[1]胡道军,史文杰,孙 敏.LncRNA NEAT1/miR-23b-3p/KLF3 轴调控结直肠癌细胞的生物学功能研究[J].现代检验医学杂志,2022,37(04):1-6+80.[doi:10.3969/j.issn.1671-7414.2022.04.001]
　HU Dao-jun,SHI Wen-jie,SUN Min.LncRNA NEAT1/miR-23b-3p/ KLF3 Axis Regulates the Biological Function of Colorectal Cancer Cells[J].Journal of Modern Laboratory Medicine,2022,37(04):1-6+80.[doi:10.3969/j.issn.1671-7414.2022.04.001]
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LncRNA NEAT1/miR-23b-3p/KLF3 轴调控结直肠癌细胞的生物学功能研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第37卷
期数:
2022年04期
页码:
1-6+80
栏目:
论著
出版日期:
2022-07-15

文章信息/Info

Title:
LncRNA NEAT1/miR-23b-3p/ KLF3 Axis Regulates the Biological Function of Colorectal Cancer Cells
文章编号:
1671-7414（2022）04-001-07
作者:
胡道军1; 史文杰2; 孙 敏3
1. 上海市新华医院崇明分院检验科，上海 202150；2. 桂林市中医医院乳腺外科，广西桂林 541002；3. 湖北医药学院附属医院太和医院普外科，湖北十堰 442000
Author(s):
HU Dao-jun1;  SHI Wen-jie2;  SUN Min3
1.Department of Laboratory Medicine, Xinhua Hospital Chongming Branch of Shanghai, Shanghai 202150, China; 2. Department of Breast Surgery, Guilin Municipal Hospital of Traditional Chinese Medicine, Guangxi Guilin 541000, China; 3. Department of General Surgery, Taihe Hospital, Affiliated Hospital of Hubei University of Medicine, Hubei Shiyan 442000, China
关键词:
结直肠癌; 长链非编码核糖核酸; 竞争性内源核糖核酸; 细胞增殖; 细胞迁移
分类号:
R735.3；R730.43
DOI:
10.3969/j.issn.1671-7414.2022.04.001
文献标志码:
A
摘要:
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（humanembryonic 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=6 677, P=0.001；U=28 257.5, 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=2 906, 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KLF3 3’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。
Abstract:
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=6 677, P=0.001；U=28 257.5, 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=2 906, 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 KLF3 3’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.

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备注/Memo

备注/Memo:
基金项目：国家自然科学基金（819024698）；上海市崇明区“可持续发展科技创新行动计划”（CKY2018-11）。
作者简介：胡道军（1981-），男，硕士，主管技师，研究方向：临床检验与肿瘤医学大数据分析，E-mail: 996978851@qq.com。
通讯作者：孙敏（1984-），男，博士，副主任医师，研究方向：肿瘤大数据和精准医学，E-mail: sunmin-0715@163.com。
更新日期/Last Update: 1900-01-01