慢病毒载体介导的类固醇生成因子-1沉默调控子宫内膜基质细胞蜕膜化与自噬

doi:10.16098/j.issn.0529-1356.2024.02.009

摘要/Abstract

摘要：

目的探讨慢病毒载体介导的类固醇生成因子-1（SF-1）沉默对人子宫内膜基质细胞（hESCs）蜕膜化进程的影响及机制。方法收集子宫内膜异位症（EM）患者与正常妊娠者的子宫内膜组织，通过Real-time PCR和免疫组织化学染色检测SF-1的差异表达情况。从正常妊娠者的子宫内膜组织中分离并鉴定hESCs，将hESCs分为对照组、雌二醇（E₂+孕酮（P4）组、短发夹RNA（shRNA,sh）-正常对照（NC+E₂+P4组、sh-SF-1+E₂+P4组，进行对应处理后，倒置显微镜下观察hESCs形态变化，Real-time PCR和Western blotting分别检测细胞内人胰岛素样生长因子结合蛋白1（IGFBP-1）、泌乳素（PRL）的mRNA表达水平和蛋白表达水平，流式细胞术测定细胞周期分布，免疫荧光染色观察细胞内自噬标记物微管相关蛋白轻链3（LC3）表达情况，Western blotting测定细胞内自噬相关蛋白LC3-Ⅱ、LC3-Ⅰ、Beclin-1表达水平。结果 EM患者子宫内膜组织中SF-1 mRNA相对表达量和SF-1蛋白阳性率均高于正常妊娠者子宫内膜组织（P<0.05）。与sh-NC+E₂+P4组比较，sh-SF-1+E₂+P4组细胞多为长梭形，未见明显蜕膜化，IGFBP1、PRL的 mRNA相对表达量与蛋白相对表达量均显著下调（P<0.05），G₀/G₁期细胞比例显著减少而S期细胞比例显著增加（P<0.05），细胞内LC3荧光表达增强，LC3-Ⅱ/LC3-Ⅰ比值显著升高，Beclin-1蛋白相对表达量也显著上调（P<0.05）。结论 EM 患者子宫内膜组织内SF-1表达升高，通过慢病毒载体介导的SF-1沉默能够抑制hESCs蜕膜化过程，该作用可能与调控细胞自噬水平相关。

关键词: 子宫内膜基质细胞, 类固醇生成因子-1, 蜕膜化, 自噬, 免疫印迹法, 人

Abstract:

Objective To explore the influence and mechanism of lentiviral vector-mediated steroidogenic factor-1 (SF-1) silence on decidual process of human endometrial stromal cells (hESCs). Methods The endometrial tissues of patients with endometriosis (EM) and normal pregnant women were collected, and the differential expression of SF-1 was detected by Real-time PCR and immunohistochemical staining. hESCs were isolated from the endometrial tissue of normal pregnancy and identified, hESCs were divided into control group, estradiol (E₂) + progesterone (P4) group, short hairpin RAN(shRNA, sh)-normal control(NC)+E₂+P4 group, sh-SF-1+E₂+P4 group, after the corresponding processing, the morphological changes of hESCs were observed under an inverted microscope. Real-time PCR and Western blotting were used to detect the mRNA expression levels and protein levels of human insulin-like growth factor-binding protein-1 (IGFBP-1) and prolactin (PRL) in cells, respectively, flow cytometry was used to determine the cycle distribution of the cells, immunofluorescence staining was used to observe the expression of the intracellular auto-bid microtubule-associated protein light chain 3(LC3), Western blotting was used to determine the protein level of intracellular autophagy-related proteins LC3-Ⅱ, LC3-Ⅰ and Beclin-1. Results The relative expression of SF-1 mRNA and the positive rate of SF-1 protein in endometrial tissue of EM patients were higher than those of normal pregnancy endometrial tissue (P<0.05). Compared with the sh-NC+E₂+P4 group, the cells in the sh-SF-1+E₂+P4 group were mostly long-spindle, and there was no obvious decidualization. The relative mRNA expression and protein expression of IGFBP1 and PRL were significantly down-regulated (P<0.05), the proportion of cells in G₀/G₁ phase was significantly decreased, the proportion of cells in S phase was significantly increased (P<0.05), the ratio of LC3-Ⅱ/LC3-Ⅰ was significantly increased, and the relative expression of Beclin-1 protein was also significantly up-regulated (P<0.05). Conclusion The expression of SF-1 in the endometrial tissue of EM patients is increased, and SF-1 silencing mediated by lentiviral vector can inhibit the decidualization process of hESCs, which may be related to regulating the level of autophagy.

Key words: Endometrial stromal cell, Steroidogenic factor-1, Decidualization, Autophagy, Western blotting, Human

中图分类号:

R711

任健陈晓燕. 慢病毒载体介导的类固醇生成因子-1沉默调控子宫内膜基质细胞蜕膜化与自噬[J]. 解剖学报, 2024, 55(2): 188-195.

REN Jian CHEN Xiao-yan. Lentiviral vector mediated steroidogenic factor-1 silencing regulating decidualization and autophagy of endometrial stromal cells[J]. Acta Anatomica Sinica, 2024, 55(2): 188-195.

参考文献

［1］Lai ZZ, Yang HL, Shi JW, et al. Protopanaxadiol improves endometriosis associated infertility and miscarriage in sex hormones receptors-dependent and independent manners［J］. Int J Biol Sci, 2021, 17(8):1878-1894.

［2］Koninckx PR, Fernandes R, Ussia A, et al. Pathogenesis based diagnosis and treatment of endometriosis［J］. Front Endocrinol (Lausanne), 2021, 12:745548.

［3］McKinnon BD, Lukowski SW, Mortlock S, et al. Altered differentiation of endometrial mesenchymal stromal fibroblasts is associated with endometriosis susceptibility［J］. Commun Biol, 2022, 5(1):600.

［4］Pan-Castillo B, Gazze SA, Thomas S, et al. Morphophysical dynamics of human endometrial cells during decidualization［J］. Nanomedicine, 2018, 14(7):2235-2245.

［5］Meinsohn MC, Smith OE, Bertolin K, et al. The orphan nuclear receptors steroidogenic factor-1 and liver receptor homolog-1: structure, regulation, and essential roles in mammalian reproduction［J］. Physiol Rev, 2019, 99(2):1249-1279.

［6］Yilmaz BD, Bulun SE. Endometriosis and nuclear receptors［J］. Hum Reprod Update, 2019, 25(4):473-485.

［7］Ng SW, Norwitz GA, Pavlicev M, et al. Endometrial decidualization: the primary driver of pregnancy health［J］. Int J Mol Sci, 2020, 21(11):4092.

［8］Sang Y, Li Y, Xu L, et al. Regulatory mechanisms of endometrial decidualization and pregnancy-related diseases［J］. Acta Biochim Biophys Sin (Shanghai), 2020, 52(2):105-115.

［9］Ashary N, Laheri S, Modi D. Homeobox genes in endometrium: from development to decidualization［J］. Int J Dev Biol, 2020, 64(1-2-3):227-237.

［10］Conrad KP, Rabaglino MB, Post Uiterweer ED. Emerging role for dysregulated decidualization in the genesis of preeclampsia［J］. Placenta, 2017, 60:119-129.

［11］Wang ShY, Shi QF, Yang GY, et al. Correlation between SF-1 gene polymorphism and adrenal dysfunction in sepsis ［J］. Clinical Research and Practice, 2019,4(34):7-9,12. (in Chinese)

王树云, 石齐芳, 杨光耀, 等. SF-1基因多态性与脓毒症肾上腺皮质功能不全的相关性研究［J］.临床医学研究与实践, 2019,4(34):7-9,12.

［12］Tanaka T, Aoyagi C, Mukai K, et al. Extension of survival in bilaterally adrenalectomized mice by implantation of SF-1/Ad4BP-induced steroidogenic cells［J］. Endocrinology, 2020, 161(3):bqaa007.

［13］Guntiboina VA, Sengupta M, Islam N, et al. Diagnostic and prognostic utility of SF1, IGF2 and p57 immunoexpression in pediatric adrenal cortical tumors［J］. J Pediatr Surg, 2019, 54(9):1906-1912.

［14］Wu KY, Zhang Q, Xu QL. Expression of ER, C-erbB-2 oncoprotein and SF-1 in endometrial polyps and their significance ［J］. Chinese Journal of Laboratory Diagnosis, 2019, 23(10):1760-1761. (in Chinese)

吴坤英, 张青, 徐庆丽. ER、C-erbB-2癌蛋白、SF-1在子宫内膜息肉中的表达及其意义［J］.中国实验诊断学, 2019, 23(10):1760-1761.

［15］Komada M, Takahashi M, Ikeda Y. Involvement of SF-1 in neurogenesis and neuronal migration in the developing neocortex［J］. Neurosci Lett, 2015, 600:85-90.

［16］Wang X, Wu P, Li X, et al. Extracellular vesicles inhibit proliferation and invasion of ovarian endometrial stromal cells and their expression of SF-1, ERβ, and Aromatase［J］. Front Endocrinol (Lausanne), 2021, 12:666195.

［17］Saito R, Kajihara T, Takamura M, et al. High stretch cycling inhibits the morphological and biological decidual process in human endometrial stromal cells［J］. Reprod Med Biol, 2020, 19(4):378-384.

［18］Jozaki K, Tamura I, Takagi H, et al. Glucose regulates the histone acetylation of gene promoters in decidualizing stromal cells［J］. Reproduction, 2019, 157(5):457-464.

［19］Liu KY, Liu HW. Role and mechanism of autophagy in endometriosis ［J］. Journal of International Obstetrics and Gynecology, 2022,49(3):241-245. (in Chinese)

刘可伊,刘恒炜.自噬在子宫内膜异位症中的作用及机制［J］.国际妇产科学杂志,2022,49(3):241-245.

［20］Oestreich AK, Chadchan SB, Popli P, et al. The autophagy gene Atg16L1 is necessary for endometrial decidualization［J］. Endocrinology, 2020, 161(1):bqz039.

［21］Zhang Y, Gao R, Zhang L, et al. AMPK/mTOR downregulated autophagy enhances aberrant endometrial decidualization in folate-deficient pregnant mice［J］. J Cell Physiol, 2021, 236(11):7376-7389.

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