miR-32-5p和FKBP14基因转染对SPC-A1细胞增殖、迁移、侵袭的影响及其靶向关系验证

Effects of miR-32-5p and FKBP14 gene transfection on proliferation,migration and invasion of SPC-A1 cells and verification of their targeting relationship

目的观察miR-32-5p和FK506结合蛋白14(FKBP14)基因转染对人非小细胞肺癌(NSCLC)细胞系SPC-A1增殖、迁移、侵袭能力的影响,并验证两者之间靶向关系。方法选取SPC-A1细胞,将细胞为A、B、C、D、E、F组,分别转染miR-32-5p+pcDNA-FKBP14、miR-32-5p+pcDNA、miR-32-5p、miR-con、pcDNA-FKBP14、pcDNA-con,采用MTT法检测细胞吸光度值,并计算细胞存活率;Transwell实验检测细胞迁移数、侵袭数,Western blotting法检测细胞CyclinD1、MMP-2、MMP-9蛋白相对表达量。取SPC-A1细胞,将细胞分为G、H、I、J组,分别转染FKBP14-WT+miR-con、FKBP14-WT+miR-32-5p、FKBP14-MUT+miR-con、FKBP14-MUT+miR-32-5p,采用荧光素酶活性检测试剂盒检测细胞荧光素酶活性。取SPC-A1细胞,将细胞分为K、L、M、N组,分别转染miR-32-5p、miR-con、anti-miR-32-5p、anti-miR-con,Western blotting法检测细胞中KBP14蛋白相对表达量。结果与B组比较,A组细胞存活率、迁移细胞数、侵袭细胞数及CyclinD1、MMP-2、MMP-9蛋白相对表达量增加(P均<0.05);与D组比较,C组细胞存活率、迁移细胞数、侵袭细胞数及CyclinD1、MMP-2、MMP-9蛋白相对表达量减少(P均<0.05);与F组比较,E组SPC-A1细胞存活率、迁移细胞数、侵袭细胞数及CyclinD1、MMP-2、MMP-9蛋白相对表达量增加(P均<0.05)。与G组比较,H组细胞荧光素酶活性减弱(P<0.05);与I组比较,J组细胞荧光素酶活性无显著变化(P>0.05)。与L组比较,K组FKBP14蛋白相对表达量减少(P<0.05);与N组比较,M组FKBP14蛋白相对表达量增加(P<0.05)。结论过表达miR-32-5p或敲减FKBP14后SPC-A1细胞存活率降低,迁移细胞数、侵袭细胞数减少,miR-32-5p与FKBP14存在靶向调控关系。

Objective To observe the effects of miR-32-5 p and FK506 binding protein 14(FKBP14)gene transfection on the proliferation,migration and invasion of non-small-cell lung cancer(NSCLC)cell line SPC-A1,and to verify the targeting relationship between them.Methods SPC-A1 cells were were divided into the groups A,B,C,D,E,and F,and were transfected with miR-32-5 p+pcDNA-FKBP14,miR-32-5 p+pcDNA,miR-32-5 p,miR-con,pcDNA-FKBP14,and pcDNA-con;MTT method was used to detect cell absorbance value and we calculated the cell survival rate;Transwell test was used to detect the number of migrating cells and the number of invasive cells;Western blotting was used to detect the relative expression levels of CyclinD1,MMP-2,and MMP-9 proteins in cells.SPC-A1 cells were divided into the groups G,H,I,and J,and were transfected with FKBP14-WT+miR-con,FKBP14-WT+miR-32-5 p,FKBP14-MUT+miR-con,and FKBP14-MUT+miR-32-5 p;luciferase activity detection kit was used to detect the cellular luciferase activity.SPC-A1 cells were divided into the groups K,L,M,and N,and were transfected with miR-32-5 p,miR-con,anti-miR-32-5 p,and anti-miR-con;Western blotting was used to detect the relative expression of KBP14 protein.Results Compared with the group B,the cell survival rate,the number of migrating cells,the number of invasive cells,and relative expression of CyclinD1,MMP-2 and MMP-9 protein in the A group increased(P<0.05);compared with the D group,the cell survival rate,the number of migrating cells,the number of invasive cells,and relative expression of CyclinD1,MMP-2 and MMP-9 protein in the group C decreased(all P<0.05);compared with the group F,the cell survival rate,the number of migrating cells,the number of invasive cells,and relative expression of CyclinD1,MMP-2 and MMP-9 protein in the group E increased(all P<0.05).Compared with the group G,the luciferase activity of group H was weakened(P<0.05);compared with the group I,the luciferase activity of group J had no significant change(P>0.05).Compared with the group L,the relative expression of FKBP14 protein in the group K decreased(P<0.05);compared with the group N,the relative expression of FKBP14 protein in the group M decreased(P<0.05).Conclusion After over-expressing miR-32-5 p or knocking down FKBP14,the survival rate of SPC-A1 cells,the number of migrating cells,and the number of invasive cells decreased,and there was a targeted regulation relationship between miR-32-5 p and FKBP14.