Talin1对人输卵管上皮细胞黏附功能的调控作用

doi:10.12280/gjfckx.20240410

摘要/Abstract

摘要：

目的：探讨踝蛋白1（Talin1）对人输卵管上皮细胞黏附功能的影响及潜在的分子机制。方法：人正常输卵管上皮细胞内转染Talin1过表达质粒以构建过表达Talin1稳定细胞株，以人正常输卵管上皮细胞为对照，采用细胞黏附实验检测过表达Talin1人输卵管上皮细胞黏附力变化，免疫荧光法观察过表达Talin1人输卵管上皮细胞中细胞骨架变化及纤毛动力蛋白的表达；实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction，qRT-PCR）及蛋白质印迹法（Western blotting）分别检测过表达Talin1人输卵管上皮细胞中黏附因子以及细胞微丝骨架蛋白的mRNA及蛋白表达。结果：人正常输卵管上皮细胞内转染Talin1过表达质粒后，Talin1的mRNA和蛋白表达量均升高（P<0.05），成功构建了过表达Talin1稳定细胞株；Talin1使输卵管上皮细胞的黏附力显著增强（P<0.01），细胞骨架F-肌动蛋白（F-actin）、纤毛动力蛋白中轴丝动力蛋白重链5（dynein axonemal heavy chain 5，DNAH5）、叉头框转录因子J1（forkhead box J1，FOXJ1）和放射状的辐条结构头蛋白9（radial spoke head component 9，RSPH9）的平均荧光强度明显增强（均P<0.01）；Talin1可下调黏附因子E-钙黏蛋白（E-cadherin）的mRNA及蛋白表达（均P<0.001），上调黏附因子β-连环蛋白（β-catenin）、P120连环蛋白（P120ctn）、细胞间黏附分子-1（intercelluar adhesion molecule-1，ICAM-1）的mRNA及蛋白表达（均P<0.05）和桩蛋白（Paxillin）、β-微管蛋白（β-tubulin）、角蛋白（Keratin，Krt）的蛋白表达（均P<0.000 1）。结论：输卵管妊娠的发生可能与Talin1促进细胞骨架蛋白的表达及重排，调控相关黏附分子的表达干预输卵管上皮细胞的黏附性有关。

关键词: 踝蛋白, 妊娠, 输卵管, 细胞黏附, 细胞黏附分子, 细胞骨架

Abstract:

Objective: To investigate the effect of Talin1 on the adhesion function of human fallopian tube epithelial cells and explore the potential molecular mechanism involved. Methods: Human normal fallopian tube epithelial cells were transfected with Talin1. The adhesion strength of cell line overexpressed Talin1. The adhesion strength of these cells was assessed through a cell adhesion assay, with normal fallopian tube epithelial cells serving as controls. Immunofluorescence was used to observe changes in the cytoskeleton and the expression of ciliary dynamics proteins in Talin1-overexpressing cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were employed to detect the mRNA and protein expression of adhesion factors and cytoskeletal proteins in these cells. Results: After transfection with the Talin1 overexpression plasmid, both mRNA and protein levels of Talin1 were significantly increased (P<0.05), successfully establishing a stable cell line overexpressing Talin1. Talin1 significantly enhanced the adhesion strength of fallopian tube epithelial cells (P<0.01). The average fluorescence intensity of cytoskeletal F-actin, dynein axonemal heavy chain 5 (DNAH5), forkhead box J1 (FOXJ1), and radial spoke head component 9 (RSPH9) was markedly increased (all P<0.01). Talin1 downregulated the mRNA and protein expression of the adhesion factor E-cadherin (both P<0.001), while upregulating the mRNA and protein expression of adhesion factors β-catenin, P120 catenin (P120ctn), and intercellular adhesion molecule-1 (ICAM-1) (all P<0.05), as well as the protein expression of Paxillin, β-tubulin and Keratin (Krt) (all P<0.000 1). Conclusions: The occurrence of tubal pregnancy may be associated with Talin1′s role in promoting the expression and rearrangement of cytoskeletal proteins and regulating the expression of related adhesion molecules, thereby influencing the adhesion properties of fallopian tube epithelial cells.

Key words: Talin, Pregnancy, tubal, Cell adhesion, Cell adhesion molecules, Cytoskeleton

陈丽珠, 邱嫔. Talin1对人输卵管上皮细胞黏附功能的调控作用[J]. 国际妇产科学杂志, 2024, 51(5): 503-508.

CHEN Li-zhu, QIU Pin. Regulation Role of Talin1 on the Adhesion Function of Human Fallopian Tube Epithelial Cells[J]. Journal of International Obstetrics and Gynecology, 2024, 51(5): 503-508.

图/表 6

表1 qRT-PCR引物序列

基因	引物序列（5′-3′）
Paxillin	正向：AGTGTGGAGCCTTCTTTGGT
	反向：GCGTGTTGAGGGCTGAGAT
ICAM-1	正向：GGTAGCAGCCGCAGTCATAA
	反向：TCCCTTTTTGGGCCTGTTGT
β-catenin	正向：AATTTCCAGAAGCAGGAGTCCC
	反向：TTGATCCCATCTTCCGCAGC
E-cadherin	正向：CTGGTGCCATTTCCACTCGG
	反向：GCTGTGTACGTGCTGTTCTTC
P120ctn	正向：TCTCCTTCCTGCTTCCTCCT
	反向：CCCCCTCACTTCACACTGTT
Krt	正向：CATCGAGATCGCCACCTACC
	反向：TATTCACGGCTCCCACTCCA
β-tubulin	正向：GCCTTCCTGCACTGGTACAC
	反向：TCGTCGTCTTCGTACATCTCG

图1 Talin1过表达后的mRNA及蛋白表达水平变化注：A qRT-PCR结果，B Western blotting结果。* P<0.05，**** P<0.000 1。

图2 过表达Talin1增加输卵管上皮细胞的黏附力注：** P<0.01。

图3 过表达Talin1对输卵管上皮细胞骨架相关蛋白的影响注：A Talin1过表达后细胞骨架相关蛋白荧光强度变化，B 细胞骨架相关蛋白镜下荧光图。DNAH5 轴丝动力蛋白重链5，F-actin F-肌动蛋白，FOXJ1 叉头框转录因子J1，RSPH9 放射状的辐条结构头蛋白9。DAPI是细胞核染料，蓝色代表细胞核已染色，红色代表细胞骨架相关蛋白，Merge代表细胞骨架相关蛋白和DAPI显色结果的重叠。** P<0.01，*** P<0.001。

图4 过表达Talin1对细胞骨架相关蛋白的影响注：A qRT-PCR结果，B Western blotting结果。β-tubulin β-微管蛋白，Paxillin 桩蛋白，Krt 角蛋白。**** P<0.000 1，ns P>0.05。

图5 过表达Talin1对黏附相关蛋白表达的影响注：A qRT-PCR结果，B Western blotting结果。E-cadherin E-钙黏蛋白，β-catenin β-连环蛋白，P120ctn P120连环蛋白，ICAM-1 细胞间黏附分子-1。* P<0.05，** P<0.01，*** P<0.001，**** P<0.000 1。

参考文献 19

[1]	Wang X, Lee CL, Vijayan M, et al. Adrenomedullin insufficiency alters macrophage activities in fallopian tube: a pathophysiologic explanation of tubal ectopic pregnancy[J]. Mucosal Immunol, 2020, 13(5):743-752. doi: 10.1038/s41385-020-0278-6. pmid: 32203061
[2]	吴亚玲, 王世宣, 张金金. 输卵管功能损伤因素和分子机制研究进展[J]. 实用妇产科杂志, 2022, 38(11):839-843.
[3]	黄新磊, 苏芃, 王兴仪, 等. 微管细胞骨架在小鼠脑缺血/再灌注超急性期海马CA1区组织中的表达及意义[J]. 扬州大学学报(农业与生命科学版), 2023, 44(1):86-93. doi: 10.16872/j.cnki.1671-4652.2023.01.011.
[4]	Weidner AE, Roy A, Vann K, et al. Paxillin regulates androgen receptor expression associated with granulosa cell focal adhesions[J]. Mol Hum Reprod, 2024, 30(5):gaae018. doi: 10.1093/molehr/gaae018.
[5]	Li Z, Chen J, Zhao S, et al. Discovery and validation of novel biomarkers for detection of cervical cancer[J]. Cancer Med, 2021, 10(6):2063-2074. doi: 10.1002/cam4.3799.
[6]	Balamurugan K, Poria DK, Sehareen SW, et al. Stabilization of E-cadherin adhesions by COX-2/GSK3β signaling is a targetable pathway in metastatic breast cancer[J]. JCI Insight, 2023, 8(6):e156057. doi: 10.1172/jci.insight.156057.
[7]	Liu J, Xiao Q, Xiao J, et al. Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities[J]. Signal Transduct Target Ther, 2022, 7(1):3. doi: 10.1038/s41392-021-00762-6.
[8]	李明艳, 刘学博. 芪贞归脾汤辅助治疗对食管癌伴锁骨上淋巴结转移患者细胞E-cadherin表达的影响[J]. 辽宁中医杂志, 2024, 51(3):74-78. doi: 10.13192/j.issn.1000-1719.2024.03.021.
[9]	Lai TH, Chen HT, Wu WB. Trophoblast coculture induces intercellular adhesion molecule-1 expression in uterine endometrial epithelial cells through TNF-α production: Implication of role of FSH and ICAM-1 during embryo implantation[J]. J Reprod Immunol, 2022,152:103650. doi: 10.1016/j.jri.2022.103650.
[10]	Guan Y, Yang H, Yao X, et al. Clinical and Genetic Spectrum of Children With Primary Ciliary Dyskinesia in China[J]. Chest, 2021, 159(5):1768-1781. doi: 10.1016/j.chest.2021.02.006. pmid: 33577779
[11]	Kang M, Otani Y, Guo Y, et al. The focal adhesion protein talin is a mechanically gated A-kinase anchoring protein[J]. Proc Natl Acad Sci U S A, 2024, 121(13):e2314947121. doi: 10.1073/pnas.2314947121.
[12]	Xu C, Yang K, Xuan Z, et al. BCKDK regulates breast cancer cell adhesion and tumor metastasis by inhibiting TRIM21 ubiquitinate talin1[J]. Cell Death Dis, 2023, 14(7):445. doi: 10.1038/s41419-023-05944-4. pmid: 37460470
[13]	邱嫔, 林欣仪, 邓高丕. Talin1在输卵管妊娠患者的输卵管和绒毛中高表达并促进妊娠滋养细胞的侵袭[J]. 南方医科大学学报, 2022, 42(4):610-617. doi: 10.12122/j.issn.1673-4254.2022.04.19.
[14]	Wang J, Song J, An C, et al. A 130-kDa protein 4.1B regulates cell adhesion, spreading, and migration of mouse embryo fibroblasts by influencing actin cytoskeleton organization[J]. J Biol Chem, 2014, 289(9):5925-5937. doi: 10.1074/jbc.M113.516617. pmid: 24381168
[15]	Chen S, Liu B, Li J, et al. Talin1 regulates endometrial adhesive capacity through the Ras signaling pathway[J]. Life Sci, 2021,274:119332. doi: 10.1016/j.lfs.2021.119332.
[16]	Dubernard G, Galtier-Fougairolles M, Cortez A, et al. Immunohistochemistry of adhesion molecules, metalloproteinases and NO-synthases in extravillous trophoblast of tubal pregnancy[J]. Cell Mol Biol(Noisy-le-grand), 2005, 51(Suppl):OL829-837.
[17]	Latour YL, Allaman MM, Barry DP, et al. Epithelial talin-1 protects mice from citrobacter rodentium-induced colitis by restricting bacterial crypt intrusion and enhancing t cell immunity[J]. Gut Microbes, 2023, 15(1):2192623. doi: 10.1080/19490976.2023.2192623.
[18]	程秀娟, 熊国平, 汪琳. β-tubulin在输卵管妊娠着床部位与非着床部位的表达[J]. 中国组织化学与细胞化学杂志, 2013, 22(2):132-134. doi: 10.3870/zgzzhx.2013.02.009.
[19]	程秀娟, 熊国平, 杨勇, 等. E-cadherin在人输卵管妊娠蜕膜组织中的表达[J]. 中国组织化学与细胞化学杂志, 2006, 15(2):153-155. doi: 10.3870/j.issn.1004-1850.2006.02.009.