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

Effects of lentivirus-mediated siRNA targeting IGF-1R on migration and invasion abilities of endometrial carcinoma cells

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。

AIM： To investigate the down - regulation of insulin - like growth factor tgpe 1 receptor （ IGF - 1 R） on the migration and invasion abilities of human endometrial cancer cell HEC - 1B. METHODS： The siRNAs targe- ting IGF - 1R gene were synthesized, cloned into a lentivirus expression vector and transfected into endometrial cancer HEC - 1 B ceils （ HEC - 1 B - KD group）. The control cells （ without virus transfection, HEC - 1 B - CON group） and negative virus transfection control cells （ HEC - 1B - NC group） were also set up. The gene silencing effect of siRNA targeting IGF -1R was determined by real -time PCR and Western blotting at mRNA and protein levels, respectively. The proliferation rate was detected by colony formation assay. The cell migration and invasion abilities were determined by Transwell experi- ment. The mRNA levels of matrix metalloproteinase （MMP） -2 and MMP -9 were measured by real -time PCR. RE- SULTS： The mRNA and protein levels of IGF - 1R in HEC - 1B - KD cells were significantly reduced by 81% and 91.5%, respectively （P 〈0.05 ）. In anchorage -dependent growth by colony formation assay, HEC -1B -KD cells showed much less colonies than HEC - 1B - CON cells and HEC - 1B - NC cells. Compared with the control cells, knock- down of IGF - 1R in HEC - 1B cells resulted in significant reduction of cell motility. Down - regulation of IGF - 1R in HEC - 1B cells also significantly reduced the invasion potential （ P 〈 0.05 ）. Down - regulation of IGF - 1R substantially re- duced the expression of MMP -2 and MMP- 9 compared with the control cells. CONCLUSION： Knockdown of IGF- 1R reduces the migration and invasion abilities of human endometrial cancer ceils in vitro accompanied with a decrease in MMP -2 and MMP- 9 expression.