCF-7 cells was increased,the number of cell invasion was increased,and the apoptosis rate was decreased(P<0.05).After pcDNA-LOXL1-AS1 and miR-28-5p mimics were co-transfected into MCF-7 cells,the proliferation activity of MCF-7 cells was increased,the number of invasion was increased,and the apoptosis rate was decreased(P<0.05).Conclusion LncRNA LOXL1-AS1 expression is up-regulated in MCF-7 cells,and down-regulating lncRNA LOXL1-AS1 expression can inhibit the proliferation and invasion of breast cancer by the miR-28-5p/IGF-1 axis.