LKB1基因与Hedgehog信号通路对乳腺癌MDA-MB-231细胞抑制机制的探讨

Inhibition effect of LKB1gene on MDA-MB-231cell growth by hedgehog signaling pathways

目的:探讨乳腺癌细胞中LKB1基因与Hedgehog信号转导途径的关系。方法:以乳腺癌MDA-MB-231细胞为研究对象,LKB1基因转染或干扰处理后,反转录-聚合酶链反应和蛋白质印迹法分别检测Hedge-hog信号通路中Shh、Sufu、Ptch、Smo、Gli 1、Hip mRNA和蛋白表达的变化。建立LKBl基因表达抑制的细胞移植瘤模型,验证体外实验结果并进行对比分析。结果:Hedgehog信号通路激动因子Ptch的mRNA和蛋白的表达在各组细胞中差异无统计学意义。而激动因子Shh、Gli1和Smo的mRNA和蛋白的表达在MDA-MB-231正常细胞及其空质粒细胞、siRNA无关序列细胞中差异无统计学意义;在LKB1转染细胞中降低(F=29.56,P<0.05),在LKBl转染siRNA细胞中升高(F=32.15,P<0.05)。而抑制因子Sufu、Hip的mRNA和蛋白的表达正好相反,在LKB1转染细胞中升高,在LKBl转染siRNA细胞中降低,F=30.25,P<0.05。移植瘤检测Shh、Su-fu、Smo、Gli 1、Hip蛋白表达与体外实验相似,F=8.40,P<0.01。而且发现LKBl转染siRNA细胞组裸鼠肿瘤平均体积显著大于对照组(F=4.68,P<0.05),生存时间显著短于对照组,F=3.50,P<0.01。LKB1基因与Hedgehog信号通路相关因子表达呈负相关,r=0.232,P<0.05。结论:LKB1基因与Hedgehog信号通路相关因子表达在乳腺癌细胞中呈负相关,LKB1基因可能通过Hedgehog信号通路抑制乳腺癌细胞的发生,两者之间的相互作用对乳腺癌的诊治可能有重要影响。

OBJECTIVE：To detect the relationship of LKB1 gene and the correlation factor of hedgehog signaling pathways in MDA-MB-231 cell. METHODS：RT-PCR and Western blot assay were used to detect the mRNA and protein expression of Shh, Sufu, Ptch, Smo, Gli 1 and Hip in the MDA-MB-231 celI, which transfected with LKBI gene plasmids and silenced LKB1 gene with the small interfering RNA （siRNA）. Xenografted tumor model was established in nude mice by subcutaneous inoculation of MDA-MB-231 cells to proof the in vitro result. RESULTS; The mRNA and protein expressions of the exciting factor of hedgehog signaling pathways, Ptch level was not changed in all the groups. But other exciting factors, the levels of Shh, Glil and Smo were all significant decrease in the MDA-MB 231/I.KBI cells （F= 29.56,P^0.05） and increase in the MDA MB-231/siRNALKB1 ceils （F--32.15,P＇Q0.05）. And the mRNA and protein expres- sions of the inhibiting factor of hedgehog signaling pathways, both sufu and hip were significant decrease in the MDA-MB 231/ siRNALKB1 cells and increase in the MDA-MB 231/LKB1 cells （F--30.25,P〈0.05）. The protein contents of Shh, Sufu, Smo, Gli 1 and Hip in transplanted tumors was similar to the result in vitro（F= 8. 40,P〈0. 01）. The average volume of the nude mouse tumor in MDA-MB-231/siRNALKB1 group was higher than control group （F=4.68, P%0.05）, and survival time was less than the control group （F 3.50, P〈0.01）. The expression of the correlation factor between LKBI and hedgehog signaling path- ways was negative （r 0. 232, P^0. 05）. CONCLUSIONS： The ex- pressions of LKB1 and the correlation factors in hedgehog signaling pathways is Negative correlation. LKB1 gene inhibits cell growth by hedgehog signaling pathways in MDA-MB-231. The crosstalk be tween these pathways may play an important role in the breast cancer growth.