长链非编码RNA KCNQ1OT1靶向调控miR-218-5p对结肠癌细胞生物学行为的影响
| 作者: |
1张祎,
1陈畅
1 宜昌市第一人民医院消化内科，湖北 宜昌 443000 |
| 通讯: |
陈畅
Email: cc20190411@sina.com |
| DOI: | 10.3978/j.issn.2095-6959.2020.03.001 |
摘要
目的：探讨长链非编码RNA KCNQ1重叠转录物1(lncRNA KCNQ1OT1)调控micro RNA-218-5p(miR-218-5p)对结直肠癌细胞恶性生物学行为的影响及其可能机制。方法：收集2019年1月至4月在宜昌市第一人民医院行根治性结肠癌切除术的30例肿瘤组织和相应的癌旁正常组织。采用实时荧光定量PCR法(qRT-PCR)检测lncRNA KCNQ1OT1，miR-218-5p在结肠癌组织、癌旁正常组织、5种结直肠癌细胞系(HCT-116，SW480，SW620，DLD-1，HT29)及正常结肠上皮细胞系CCD841中的表达水平；干预结肠癌细胞系HCT-116和SW480中lncRNA KCNQ1OT1和miR-218-5p的表达，采用CCK-8法检测结肠癌细胞的增殖，Transwell法检测结肠癌细胞的迁移和侵袭；采用流式细胞术检测细胞周期分布及细胞凋亡率；用Starbase数据库预测lncRNA KCNQ1OT1的靶向miRNA，并用qRT-PCR、双荧光素酶报告基因法验证lncRNA KCNQ1OT1与miR-218-5p的靶向关系。结果：与癌旁正常组织和正常结肠上皮细胞系相比，结直肠癌组织和结直肠癌细胞系中lncRNA KCNQ1OT1的表达水平显著上调，miR-218-5p的表达显著下调(P<0.05)；过表达lncRNA KCNQ1OT1促进SW480细胞的增殖、迁移和侵袭，且减少处于G0/G1期的细胞比率，抑制细胞凋亡；在结直肠癌组织中lncRNA KCNQ1OT1的表达水平与miR-218-5p的表达水平呈负相关(r2=0.437，P<0.001)；双荧光素酶报告基因法分析证实lncRNA KCNQ1OT1能特异性结合miR-218-5p，并能降低其表达；过表达miR-218-5p抑制HCT-116细胞的增殖、迁移和侵袭，增加处于G0/G1期的细胞比率，促进细胞凋亡，且miR-218-5p的表达可以抑制由lncRNA KCNQ1OT1过表达引起的细胞增殖、迁移和侵袭能力的增加及凋亡的减少。结论：LncRNA KCNQ1OT1通过靶向调控miR-218-5p表达影响结直肠癌细胞的增殖、迁移和侵袭，促进结直肠癌的发展。
关键词：
lncRNA KCNQ1OT1；miR-218-5p；结直肠癌；HCT-116；增殖；迁移；侵袭
Effect of long non-coding RNA KCNQ1OT1 regulating miR-218-5p on biological behaviors of colon cancer cells
CorrespondingAuthor: CHEN Chang Email: cc20190411@sina.com
DOI: 10.3978/j.issn.2095-6959.2020.03.001
Abstract
Objective: To investigate the effect of long non-coding RNA KCNQ1 overlapping transcript 1 (lncRNA KCNQ1OT1) on malignant biological behavior of colorectal cancer cells via micro RNA-218-5p (miR-218-5p) and its underlying mechanism. Methods: Thirty cases of tumor tissues and corresponding adjacent normal tissues were collected from patients underwent radical resection of colorectal cancer in the First People’s Hospital of Yichang from January 2019 to April 2019. Real-time quantitative PCR (qRT-PCR) was performed to detect the expressions of lncRNA KCNQ1OT1 and miR-218-5p in colorectal cancer tissues, adjacent tissues, five colorectal cancer cell lines (HCT-116, SW480, SW620, DLD-1 and HT29) and normal colon epithelial cell line CCD841. After the interference with the expressions of lncRNA KCNQ1OT1 and miR-218-5p in HCT-116, CCK-8 assay was conducted to monitor the proliferation, and the migration and invasion were detected by Transwell assay. Cell cycle distribution and apoptosis rate were detected by flow cytometry. Starbase database predicted the targeted miRNA of lncRNA KCNQ1OT1 and the targeting relationship between lncRNA KCNQ1OT1 and miR-218-5p was verified by qRT-PCR and dual luciferase reporter gene assay. Results: Compared with adjacent tissues and normal colon epithelial cell lines, the expression of lncRNA KCNQ1OT1 was significantly up-regulated while the expression of miR-218-5p was down-regulated in colorectal cancer tissues and cells. Overexpressed lncRNA KCNQ1OT1 promoted the proliferation, migration and invasion of SW480 cells, and the cell ratio in the G0/G1 phase was reduced and apoptosis was inhibited. The expression level of lncRNA KCNQ1OT1 was negatively correlated with the expression of miR-218-5p in colorectal cancer (r2=0.437, P<0.001). Dual luciferase reporter gene assay confirmed that lncRNA KCNQ1OT1 could specifically bind to miR-218-5p, and reduce its expression. Overexpressed miR-218-5p inhibited the proliferation, migration and invasion of HCT-116 cells, and the expression of miR-218-5p could impede the increase of proliferation, migration and invasion and the decrease of apoptosis of SW480 cells induced by overexpressed lncRNA KCNQ1OT1. Conclusion: LncRNA KCNQ1OT1 modulates the proliferation, migration and invasion of colorectal cancer cells by targeted regulation of miR-218-5p expression, thus promoting the progression of colorectal cancer.
Keywords：
lncRNA KCNQ1OT1; miR-218-5p; colorectal cancer; HCT-116; proliferation; migration; invasion