去氧鬼臼毒素对结肠癌细胞增殖、迁移及侵袭的影响

Effects of deoxypodophyllotoxin on the proliferation,migration and invasion of colon cancer cells

目的探讨去氧鬼臼毒素(DPPT)是否通过调控长链非编码RNA ZFPM2-AS1(LncRNA ZFPM2-AS1)/微小RNA-515-5p(miR-515-5p)分子轴而抑制结肠癌细胞增殖、迁移及侵袭。方法体外培养人结肠癌LoVo细胞,随机分为Control组、DPPT-L组、DPPT-M组、DPPT-H组。甲基噻唑基四唑(MTT)检测细胞增殖;Transwell小室实验检测迁移及侵袭;实时荧光定量聚合酶链反应(qRT-PCR)检测ZFPM2-AS1、miR-515-5p的表达水平;LoVo细胞中转染pcDNA-ZFPM2-AS1,采用上述方法检测细胞增殖、迁移及侵袭;双荧光素酶报告实验验证ZFPM2-AS1、miR-515-5p的靶向关系;蛋白免疫印迹法(Western blot)检测细胞周期蛋白1(CyclinD1)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、P21的表达量。结果与Control组比较,DPPT-L组、DPPT-M组、DPPT-H组细胞存活率降低(P<0.05),迁移细胞数、侵袭细胞数减少(P<0.05),CyclinD1、MMP-2、MMP-9蛋白水平降低(P<0.05),P21蛋白水平升高(P<0.05),ZFPM2-AS1表达水平降低(P<0.05),miR-515-5p表达水平升高(P<0.05),且DPPT-L组、DPPT-M组、DPPT-H组上述指标比较,差异均有统计学意义(P<0.05);ZFPM2-AS1可靶向结合miR-515-5p;与DPPT-H+pcDNA组比较,DPPT-H+pcDNA-ZFPM2-AS1组细胞存活率升高(P<0.05),迁移细胞数、侵袭细胞数增多(P<0.05),CyclinD1、MMP-2、MMP-9蛋白水平升高(P<0.05),P21蛋白水平降低(P<0.05)。结论DPPT可能通过下调ZFPM2-AS1的表达及上调miR-515-5p的表达从而抑制结肠癌细胞增殖、迁移及侵袭。

Objective To investigate whether deoxypodophyllotoxin(DPPT)inhibits proliferation,migration and invasion of colon cancer cells by regulating the long non-coding RNA ZFPM2-AS1/microRNA-515-5p(miR-515-5p)molecular axis.Methods Human colon cancer LoVo cells were cultured in vitro and randomly divided into the control group,DPPT-L group,DPPT-M group,and DPPT-H group.Methylthiazolyl tetrazole(MTT)assay was used to assess cell proliferation;Transwell chamber assay was used to detect cell migration and invasion;real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression levels of ZFPM2-AS1 and miR-515-5p.Using these tests,LoVo cells were transfected with pcDNA-ZFPM2-AS1,and examined for proliferation,migration and invasion.Dual luciferase reporter assay was used to determine the targeting relationship between ZFPM2-AS1 and miR-515-5p.Western blotting was used to detect the expression of Cyclin 1,matrix metalloproteinase(MMP)-2,MMP-9,and P21 in the cells.Results Compared with the control group,the DPPT-L,DPPT-M,and DPPT-H groups showed decreased cell viability(P<0.05),fewer migrating cells and invasive cells(P<0.05),lower expression levels of CyclinD1,MMP-2,and MMP-9 proteins(P<0.05),higher P21 protein level(P<0.05),lower ZFPM2-AS1 expression(P<0.05),and higher miR-515-5p expression(P<0.05).These measurements did not differ significant among DPPT-L,DPPT-M and DPPT-H groups(P<0.05).ZFPM2-AS1 was shown to target miR-515-5p.Compared with the DPPT-H+pcDNA group,the DPPT-H+pcDNA-ZFPM2-AS1 group showed increased cell viability(P<0.05),more migrating cells and invasive cells(P<0.05),higher protein expression of CyclinD1,MMP-2 and MMP-9(P<0.05),and lower protein expression of P21(P<0.05).Conclusion DPPT may inhibit the proliferation,migration and invasion of colon cancer cells by down-regulating the expression of ZFPM2-AS1 and up-regulating the expression of miR-515-5p.