miR-200a-3p靶向SIRT1/NF-κB/NLRP3通路参与大鼠先天性肛门直肠畸形发生的作用机制-东南大学学报医学版

miR-200a-3p靶向SIRT1/NF-κB/NLRP3通路参与大鼠先天性肛门直肠畸形发生的作用机制

单位：1. 郑州大学附属儿童医院/河南省儿童医院郑州儿童医院小儿普外科, 河南郑州 450018;
2. 郑州大学附属儿童医院/河南省儿童医院郑州儿童医院新生儿外科, 河南郑州 450018

关键词：先天性肛门直肠畸形 miR-200a-3p 沉默信息调节因子1 增殖炎症 Wistar孕鼠

分类号：R726.5

出版年·卷·期（页码）：2025·44·第五期（739-746）

摘要：

目的:探讨miR-200a-3p靶向沉默信息调节因子1(SIRT1)/核因子κB(NF-κB)/含NLR家族Pyrin域蛋白3(NLRP3)通路参与大鼠先天性肛门直肠畸形(ARM)发生的作用机制。方法:6只Wistar孕鼠在妊娠10 d时灌胃125 mg·kg^-1 1%乙烯硫脲,另外6只Wistar孕鼠接受等体积的生理盐水作为对照。采用妊娠14~16 d剖宫产术,共获得乙烯硫脲诱导的ARM胎鼠72例(ARM组),生理盐水处理的正常胎鼠84例(对照组)。取ARM组、对照组胎鼠各72例用于指标检测。qRT-PCR检测妊娠14、15、16 d胎鼠后肠组织miR-200a-3p表达;荧光原位杂交(FISH)检测miR-200a-3p在妊娠14、15、16 d胎鼠后肠组织中的表达;验证miR-200a-3p与SIRT1靶向关系;Western blotting检测妊娠14、15、16 d胎鼠后肠组织SIRT1蛋白表达;免疫组化染色检测SIRT1蛋白在妊娠14、15、16 d胎鼠后肠组织中的表达。对IEC-6细胞进行过表达miR-200a-3p、沉默SIRT1或同时上调miR-200a-3p和SIRT1表达处理,qRT-PCR检测细胞miR-200a-3p表达;Western blotting检测细胞SIRT1、p-NF-κB p65、NLRP3蛋白;CCK-8检测细胞增殖;ELISA检测细胞上清中白细胞介素(IL)-18、IL-1β水平。结果:与对照组相比,ARM组妊娠14、15、16 d胎鼠后肠组织中miR-200a-3p表达升高(P＜0.05);对照组胎鼠后肠组织中的尿道、肠道处有较强的miR-200a-3p荧光信号,而ARM组胎鼠后肠组织中的尿道、肠道、直肠尿道瘘、尿道直肠隔、泄殖腔膜处均有较强的miR-200a-3p荧光信号;miR-200a-3p靶向调控SIRT1;与对照组相比,ARM组妊娠14、15、16 d胎鼠后肠组织中SIRT1蛋白表达降低(P＜0.05);在对照组胎鼠后肠组织中的直肠、尿道、直肠尿道瘘、尿道直肠隔、泄殖腔膜处有大量SIRT1阳性细胞,而ARM组胎鼠后肠组织中的直肠、尿道有少量SIRT1阳性细胞。过表达miR-200a-3p或沉默SIRT1均可抑制IEC-6细胞增殖,促进炎症,而过表达SIRT1可减弱上调miR-200a-3p对IEC-6细胞增殖的抑制以及对炎症的促进作用。结论:miR-200a-3p和SIRT1可能通过影响NF-κB/NLRP3通路参与ARM的发生。

Objective: To explore the mechanism by which miR-200a-3p participates in the occurrence of congenital anorectal malformation(ARM) in rats by targeting silent information regulator 1(SIRT1)/nuclear factor-kappa B(NF-κB)/NLR family pyrin domain containing protein 3(NLRP3) pathway. Methods: Six pregnant Wistar rats were orally administered 125 mg·kg^-1 1% ethylene thiourea at 10 days of gestation, while the other 6 pregnant Wistar rats were received an equal volume of physiological saline as a control. Totally 72 cases of ARM fetal rats induced by ethylene thiourea(ARM group) and 84 cases of normal fetal rats treated with physiological saline(control group) were obtained by cesarean section during the 14th to 16th day of pregnancy. The 72 fetal rats from the ARM group and 72 fetal rats from the control group were collected for indicator detection. qRT-PCR was used to detect miR-200a-3p in the hindgut tissue of pregnant rats on 14, 15 and 16 d. Fluorescence in situ hybridization(FISH) was used to detect the spatiotemporal expression of miR-200a-3p in the hindgut tissue of pregnant rats on 14, 15 and 16 d. The targeting relationship between miR-200a-3p and SIRT1 was validated. Western blotting was used to detect SIRT1 protein in the hindgut tissue of pregnant rats on 14, 15 and 16 d. Immunohistochemical staining was used to detect the spatiotemporal expression of SIRT1 protein in the hindgut tissue of pregnant rats on 14, 15 and 16 d. IEC-6 cells were treated with overexpression of miR-200a-3p, silencing of SIRT1, or simultaneous upregulation of miR-200a-3p and SIRT1. qRT-PCR was used to measure miR-200a-3p in cells. Western blotting was performed to measure SIRT1, p-NF-κB p65, and NLRP3 proteins in cells. CCK-8 was performed to detect cell proliferation. ELISA was used to measure interleukin(IL) -18 and IL-1β in cell supernatant. Results: Compared with the control group, the miR-200a-3p in the hindgut tissue of pregnant rats on 14, 15 and 16 d in the ARM group was increased(P<0.05). There were strong miR-200a-3p fluorescence signals in the urethra and intestine in hindgut tissue of fetal rats in control group, while there were strong miR-200a-3p fluorescence signals in the urethra, intestine, rectovaginal fistula, urethral rectal septum, and cloacal membrane in hindgut tissue of fetal rats in ARM group. MiR-200a-3p targeted and regulated SIRT1. Compared with the control group, the expression of SIRT1 protein in the hindgut tissue of pregnant rats on 14, 15 and 16 d in the ARM group was reduced(P<0.05). There were a large number of SIRT1 positive cells in the rectum, urethra, rectovaginal fistula, urethral rectal septum, and cloacal membrane of fetal rats in the control group, while there were a small number of SIRT1 positive cells in the rectum and urethra of fetal rats in the ARM group. Overexpression of miR-200a-3p and silencing of SIRT1 could both inhibit the proliferation of IEC-6 cells and promote inflammation, while overexpression of SIRT1 could weaken the inhibitory effect of upregulation of miR-200a-3p on IEC-6 cell proliferation and its promoting effect on inflammation. Conclusion: MiR-200a-3p and SIRT1 may participate in the occurrence of ARM by affecting the NF-κB/NLRP3 pathway.

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