作者：王晓龙1，陈亮1，曹洪涛1，曹天生1，张文1，林波1，汤婷婷1，马克强1，杨建荣1，何志军2，汪彪2，张健2

单位：1.南方医科大学附属花都医院 胃肠外科， 广东 广州 510800；2.湖北医药学院附属人民医院 胃肠外科， 湖北 十堰 420000

Authors: Wang Xiaolong1，Chen Liang1， Cao Hongtao1，Cao Tiansheng1，Zhang Weiwen1，Lin Bo1，Tang Tingting1，Ma Keqiang1，Yang Jianrong1，He Zhijun2， Wang Biao2

，Zhang Jian2

Unit: 1.Gastrointestinal Surgery, Huadu Hospital Affiliated to Southern Medical University, Guangzhou 510800, Guangdong, China；2.Gastrointestinal Surgery, People's Hospital Affiliated to Hubei University of Medicine, Shiyan 420000, Hubei, China

摘要：

目的 探讨长链非编码RNA NPPA-AS1 (lncRNA NPPA-AS1)对结直肠癌SW480细胞增殖和凋亡的影响及其分子机制。方法 选取南方医科大学附属花都医院和湖北医药学院附属人民医院2017年1月至2019年1月期间的30对结直肠癌组织及匹配的癌旁组织；将结直肠癌细胞（SW480细胞）进行体外培养并单独或者联合转染pcDNA-3.1载体（pcDNA）、miRNA抑制剂阴性对照（anti-miR-NC）、NPPA-AS1过表达载体（pcDNA-NPPA-AS1）、 miR-372-3p抑制剂（anti-miR-372-3p）、miRNA模拟物阴性对照（miR-NC）和miR-372-3模拟物（miR-372-3p）以构建pcDNA组、pcDNA-NPPA-AS1组、anti-miR-NC组、anti-miR-372-3p组、pcDNA-NPPA-AS1+miR-NC组和pcDNA-NPPA-AS1+miR-372-3p组；实时荧光定量PCR(RT-qPCR)验证lncRNA NPPA-AS1和miR-372-3p的表达；四甲基偶氮唑盐比色法（MTT）比较细胞活力；蛋白质印迹法比较蛋白表达；流式细胞术比较凋亡情况。结果 结直肠癌的lncRNA NPPA-AS1对比癌旁组织表达更低，但miR-372-3p对比癌旁组织表达更高（P<0.05）；过表达lncRNA NPPA-AS1显著降低SW480细胞的活力，使细胞的凋亡率增加，并降低了B淋巴细胞瘤/白血病-2(B-cell lymphoma-2;Bcl-2)和cyclinD1的表达，提高了B淋巴细胞瘤/白血病2相关X蛋白（BCL2-associated x protein;Bax）和p21的表达（P<0.05）。lncRNA NPPA-AS1靶向调控miR-372-3p表达。抑制miR-372-3p显著降低SW480细胞的活力，使细胞的凋亡率增加，并降低了Bcl-2和cyclinD1的表达，提高了Bax和p21的表达（P<0.05）。上调miR-372-3p表达逆转了过表达lncRNA NPPA-AS1对SW480细胞的影响。结论 过表达lncRNA NPPA-AS1通过靶向下调miR-372-3p表达调控结直肠癌SW480细胞的增殖及凋亡。

关键词：lncRNA NPPA-AS1；miR-372-3p；增殖；凋亡

Abstract：

Objective This study aims to investigate the effects and molecular mechanism of long non-coding RNA NPPA-AS1 （lncRNA NPPA-AS1） regulating the proliferation and apoptosis of colorectal cancer cells （SW480 cells ）. Method Thirty pairs of colorectal cancer tissues and the matched adjacent tissues were selected from our hospital during January 2017 to January 2019. SW480 cells were cultured in vitro and transfected with pcDNA-3.1 vector （pcDNA）, NPPA-AS overexpression plasmid （pcDNA-NPPA-AS ）, miRNA inhibitor negative control （anti -miR -NC）, miR -372 -3p inhibitor （anti -miR -372 -3p ）, miRNA mimic negative control （miR-NC） and miR-372-3p mimic （miR-372-3p） alone or jointly, generating pcDNA group, pcDNA -NPPA -AS group, anti -miR -NC group, anti -miR -372 -3p group, pcDNA -NPPA - AS1+miR-NC group, and pcDNA-NPPA-AS1+miR-372-3p group. Then, real-time fluorescence quantitative PCR （RT- qPCR） was used to detect the expression levels of lncRNA NPPA - AS1 and miR - 372 - 3p . 4,5-Dimethylthazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） method was conducted to compare cell activity. Western blot was performed to detect protein expression. Flow cytometry was utilize to analyze cell apoptosis . Result Compared with adjacent tissues , lncRNA NPPA - AS1 expression was significantly decreased, while miR-372-3p expression was increased in colorectal cancer tissues （P＜0.05）. Overexpression of lncRNA NPAA -AS1 significantly reduced the cell activity and increased the apoptosis rate of SW480, accompanied by the decreases in Bcl -2 and CyclinD1 expression and increases in Bax and p21 expression （P<0.05）. LncRNA NPAA-AS1 targets miR-372-3p, and inhibition of miR-372-3p significantly reduced the cell activity and promoted the apoptosis rate of SW480. In addition, miR-372-3p depletion decreased the expression of Bcl -2 and CyclinD1 and increased the expression of Bax and p21 （P<0.05 ）. Further, up-regulation of miR-372-3p reversed the effects of lncRNA NPAA-AS1 overexpression in SW480 cells. Conclusion Overexpression of lncRNA NPAA-AS1 regulates proliferation and apoptosis of colorectal can[1]cer SW480 cells through the down-regulation of miR-372-3p.

Key Words: lncRNA NPPA-AS1；miR-372-3p；Proliferation；Apoptosis