慢病毒介导的靶向沉默胰岛素样生长因子1型受体的siRNA对人子宫内膜癌细胞迁移和侵袭能力的影响

摘要

AIM: To invesligale the down — regulation of insulin — like growth factor Lgpe 1 receplor(IGF — 1R) on the migration and invasion abilities of human endomelrial cancer cell HEC — 1B. METHODS; The siRNAs Large-Ling IGF — 1R gene were synLhesized, cloned inLo a lenLivirus expression vecLor and LransfecLed inLo endomeLrial cancer HECrn— 1B cells (HEC — 1B — KD group) . The conLrol cells ( wiLhouL virus LransfecLion, HEC — 1B — CON group) and negaLive virus LransfecLion conLrol cells ( HEC — 1B — NC group) were also seL up. The gene silencing effecL of siRNA LargeLing IGFrn— 1R was deLermined by real — Lime PCR and WesLern bloLLing aL mRNA and proLein levels,respeclively. The proliferaLion rate was deLecLed by colony formaLion assay. The cell migraLion and invasion abiliLies were deLermined by Transwell experi-menL. The mRNA levels of maLrix metalloproLeinase ( MMP) — 2 and MMP — 9 were measured by real — Lime PCR. RESULTS; The mRNA and protein levels of IGF — 1R in HEC — 1B — KD cells were significanLly reduced by 81% and 91.5% , respectively (P<0. 05). In anchorage — dependenL growLh by colony formaLion assay, HEC — 1B — KD cells showed much less colonies Lhan HEC — 1B — CON cells and HEC — 1B — NC cells. Compared wiLh Lhe conLrol cells, knockdown of IGF — 1R in HEC — 1B cells resulted in significanL reduction of cell moLiliLy. Down — regulaLion of IGF — 1R in HECrn— 1B cells also significanLly reduced Lhe invasion poLenLial ( P < 0. 05 ) . Down — regulaLion of IGF — 1R subsLanLially reduced the expression of MMP -2 and MMP -9 compared wiLh Lhe conLrol cells. CONCLUSION; Knockdown of IGF - 1R reduces Lhe migraLion and invasion abiliLies of human endomelrial cancer cells in vitro accompanied wiLh a decrease in MMPrn— 2 and MMP - 9 expression.%目的:探讨慢病毒介导的靶向沉默胰岛素样生长因子1型受体(IGF-1R)的siRNA对人子宫内膜癌细胞迁移和侵袭功能的影响和可能机制.方法:针对IGF-1R基因设计siRNA,筛选出最有效的siRNA进行慢病毒包装扩增.转染人子宫内膜癌HEC-1B细胞后分别用real-time PCR和Western blotting方法验证其沉默效率,平板克隆法检测其克隆形成情况,Transwell小室方法检测细胞迁移和侵袭情况,real-time PCR检测基质金属蛋白酶2(MMP-2)和MMP-9 mRNA水平的变化.结果:筛选有效siRNA转染子宫内膜癌HEC-1B细胞后,IGF-1R 的mRNA水平下降81%,蛋白表达水平下降91.5%.沉默IGF-1R 基因后,HEC-1B细胞克隆形成能力下降,迁移和侵袭能力下降,与对照组和转染阴性组相比有显著差异(P<0.05);MMP-2和MMP-9 mRNA表达水平下降(P<0.05).结论:在人子宫内膜癌HEC-1B细胞中下调IGF-1R水平能降低MMP-2和MMP-9 mRNA表达,抑制细胞迁移和侵袭能力.