长链非编码RNA FTX靶向miR-21-3p对肝癌细胞生长侵袭和迁移调控作用

Long noncoding RNA FTX targeting miR-21-3p regulates the proliferation,invasion and migration of liver cancer cells

目的在发展中国家肝癌是癌症致死的第二大原因。本研究旨在探讨长链非编码RNA(long noncoding RNA,LncRNA)FTX靶向miR-21-3p对肝癌细胞生长、侵袭和迁移的影响及其机制。方法实时定量PCR(quantitative realtime reverse transcription PCR,qRT-PCR)检测lncRNA FTX和miR-21-3p表达,通过慢病毒载体过表达lncRNA FTX,将HepG2细胞分为对照组(LV-Ctrl)、过表达组(LV-FTX)、LV-FTX+miR-21mock组和LV-FTX+miR-21mimic组4组,荧光素酶实验分析靶向关系,MTT法检测细胞增殖,Transwell检测细胞侵袭,划痕实验分析细胞迁移,蛋白质印迹法检测PCNA、Cyclin D1、Ki-67、MMP-9、MMP-14和VEGF蛋白表达。结果在肝癌组织和肝癌细胞株中FTX低表达,而miR-21-3p高表达。miR-21mimic缓解lncRNA FTX对miR-21表达的抑制,P<0.05。与对照组相比,LV-FTX组和LV-FTX+miR-21 mock组细胞增殖倍数降低,差异有统计学意义,P<0.05。与LV-FTX组相比,LV-FTX+miR-21mimic组肝癌细胞增殖倍数升高,差异有统计学意义,P<0.05。LV-FTX组单视野下侵袭细胞数目(38±11)和划痕愈合率[(38±7)%]降低(P<0.05)。LV-FTX+miR-21mimic组单视野下侵袭细胞数目(128±32)和划痕愈合率[(59±6)%]高于LV-FTX组(P<0.05)。LV-FTX组PCNA、Cyclin D1、Ki-67、MMP-9、MMP-14和VEGF的mRNA水平(0.39±0.07、0.35±0.06、0.24±0.03、0.31±0.07、0.26±0.04和0.36±0.06)和蛋白水平(0.33±0.04、0.32±0.02、0.26±0.02、0.21±0.06、0.15±0.05和0.32±0.04)低于对照组,P<0.05。与LV-FTX组相比,LV-FTX+miR-21mimic组PCNA、Cyclin D1、Ki-67、MMP-9、MMP-14、VEGF的mRNA水平(0.76±0.08,0.87±0.09、0.85±0.03、0.75±0.09、0.72±0.08、0.79±0.11)和蛋白水平(0.93±0.05、1.25±0.06、1.13±0.04、0.76±0.08、0.45±0.05和0.66±0.06)升高,差异有统计学意义,P<0.05。结论LncRNA FTX靶向miR-21-3p抑制肝癌细胞增殖、侵袭和迁移。

OBJECTIVE Liver cancer is the second leading cause of cancer deaths in developing countries.This study aimed to explore the effect of long noncoding RNA FTX targeting miR-21-3 p on the proliferation,invasion and migration of liver cancer cells.METHODS The expression of lncRNA FTX and miR-21-3 p was detected by qRT-PCR.LncRNA FTX was overexpressed via lentiviral vector.HepG2 cells were divided into four groups:cntrol group(LV-Ctrl),overexpression group(LV-FTX),LV-FTX+miR-21 mock group and LV-FTX+miR-21 mimic group.The target relationship was confirmed by luciferase experiment.Cell proliferation was tested by MTT.Cell invasion was measured through transwell.Cell migration was tested by wound healing.The expressions of PCNA,Cyclin D1,Ki-67,MMP-9,MMP-14 and VEGF were detected by western blot.RESULTS The expression of FTX was low in hepatocellular carcinoma tissues and HCC cell lines,while miR-21-3 p was highly expressed.MiR-21 mimic reversed the inhibition of lncRNA FTX on the expression of miR-21(P<0.05).LncRNA FTX directly targeted mir-21-3 p.The proliferation folds in LV-FTX group and LV-FTX+miR-21 mock group were lower than that in control group(P<0.05).Compared with LV-FTX group,the proliferation folds in LV-FTX+miR-21 mimic group were increased(P<0.05).Compared with control group,the number of invasive cells per field(38±11)and wound closure rate(38±7)%in LV-FTX group were alleviated(P<0.05).The number of invasive cells per field(128±32)and wound closure rate(59±6)%in LV-FTX+miR-21 mimic group were higher than that in LV-FTX group(P<0.05).The mRNA(0.39±0.07,0.35±0.06,0.24±0.03,0.31±0.07,0.26±0.04,0.36±0.06)and protein(0.33±0.04,0.32±0.02,0.26±0.02,0.21±0.06,0.15±0.05,0.32±0.04)levels of PCNA,Cyclin D1,Ki-67,MMP-9,MMP-14 and VEGF in LV-FTX group were lower than that in control group(P<0.05).Compared with LV-FTX group,the mRNA(0.76±0.08,0.87±0.09,0.85±0.03,0.75±0.09,0.72±0.08,0.79±0.11)and protein(0.93±0.05,1.25±0.06,1.13±0.04,0.76±0.08,0.45±0.05,0.66±0.06)levels of PCNA,Cyclin D1,Ki-67,MMP-9,MMP-14 and VEGF in LV-FTX+miR-21 mimic group were enhancive(P<0.05).CONLUSION LncRNA FTX attenuates the proliferation,invasion and migration of liver cancer cells via targeting miR-21-3p.