Janjusevic Milijana, Greco Stefania, Islam Md Soriful, Castellucci Clara, Ciavattini Andrea, Toti Paolo, Petraglia Felice, Ciarmela Pasquapina
Department of Experimental and Clinical Medicine, Faculty of Medicine, Università Politecnica delle Marche, Ancona, Italy.
Department of Experimental and Clinical Medicine, Faculty of Medicine, Università Politecnica delle Marche, Ancona, Italy; Biotechnology and Microbiology Laboratory, Department of Botany, University of Rajshahi, Rajshahi, Bangladesh.
Fertil Steril. 2016 Nov;106(6):1530-1538.e1. doi: 10.1016/j.fertnstert.2016.08.010. Epub 2016 Aug 24.
To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells.
Laboratory study.
Human myometrium and leiomyoma.
PATIENT(S): Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy.
INTERVENTION(S): Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry.
MAIN OUTCOME MEASURE(S): The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells.
RESULT(S): RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells.
CONCLUSION(S): Our results indicate that RKIP may be involved in leiomyoma pathophysiology.
研究Raf激酶抑制蛋白（RKIP）在人子宫肌层和平滑肌瘤中的存在情况，并确定洛考他汀（RKIP抑制剂）对人子宫肌层和平滑肌瘤细胞外基质（ECM）产生、增殖及迁移的影响。
实验室研究。
人子宫肌层和平滑肌瘤。
30名因子宫肌瘤切除术或子宫切除术入院的绝经前女性。
采用免疫组织化学和蛋白质印迹法，利用子宫肌层和平滑肌瘤组织研究RKIP的定位及表达水平。用洛考他汀（10 μM）处理子宫肌层和平滑肌瘤细胞，通过实时聚合酶链反应检测ECM表达，蛋白质印迹法检测糖原合成酶激酶3β（GSK3β）表达，划痕实验检测细胞迁移，MTT法和免疫细胞化学检测细胞增殖。
RKIP在人子宫肌层和平滑肌瘤组织中的表达；子宫肌层和平滑肌瘤细胞中ECM成分、GSK3β表达、迁移及增殖情况。
RKIP在人子宫肌层和平滑肌瘤组织中表达。洛考他汀处理导致丝裂原活化蛋白激酶（MAPK）信号通路激活（细胞外信号调节激酶磷酸化），有力验证了我们的靶向方案。此外，洛考他汀抑制RKIP可减少ECM成分。而且，洛考他汀抑制RKIP会损害平滑肌瘤细胞和子宫肌层细胞的增殖及迁移。最后，洛考他汀处理降低了GSK3β表达。因此，即使MAPK通路激活会增加增殖和迁移，但GSK3β的失稳会导致子宫肌层和平滑肌瘤细胞增殖及迁移减少。
我们的结果表明RKIP可能参与了子宫肌瘤的病理生理过程。
