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目的:雌激素和孕激素在子宫肌瘤发病中起重要作用。但miRNA在子宫肌瘤发病中的作用还知之甚少,我们前期已证实mir-26a在子宫肌瘤中低表达,本实验进一步探讨mir-26a在体外对子宫肌瘤中孕激素受体a(PRa)、雌激素受体α(ERα)表达的调控。方法:利用TargetScan软件预测mir-26a的潜在靶基因,找出靶基因3’UTR区片段,插入PmirGLO绿色荧光蛋白编码区下游,构建报告基因载体,同时原代培养子宫肌瘤平滑肌细胞。将报告基因载体与mir-26a共转染入原代培养的子宫肌瘤平滑肌细胞,引入双荧光素酶报告基因系统对mir-26a的靶基因进行验证。转染mir-26amimics于子宫肌瘤平滑肌细胞,westernblotting检测子宫肌瘤平滑肌细胞中mir-26a靶蛋白表达水平。结果:用TargetScan软件和双荧光素酶报告基因系统证实ERα、PRa为mir-26a的靶基因。蛋白水平进一步验证,mir-26amimics的转染量不同,ERα、PRa的蛋白表达水平下调不同。结论:Mir-26a通过结合靶基因的3’-UTR区调控靶基因的mRNA水平。Mir-26a抑制雌激素受体α(ERα)、孕激素受体a(PRa)在子宫肌瘤中的表达。Mir-26a可能通过调控雌激素受体α(ERα)、孕激素受体a(PRa)影响子宫肌瘤的发展。本实验通过确定mir-26a对子宫肌瘤的作用机制,有望进一步提高子宫肌瘤的治疗技术,减少手术治疗的创伤。
Objective: Estrogen and progesterone play an important role in the pathogenesis of uterine fibroids. However, the role of miRNA in the pathogenesis of uterine fibroids is also poorly understood, we have confirmed earlier mir-26a low expression in uterine fibroids, this study further explore the mir-26a in vitro uterine fibroids progesterone receptor a ( PRa), estrogen receptor alpha (ERa) expression. Methods: The potential target gene of mir-26a was predicted by using TargetScan software. The 3’UTR region of target gene was identified and inserted into downstream of coding region of PmirGLO to construct reporter gene vector. At the same time, primary smooth muscle cells of uterine fibroids were cultured. The reporter gene vector was co-transfected into mir-26a primary cultured myofibroblasts smooth muscle cells, and the dual luciferase reporter gene system was introduced to verify the target gene of mir-26a. Transfection mir-26 amimics in uterine fibroids smooth muscle cells, western blotting detection of uterine fibroids smooth muscle cells mir-26a target protein expression levels. Results: ERα was confirmed by TargetScan software and dual luciferase reporter gene system and PRa was the target gene of mir-26a. Further validation of protein levels, mir-26amimics transfected different, ERα, PRa protein expression levels down. Conclusion: Mir-26a regulates the mRNA level of the target gene by binding to the 3’-UTR region of the target gene. Mir-26a inhibits the expression of estrogen receptor alpha (ERa) and progesterone receptor alpha (PRa) in uterine fibroids. Mir-26a may affect the development of uterine fibroids through the regulation of estrogen receptor α (ERα) and progesterone receptor a (PRa). The experiment by determining the role of mir-26a on the mechanism of uterine fibroids, is expected to further improve the treatment of uterine fibroids and reduce surgical treatment of trauma.