下调长链非编码RNA THUMPD3-AS1靶向miR-185抑制前列腺癌细胞C4-2迁移、侵袭

Down-regulating IncRNA THUMPD3-AS1 targeting miR-185 inhibits the migration and invasion of prostate cancercell C4-2

目的研究下调长链非编码RNA(lncRNA)THUMPD3-AS1靶向miR-185对前列腺癌细胞C4-2迁移、侵袭的影响。方法采用qRT-PCR方法分析前列腺癌细胞22RV1、DU-145、LNcap、C4-2和正常前列腺细胞RWPE-2中THUMPD3-AS1的表达变化。将前列腺癌细胞C4-2分成对照组、si-NC组(转染siRNA对照剂)、si-THUMPD3-AS1组(转染THUMPD3-AS1 siRNA)、si-THUMPD3-AS1+Anti-miR-NC组(共转染抑制物对照剂、THUMPD3-AS1 siRNA)、si-THUMPD3-AS1+Anti-miR-185组(共转染miR-185抑制物、THUMPD3-AS1 siRNA),采用噻唑蓝(MTT)方法分析细胞的增殖活性,采用Transwell小室检测细胞的迁移和侵袭能力,采用Western blot检测上皮性钙黏附素(E-cadherin)、波形蛋白(vimentin)、基质金属蛋白酶-9(MMP-9)、基质金属蛋白酶-2(MMP-2)蛋白的表达变化。采用生物信息学软件预测THUMPD3-AS1的靶基因,采用荧光素酶报告系统鉴定二者的靶向关系。结果与正常前列腺细胞RWPE-2比较,前列腺癌细胞22RV1、DU-145、LNcap、C4-2中的THUMPD3-AS1表达水平明显升高(均P<0.05);与前列腺癌细胞22RV1、DU-145、LNcap比较,前列腺癌细胞C4-2中的THUMPD3-AS1表达水平明显升高(均P<0.05)。与对照组、si-NC组比较,si-THUMPD3-AS1组的前列腺癌细胞C4-2细胞增殖活性降低,细胞迁移数目、细胞侵袭数目也明显减少,vimentin、MMP-2、MMP-9蛋白表达减少,E-cadherin蛋白表达增多,差异均有统计学意义(均P<0.05)。与si-THUMPD3-AS1+Anti-miR-NC组比较,si-THUMPD3-AS1+Anti-miR-185组的前列腺癌细胞C4-2细胞增殖活性明显升高,细胞迁移数目、细胞侵袭数目增多,vimentin、MMP-2、MMP-9蛋白表达水平升高,E-cadherin蛋白表达水平降低,差异均有统计学意义(均P<0.05)。与模拟物对照剂、WT共转染相比,miR-185模拟物、WT共转染后的前列腺癌细胞C4-2的荧光素酶活性降低(P<0.001)。THUMPD3-AS1和miR-185互为靶向关系。与对照组、si-NC组比较,si-THUMPD3-AS1组前列腺癌细胞C4-2中miR-185表达水平明显升高(P<0.001)。结论下调lncRNA THUMPD3-AS1可靶向miR-185抑制前列腺癌细胞C4-2的迁移、侵袭。

Objective To study the effect of down-regulating long non-coding RNA(lncRNA)THUMPD3-AS1 targeting miR-185 on the migration and invasion of prostate cancer cell C4-2.Methods qRT-PCR method was used to analyze the expression of THUMPD3-AS1 in prostate cancer cells 22RV1,DU-145,LNcap,C4-2 and normal prostate cells RWPE-2.Prostate cancer cells C4-2 were divided into control group,si-NC group,si-THUMPD3-AS1 group,si-THUMPD3-AS1+Anti-miR-NC group,si-THUMPD3-AS1+Anti-miR-185 group,the cell proliferation activity was analyzed by Methylthiazolyldiphenyl-tetrazolium bromide(MTT)method,and the cell migration and invasion ability was detected by Transwell chamber.Western blot detection of epithelical cadherin(E-cadherin),vimentin,matrix metalloproteinase-9(MMP-9),matrix metalloproteinase-2(MMP-2)protein expression changes.Bioinformatics software predicted the target genes of THUMPD3-AS1,and the luciferase reporter system identified the target relationship between the two.Results Compared with normal prostate cells RWPE-2,the expression levels of THUMPD3-AS1 in prostate cancer cells 22RV1,DU-145,LNcap and C4-2 were significantly increased.Compared with prostate cancer cells 22RV1,DU-145,and LNcap,the expression level of THUMPD3-AS1 in prostate cancer cell C4-2 was significantly increased.Compared with the control and si-NC groups,the si-THUMPD3-AS1 group prostate cancer cell C4-2 cell proliferation activity decreased,the number of cell migration and cell invasion were also significantly reduced,vimentin,MMP-2,MMP-9 protein expression decreased,and the E-cadherin protein expression increased.Compared with the si-THUMPD3-ASI+Anti-miR-NC group,the si-THUMPD3-AS1+Anti-miR-185 group prostate cancer cell C4-2 cell proliferation activity was significantly increased,the number of cell migration,cell invasion number increased,the expression level of vimentin,MMP-2,MMP-9 protein increased,and the expression level of E-cadherin protein decreased.Compared with mimics control and WT co-transfection,the luciferase activity of C4-2 prostate cancer cells after miR-185 mimics and WT co-transfection was decreased(P<0.001).THUMPD3-AS1 and miR-185 were mutually targeted.Compared with the control group and si-NC group,the expression level of miR-185 in C4-2 prostate cancer cells in si-THUMPD3-ASl group was significantly increased(P<0.001).Conclusions Down-regulating IncRNA THUMPD3-AS1 targeting miR-185 inhibits the migration and invasion of prostate cancer cell C4-2.