Song Xujun, Wu Rong, Tao Qingsong, Zhao Liangtao, Ji Zhenling
Department of General Surgery, Zhongda Hospital, Medical School of Southeast University, Nanjing, Jiangsu, China.
Cell Mol Biol (Noisy-le-grand). 2019 Jul 31;65(6):52-55.
The aim of this study was to investigate the effect of microRNA-532 (miR-532) on invasion and metastasis of colorectal cancer (CRC) cells, and the underlying mechanism. Human CRC cell line (HCT116) and normal colon (FHC) cells were used for this study. The cells were transfected with naked cuticle homolog 1 (NKD1) overexpression plasmid, miR-532 mimics, miR-532 inhibitor or miR-532 non-homologous sequence using lipofectamine 2000. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression of miR-532 in CRC cells, and a combination of scratch and Transwell assays was used to assess the effect of miR-532 on migration and invasion of CRC cells. Western blotting was used to determine the effect of miR-532 on NKD1 expression in CRC cells. Bioinformatics analysis and luciferase reporter gene assay were used to assess the regulatory effect of miR-532 on NKD1. The expression of miR-532 was upregulated in CRC cells relative to normal colon cells (p < 0.05). The HCT116 cells transfected with miR-532 mimics migrated faster than those of miR-532 negative control (miR532-NC) group (p < 0.05). The migration ability of HCT116 cells transfected with miR-532 inhibitor was significantly reduced, when compared with that of miR532-NC group (p < 0.05). The invasive ability of HCT116 cells transfected with miR-532 mimics was significantly higher than that of miR532-NC cells (p < 0.05). However, inhibition of miR-532 expression significantly reduced the invasive ability of HCT116 cells (p < 0.05). Results of bioinformatics showed that miR-532 had specific binding sequence with the 3'UTR region of NKD1. After cloning the sequence into the luciferase reporter plasmid, miR-532 significantly inhibited the expression of NKD1 (p < 0.05). However, miR-532 had no inhibitory effect on mutated NKD1 3'UTR (p > 0.05). Results of Western blotting showed that increased miR-532 expression significantly reduced the expression of NKD1, while decreased miR-532 expression promoted the expression of NKD1 (p < 0.05). Overexpression of NKD1 significantly down-regulated miR-532 overexpression and promoted CRC cell invasion and metastasis (p < 0.05). miR-532 is highly expressed in CRC cells and directly inhibits NKD1 expression, while enhancing invasion and metastasis of CRC cells. It promotes the development of CRC by inhibiting the expression of NKD1.
本研究旨在探讨微小RNA-532（miR-532）对结直肠癌（CRC）细胞侵袭和转移的影响及其潜在机制。本研究使用了人CRC细胞系（HCT116）和正常结肠（FHC）细胞。使用脂质体2000将裸角质同源物1（NKD1）过表达质粒、miR-532模拟物、miR-532抑制剂或miR-532非同源序列转染至细胞。采用实时定量聚合酶链反应（qRT-PCR）检测CRC细胞中miR-532的表达，并采用划痕实验和Transwell实验相结合的方法评估miR-532对CRC细胞迁移和侵袭的影响。采用蛋白质免疫印迹法检测miR-532对CRC细胞中NKD1表达的影响。采用生物信息学分析和荧光素酶报告基因实验评估miR-532对NKD1的调控作用。与正常结肠细胞相比，CRC细胞中miR-532的表达上调（p<0.05）。转染miR-532模拟物的HCT116细胞比miR-532阴性对照（miR532-NC）组细胞迁移更快（p<0.05）。与miR532-NC组相比，转染miR-532抑制剂的HCT116细胞迁移能力显著降低（p<0.05）。转染miR-532模拟物的HCT116细胞侵袭能力显著高于miR532-NC细胞（p<0.05）。然而，抑制miR-532表达显著降低了HCT116细胞的侵袭能力（p<0.05）。生物信息学结果显示，miR-532与NKD1的3'UTR区域具有特异性结合序列。将该序列克隆到荧光素酶报告质粒后，miR-532显著抑制了NKD1的表达（p<0.05）。然而，miR-532对突变的NKD1 3'UTR无抑制作用（p>0.05）。蛋白质免疫印迹法结果显示，miR-532表达增加显著降低了NKD1的表达，而miR-532表达降低则促进了NKD1的表达（p<0.05）。NKD1的过表达显著下调了miR-532的过表达，并促进了CRC细胞的侵袭和转移（p<0.05）。miR-532在CRC细胞中高表达，直接抑制NKD1表达，同时增强CRC细胞的侵袭和转移。它通过抑制NKD1的表达促进CRC的发展。
