沉默HIF-2α对低氧下接种乳腺球细胞的裸鼠移植瘤生长的影响

Silencing hypoxia inducible factor-2α gene by small interference RNA inhibits the growth of mammosphere cells in nude mice under hypoxic microenvironment

目的探讨RNA干扰（RNAi）沉默HIF-2α表达对低氧下接种乳腺球细胞的裸鼠移植瘤生长的影响。方法将慢病毒介导HIF-2α基因RNAi载体质粒和空载体质粒转染至MCF-7细胞株，G418筛选获得稳定转染细胞株。将空载体组及干扰组MCF-7细胞注射接种至裸鼠左右侧背部近后肢皮下，比较成瘤时间，肿瘤体积及瘤体重量等；Western印迹检测移植瘤组织中HIF-2α、CIM4、OCT-4及KLF4蛋白表达。结果成功构建HIF-2α基因RNAi慢病毒载体，沉默HIF-2α表达降低乳腺球的形成；与空载体组比较，干扰组移植瘤生长速率、肿瘤体积和重量均明显降低（P〈0．05）。Western印迹显示干扰组HIF-2α蛋白表达量（0．42±0．01）明显低于空载体组（0．89±0．03，P〈0．05），CD44蛋白表达量（0．21±0．01）明显低于空载体组（0．78±0．03，P〈0．05），OCT-4蛋白表达量（0．134-0．01）明显低于空载体组（0．68±0．03，P〈0．05），KLF4蛋白表达量（0．34±0．01）明显低于空载体组（0．72±0．03，P〈0．05）。结论RNAi沉默HIF-2α表达明显抑制低氧下乳腺球细胞裸鼠移植瘤生长，其机制可能通过下调干细胞标志物表达抑制乳腺癌细胞自我更新和增殖，HIF-2α有望成为乳腺癌基因治疗新的靶点。

Objective To explore the effects of silencing hypoxia inducible factor-2α（ HIF-2α by small interference RNA on the growth of mammosphere cells in nude mice under hypoxic microenvironment. Methods The empty and interference vectors were transfected into MCF-7 cell. Then G418 was added to screen the positive cells to obtain stable cell hne. The empty and interference vectors were inoculated subcutaneously into left and right back near hind limb of nude mice. The volume and weight of tumors were calculated respectively. The expressions of HIF-2α, CD44, OCT4 and KLF4 protein in xenograft tumor tissues were detected by Western blot. Results The expression vector of HIF-2ct-siRNA was successfully established. The formation of mammosphere was lowered by silencing HIF-2α gene expression. In contract to empty vector group cell, there were obvious decreases in the volumes and weights of tumors in interference group （P 〈0. 05）. The expression of HIF-2αprotein of interference group （0. 42 ± 0. 01 ） was much lower than that of the empty vector group （0. 89 ± 0. 03, P 〈 0. 05 ）, the expression of CD44 protein of interference group （0. 21 ± 0. 01 ） was much lower than the empty vector group （ 0.78± 0. 03, P 〈 0.05 ） , the expression of OCT-4 protein of interference group （0.42 ± 0.01 ） was much lower than the empty vector group （0. 68 ±0.03, P 〈0. 05 ） and the expression of KLF-4 protein of interference group （0. 34 ±0. 01 ） was much lower than the empty vector group （0.72 ± 0. 03, P 〈 0. 05 ）. Conclusion Silencing HIF-2α gene can effectively inhibit the growth of breast cancer stem cells in nude mice under hypoxic microenvironment. Its mechanism may be through inhibited capacity for self-renewal and proliferation of breast cancer stem cells in vivo through the down-regulated expressions of markers associated with stemcells. HIF-2α is expected to become a new target for gene therapy of breast cancer.