摘要目的 构建乳脂球表皮生长因子-8(MFG-E8)短发卡RNA(shRNA)慢病毒载体,稳定转染HCC1973细胞株,检测其在乳腺癌细胞HCC1973中的干扰效率及影响.方法 通过构建PLKO.1-MFG-E8重组慢病毒载体,并将包装好的重组慢病毒感染乳腺癌细胞HCC1973,实时荧光定量聚合酶链反应(FQ-PCR)检测MFG-E8基因的表达,蛋白质印迹法(Western blot)法检测MFG-E8蛋白的表达;同时采用噻唑蓝(MTT)检测细胞的增殖能力、流式细胞技术(FCM)检测细胞的周期及凋亡变化、Transwell检测细胞的侵袭迁移能力.应用SPSS 17.0统计软件进行分析.结果 成功构建的PLKO.1-MFG-E8 shRNA重组慢病毒载体可明显降低MFG-E8基因和蛋白的表达;重组载体经包装产生的慢病毒滴度为3×108 PFU/ml;经shRNA干扰后的乳腺癌细胞,细胞增殖能力显著减弱(t =4.426,P＜0.05),Go/G1期细胞明显增加,S期细胞明显减少(t=2.264,P＜0.05),细胞侵袭、迁移能力明显降低(t =4.802,P＜0.05).结论 通过shRNA干扰MFG-E8基因和蛋白的表达,能有效抑制乳腺癌细胞向恶性生物学行为的改变,表明在乳腺癌发生的过程中,MFG-E8参与介导乳腺癌的侵袭转移及凋亡.

abstractsObjective To construct the lentiviral vector of milk fat globule-epidermal growth factor 8 (MFG-E8) short hairpin RNA (shRNA),and stably transfected the HCC1973 cell line to detect its interference efficiency and influence in breast cancer cell HCC1973.Methods The knockout gene fragment was synthesized and inserted into the shRNA lentiviral vector PLKO.1 with a cherry protein reporter.The constructed recombinant plasmids were transfected into 293T cells respectively.The lentiviruses were transfected into HCC1973 cells and stably transfected PLKO.1-MFG-E8 shRNA recombinant lentiviruses were obtained.The expression of MFG-E8 was detected by real-time PCR and Western blotting.The cell proliferation was measured by methyl thiazol tetrazolium (MTT) assay and Flow cytometry was used to detect the cell cycle and apoptosis,Transwell was used to detect the invasion and migration of cells.Statisticalanalysis of data using the statistical product and service solutions 17.0 software.Results The constructed vector of PLKO.1-MFG-E8 shRNA significantly reduced the expression of MFG-E8 gene and protein in breast cancer cell HCC1973,the lentivirus titer was 3 × 108 PFU/ml.The proliferation of breast cancer cells was significantly suppressed (t =4.426,P ＜0.05),G0/G1 phase cells increased significantly with a decrease of cells in S phase (t =2.264,P ＜ 0.05),the ability of cell invasion and migration was significantly reduced (t =4.802,P ＜ 0.05),after the lentiviral infection.Conclusion The expression of MFG-E8 gene and protein by shRNA interference can inhibit the malignant biological behavior of breast cancer cells effectively,which indicates that MFG-E8 is involved in the invasion,metastasis and apoptosis of breast cancer during the process of breast carcinogenesis.