胸腺素β4和β10表达促进小鼠乳腺癌细胞转移

Expressions of thymosin β4 and β10 promote metastasis of mouse breast cancer

目的研究胸腺素β4(Tβ4)和β10(Tβ10)对小鼠乳腺癌转移的影响。方法用对照腺病毒(pENTER组)、过表达Tβ4腺病毒(TMSB4X组)或Tβ10腺病毒(TMSB10组)感染小鼠乳腺癌细胞4T1,分别作用24,48和72 h后,MTT和甲紫(结晶紫)法体外检测Tβ4和Tβ10对4T1细胞增殖的影响;划痕法检测Tβ4和Tβ10对4T1细胞迁移能力的影响;Transwell法检测Tβ4和Tβ10对4T1细胞侵袭能力的影响;RT-PCR法检测不同腺病毒感染后4T1细胞中与细胞增殖及迁移相关基因基质金属蛋白酶2(MMP2),MMP9,信号转导与转录活化因子3(Stat3),癌基因(c-myc)和骨髓基质细胞衍生因子1(Sdf1)mRNA水平的变化。通过在雌性裸鼠乳腺脂肪垫下分别接种感染了对照腺病毒、过表达Tβ4或过表达Tβ10腺病毒的荧光素酶标记的4T1细胞(4T1-Luc),建立小鼠乳腺癌模型。造模成功后,每周于小鼠瘤体内注射10μL相应腺病毒(连续5周),每5 d测量小鼠肿瘤体积。小动物活体成像法观察小鼠肿瘤生长和转移。结果与pENTER组相比,TMSB4X和TMSB10组4T1细胞增殖抑制率及穿过小室细胞数无明显变化,提示Tβ4和Tβ10对4T1细胞的增殖和侵袭无明显影响;与pENTER组细胞迁移率(22±7)%相比,另两组细胞迁移率明显升高(P<0.05),分别为(36±6)%和(36±4)%,提示Tβ4和Tβ10促进了细胞迁移;TMSB4X和TMSB10组4T1细胞MMP2,MMP9,Sdf1和Stat3 mRNA水平显著上调(P<0.05,P<0.01)。动物实验表明,TMSB4X和TMSB10组小鼠比pENTER组更早出现乳腺癌肺转移,肺部转移灶数量在35 d时明显增多(P<0.05)。结论Tβ4和Tβ10均能促进小鼠乳腺癌细胞的迁移,体内促进小鼠乳腺癌的转移。

OBJECTIVE To study the effect of thymosin β4(Tβ4) and Tβ10 on the metastasis of breast cancer in mice. METHODS Mouse breast cancer 4 T1 cells were infected with control adenovirus(pENTER group)、Tβ4(TMSB4 X group) or Tβ10(TMSB10 group) overexpression adenovirus, and the effects of Tβ4 and Tβ10 on proliferation of 4 T1 cel s were detected by MTT assay and crystal violet viability assay(24, 48 and 72 h) in vitro. Migration assay was used to detect the effects of Tβ4 and Tβ10 on the migration ability of 4 T1 cells. The effects of Tβ4 and Tβ10 on invasion ability of 4 T1 cells were detected by transwell assay. The mRNA levels of matrix metalloprotein 2(MMP2), matrix metalloprotein 9(MMP9), signal transducers and activators of transcription 3(Stat3), c-myc, and stromal cell-derived factor-1(Sdf1) associated with cell proliferation and migration in 4 T1 cells were detected by RT-PCR. A mouse breast cancer model was established via inoculation of 4 T1-luciferase(4 T1-Luc) cells infected with different adenovirus under the fat pad of female nude mice. After successful modeling, mice were injected with 10 μL corresponding adenovirus per week for 5 weeks. Tumor volume was measured every5 d, and tumor growth and metastasis in mice were observed by bioluminescence imaging. RESULTS Compared with pENTER group, the inhibitory rate of cell proliferation and the number of cells passing through transwell in TMSB4 X and TMSB10 groups were not significantly different, indicating that Tβ4 and Tβ10 had no effect on the proliferation and invasion of 4 T1 cells. The cell migration rates of TMSB4 X and TMSB10 groups were increased(P<0.05) when compared with p ENTER group, suggesting that Tβ4 and Tβ10 promoted the migration of 4 T1 cells. The mRNA levels of MMP2, MMP9, Stat3 and Sdf1 in 4 T1 cells of TMSB4 X and TMSB10 group were significantly upregulated(P<0.05, P<0.01). Animal experiments showed that lung metastasis was increased in the mice in TMSB4 X and TMSB10 groups when compared with pENTER group, and the number of lung metastases was significantly larger than in pENTER group at 35 d after modeling(P<0.05). CONCLUSION Both Tβ4 and Tβ10 can promote metastasis of breast cancer and migration of breast cancer cells in mice.