多亮氨酸重复区免疫球蛋白样蛋白1基因表达对体外垂体瘤细胞株RC-4B／C生物学功能的影响

Effect of leucine - rich repeats and immunoglobulin - like domains 1 on proliferation and apoptosis of RC -4B/C pituitary tumor cell lines in vitro

目的观察多亮氨酸重复区免疫球蛋白样蛋白1（LRIGI）基因表达对体外垂体瘤细胞株RC-4B／C的增殖凋亡和生物学功能的影响。方法应用基因转录方法检测LRIG1基因表达射体外垂体瘤细胞株RC-4B／C的抗增殖作用和生物学功能的影响；通过免疫组织化学法和Western blot技术检测LRIGI蛋白在RC-4B／C细胞中的表达水平，反转录-聚合酶链反应（RT—PCR）测定转染后RC-4B／C细胞中Ras、Raf、蛋白激酶B（Akt）和细胞外信号调节激酶（ERK）mRNA表达变化，并通过噻唑蓝（MTF）法、划痕实验、苏木素-伊红（HE）染色、显微镜等观察比较转染LRIG1基因的RC-4B／C细胞的的迁移能力、增殖凋亡和形态学改变。结果Western blot和免疫组织化学法证实转染后RC-4B／C细胞中LRIG1稳定表达；RT—PCR证实转染后RC-4B／C细胞中Ras、Raf和AktmRNA水平显著降低（P〈0．01），分别为0．87±0．17、0．46±0．11、0．92±0．25。MTT法结果表明随着LRIG1与RC-4B／C细胞效靶比增加及作用时间延长，抑制率明显增强（P〈0．01）。划痕实验结果显示：LRIG1转染的RC-4B／C细胞迁移能力低于正常RC-4B／C细胞。LRIGI作用于垂体瘤细胞24h后，形态学观察垂体瘤细胞发生凋亡或坏死。结论LRIGI基因转染对体外垂体瘤细胞株RC-4B／C细胞具有抑制增殖和促进凋亡作用，其可能是通过抑制表皮生长因子受体（EGFR）和表皮生长因子受体Ⅲ型突变体（EGFRvm）信号传导通路中的磷酸肌醇3激酶（P13K）／Akt和Ras／Rat／ERK两条重要信号通路分子的活性来发挥作用。

Objective To explore the effect of leucine - rich repeats and immunoglobulin - like domains 1 （ LRIG1 ） gene on proliferation and apoptosis of RC - 4B/C cell lines of pituitary tumor in vitro. Methods Gene transfenction was used to detect anti -proliferation activity and biological function of LRIG1 gene on RC -4B/C cell lines of pituitary tumor. Immunohistochemistry and Western blotting were used to detect the expression level of LRIG1 in RC -4B/C cells. Reverse transcriptasepolymerase chain reaction （RT -PCR） was used to measure the expression of Ras, Raf, protein kinase B （Akt） and extra- cellular signal regulated kinase （ERK） mRNA in transfected RC -4B/C cells. Methyl thiazol tetrazolium （MTT） assay, the scratch test, hematoxylin and eosin （HE） staining, and microscopy were used to compare the migration ability, proliferation, apoptosis and morphological changes between LRIGI -transfected RC-4B/C cells and normal RC -4B/C cells. Results Stable expression of LRIGI was confirmed in LRIGI -transfected RC -4B/C cells. The expression of Ras, Raf and Akt mRNA was significantly decreased in LRIGI - transfected RC - 4B/C cells compared to normal cells （ P 〈 0. 01, 0. 87 ±0. 17, 0. 46±0. 11 and 0. 92 ±0. 25 respectively）. MTT assay showed that LRIGI could inhibit proliferation of RC - 4B/C cells. With the increase of effector target ratio and time prolongation, the inhibitory rate of proliferation was increased obviously （P 〈 0. 01 ）. The scratch test showed that LRIG1 reduced migration ability of RC -4B/C cells. At 24 h after transfection with LRIG1, the apoptosis or necrosis of RC -4B/C cells was increased compared to normal cells. Conclusion LRIG1 gene can significantly inhibit proliferation and promote apoptosis of pituitary adenoma cells in vitro via inhibition of activation of the PI3K/Akt and Ras/Raf/ERK signal, a significant downstream signal pathway of EGFR signal transduction pathway.