RNA干扰沉默HIF-1α基因对卵巢癌SKOV3细胞定植和侵袭的影响

【目的】构建针对HIF-1α基因的短发夹干忧RNA真核表达质粒，探讨低氧条件下其对卵巢癌SKOV3细胞定植和侵袭能力的影响。【方法】根据siRNA设计原则，结合psilencer 2．1-U6-neo质粒特点，针对HIF-1α基因设计并合成2条寡聚DNA片段，退火后将其克隆人DSilencer2．1-U6-neo质粒中，采用脂质体法将重组真核表达质粒转染人卵巢癌SKOV3细胞。缺氧培养后，应用实时定量PCR和Western blotting方法检测HIF-1α蛋白及mRNA表达水平；用软琼脂克隆形成实验和体外侵袭实验检测SKOV3细胞的定植和侵袭能力。【结果】成功构建的HIF-1α的短发状siRNA真核表达载体，转染卵巢癌SKOV3细胞，低氧条件下细胞HIF-1蛋白及mRNA表达水平下降；同时SKOV3细胞在软琼脂中形成的克隆数和穿透Matrigel膜的细胞数明显减少。【结论】构建的HIF-1α短发状siRNA真核表达载体，下调卵巢癌SKOV3细胞HIF-1α基因表达，卵巢癌细胞的定植和侵袭能力降低。

Effects of RNA interference targeting HIF-1α gene on the location and metastasis of SKOV3 cells

Objective] To construct the eukaryotic vector cxpressing short hairpin RNA （shRNA） of HIF-1α, and to observe its effects on location and metadtasis of SKOV3 cells under hypoxic condition . Methods] shRNA templates was designed base on HIF-1α gene sequence and was cloned into pSilencer2.1-U6-neo vector . The resultant plasmid pSilencer2.1-U6-HIF-1α was transfected into SKOV3 cells with Lipofectamine 2000. The cells were incubated under hypoxic hypoxic condition. The HIF-1α protein and mRNA were detected by Western blot and real time PCR, and the colony formation assay and Transwell cabin assay were performed to measure the location and metastasis of SKOV3 cells. Resuits] The plasmid pSilencer2. 1-U6-HIF-1α efficiently down-regulate the expression of HIF-1α, and decreased the number of colony formation in soft agar and cells penetrating matrigel of SKOV3 cells under hypoxic condition . Conclusions] The successful construction of the siRNA expressing plasmid pSilencer12.1-U6-HIF-1α could suppress the expression of HIF-1α, and reduce location and metastasis of ovarian caner aells.