RNAi技术沉默VEGF-C基因对小鼠4T1细胞生物学行为的影响

目的体外观察siRNA靶向干扰VEGF-C基因对4T1小鼠乳腺癌细胞的生物学行为影响。方法选用HifectinII真核转染试剂将化学修饰的针对小鼠VEGF-C基因的siRNA转染4T1小鼠乳腺癌细胞株,设立空白对照组(只加培养基),脂质体组(每孔加培养基与0.5μL Hifectin II真核转染试剂),SCR组(100 nM),siRNA组(100 nM)。蛋白印迹试验检测转染前后细胞中VEGF-C的蛋白水平表达的变化;半定量RT-PCR检测VEGF-C、Survivin、BCL-2、BAX的mRNA水平表达的变化;MTT比色法检测细胞的增殖;Transwell小室方法检测肿瘤细胞的侵袭性改变;Hochest33258荧光染色观察细胞凋亡。结果转染VEGF-C siRNA 48 h后4T1细胞的VEGF-C基因的mRNA和蛋白水平表达明显降低,空白对照组、脂质体组和siRNA SCR组各指标差异无显著性(P>0.05);4T1细胞生长受到抑制,Transwell小室方法检测肿瘤细胞的侵袭性降低,Hoechst 33258荧光染色显示细胞内可见凋亡小体。结论化学修饰siRNA介导的RNA干扰能下调靶基因VEGF-C的表达并能够促进4T1细胞的凋亡,抑制4T1细胞的增殖与侵袭性。

The effect of siRNA targeted VEGF-C gene on the biological behavior of mouse 4T1 breast cancer cells

Objective To observe the effect of siRNA targeted VEGF-C gene on the biological behavior of mouse 4T1 breast cancer cells in vitro.Methods Mouse 4T1 breast cancer cells were transfected with chemically modified siRNA targeted VEGF-C gene using Hifectin II,an eukaryotic transfection reagent.There were four groups were studied-the blank group(medium only),medium group(medium with Hifectin II) and SCR group(transfected with unspecific siRNA,100nM) as two negative groups and experimental group transfected with specific siRNA(100 nM).The protein level changes of VEGF-C were detected by Western blot;the mRNA levels of VEGF-C,Survivin,BCL-2,BAX were detected by semi-quantitative RT-PCR;the status of cell proliferation was detected by MTT assay;to detect the changes of invasion and apoptosis in tumor cells were detected by Transwell chamber and Hochest33258 fluorescence staining,respectively.Results The levels of mRNA and protein of VEGF-C were significantly reduced after 48 h transfected with specific siRNA.However,there was no significant difference in blank group and two negative groups(P>0.05);the growth of 4T1 cell was inhibited and its invasive ability also reduced detected by Transwell assay.Apoptotic bodies were observed in 4T1 cell from Hoechst 33258 fluorescent staining.Conclusion Chemical modified siRNA can down-regulate the expression of VEGF-C and can promote apoptosis,inhibit the proliferation and invasion of 4T1 cells.