LncRNA NEAT1/miR-23b-3p/KLF3轴调控结直肠癌细胞的生物学功能研究

LncRNA NEAT1/miR-23b-3p/KLF3 Axis Regulates the Biological Function of Colorectal Cancer Cells

目的探究长链非编码核糖核酸(long non-coding RNAs,LncRNAs)NEAT1/miR-23b-3p/KLF3调控轴对结直肠癌细胞生物学功能的影响。方法利用癌症基因组图谱(the cancer genome atlas,TCGA)数据库分析结直肠癌组织和癌旁组织NEAT1,miR-23b-3p和KLF3表达水平,并计算三者之间的相关性。以结直肠癌细胞系(HT29细胞)作为实验对象,实验被分为Control组(空白对照组)、NC组(空载体组)和si-NEAT1组(NEAT1干扰组)。CCK8检测各组细胞的增殖情况,流式细胞术检测各组细胞的凋亡、周期情况,Transwell检测各组细胞的侵袭情况,划痕实验检测各组细胞的迁移情况。在线工具starbase等预测能与miR-23b-3p靶向结合的基因,采用人胚胎肾细胞293(human embryonic kidney cells 293,HEK293)进行双荧光素酶报告基因实验验证。qRT-PCR检测NC组、si-NEAT1组、si-NEAT1+miR-23b-3p inhibitor组(共转染si-NEAT1和miR-23b-3p inhibitor)的miR-23b-3p和KLF3转录水平,免疫印记法检测以上三组KLF3蛋白质表达水平。结果结直肠癌组织NEAT1[5.29(4.55,5.95)],KLF3[4.94(4.62,5.24)]表达高于癌旁组织[4.79(4.26,5.19),4.49(4.24,4.80)],差异有统计学意义(U=6677,P=0.001;U=28257.5,P<0.001),miR-23b-3p表达低于癌旁组织[9.99(9.49,10.6)vs 10.80(10.62,10.88)],差异有统计学意义(U=2906,P=0.004)。结直肠癌组织中,NEAT1和KLF3表达呈正相关(r=0.26,P<0.01),miR-23b-3p和NEAT1,KLF3表达量均呈负相关(r=-0.14,P=0.008;r=-0.17,P=0.001)。与对照组相比,si-NEAT1组使结直肠癌细胞增殖、迁移、侵袭的能力降低,凋亡增加,细胞被阻滞在S期,结果差异均有统计学意义(均P<0.05)。starbase预测miR-23b-3p靶基因为NEAT1和KLF3。双荧光素酶报告基因实验也证实miR-23b-3p分子能互补结合NEAT1和KLF33’UTR。与NC组相比,si-NEAT1组miR-23b-3p表达上调,KLF3转录和蛋白水平下调;与si-NEAT1组相比,si-NEAT1+miR-23b-3p inhibitor组miR-23b-3p表达下调,KLF3转录和蛋白水平上调。结论NEAT1可通过直接靶向HT29细胞中的miR-23b-3p上调KLF3表达,增强结直肠癌细胞的增值、侵袭和迁移等。

Objective To explore the biological function of long non-coding RNAs(LncRNAs)NEAT1/miR-23b-3p/KLF3 regulatory axis in colorectal cancer.Methods The expression levels of NEAT1,miR-23b-3p and KLF3 in colorectal cancer tissues and adjacent tissues were analyzed by TCGA(the cancer genome atlas)database,and the correlations between the three molecular were calculated.HT29 cells were treated as the experimental object,which was divided into control group(blank control group),NC group(empty vector group)and si-NEAT1 group(NEAT1 interference group).CCK8 was used to detect cell proliferation,cell apoptosis and cell cycle were detected by flow cytometry,cell invasion was detected by Transwell,and cell migration was detected by wound-healing assay in each group.The online tool starbase etc.were used to predict the target genes of miR-23b-3p,and dual luciferase reporter gene assay was used to verify the target genes of miR-23b-3p by HEK293(human embryonic kidney 293 cells).The transcript levels of miR-23b-3p and KLF3 in NC group,si-NEAT1 group and si-NEAT1+miR-23b-3p inhibitor group(co-transfected with si-NEAT1 and miR-23b-3p inhibitor)were detected by qRT-PCR,and KLF3 protein levels were detected by western blotting.Results NEAT1[5.29(4.55,5.95)]and KLF3[4.94(4.62,5.24)]levels were higher in colorectal cancer than in paracancerous tissues[4.79(4.26,5.19),4.49(4.24,4.80)],the difference was statistically significant(U=6677,P=0.001;U=28257.5,P<0.001),and miR-23b-3p levels were lower in colorectal cancer than in paracancerous tissues[9.99(9.49,10.6)vs 10.80(10.62,10.88)],the difference was statistically significant(U=2906,P=0.004),respectively.A positive correlation exists between NEAT1 and KLF3 expression levels(r=0.26,P<0.01),and expression of miR-23b-3p was inversely correlated with NEAT1 and KLF3(r=-0.14,P=0.008;r=-0.17,P=0.001).Compared with control group,the ability of proliferation,migration and invasion was reduced,apoptosis increased,and cells were blocked in S phase in si-NEAT1 group for colorectal cancer cells,which were statistically significant(all P<0.05).The starbase,etc.predicted that the miR-23b-3p target genes were NEAT1 and KLF3.Dual luciferase reporter gene assay also confirmed that miR-23b-3p could bind NEAT1 and KLF33’UTR.In the si-NEAT1 group,the expression of miR-23b-3p was up-regulated,while the KLF3 transcript and protein levels were down-regulated through comparison with a NC group(all P<0.05).In the si-NEAT1+miR-23b-3p inhibitor group,the expression of miR-23b-3p was down-regulation,KLF3 transcript and protein levels were up-regulated through comparison with a si-NEAT1 group(all P<0.05).Conclusion NEAT1 can up-regulate the expression of KLF3 and enhance the proliferation,invasion,and migration of colorectal cancer cells by directly targeting miR-23b-3p in HT29 cells.