TCF3 knockdown inhibits the decidualization of human endometrial stromal cells

doi:10.3969/j.issn.1674-8115.2022.09.011

Abstract

Abstract:

Objective ·To investigate the effect of transcription factor 3 (TCF3) on decidualization of human endometrial stromal cells (HESCs). Methods ·HESC decidualization in vitro was induced by 8-bromoadenosine 3',5'-cyclic adenosine monophosphate (8-Br-cAMP) and medroxy progesterone acetate (MPA). Small interfering RNA (siRNA) against TCF3 was used to construct TCF3-knockdown cell model. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to confirm the knockdown efficiency of TCF3. RT-qPCR and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of decidual markers, including insulin like growth factor binding protein 1 (IGFBP1) and prolactin (PRL). The morphological changes of HESCs after decidualization and after TCF3 knockdown were detected by Alexa Fluor-labeled phalloidin staining. The HESCs were divided into three groups: non-decidualized+NC (transfected with control siRNA) group, decidualized 4 d+NC group, and decidualized 4 d+siTCF3 (transfected with TCF3 siRNA) group, and RNA sequencing was performed. In order to explore the mechanism of TCF3 regulating the decidualization of HESCs, cluster analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and gene set enrichment analysis (GSEA) were conducted in the three groups of cells. qPCR was used to confirm the results of RNA sequencing analysis. Results ·The expression level of TCF3 increased gradually after 2 d and 4 d of decidualization of HESCs. Both ELISA and RT-qPCR results showed that after decidualization for 2 d or 4 d, the expression of decidual markers IGFBP1 and PRL decreased after TCF3 knockdown. Phalloidin staining results showed that after decidualization, the cell morphology changed from a long and slender fusiform structure to a large and round cell shape, while after TCF3 was knockdown, the cell morphology returned to the long fusiform structure. RNA sequencing analysis showed that the differential genes were mainly enriched in the cytokine-cytokine receptor pathway, which was further verified by GSEA analysis. RT-qPCR results confirmed that knockdown of TCF3 down-regulated the expression of several cytokines, such as leukemia inhibitory factor (LIF), interleukin 6 (IL-6), interleukin 1 β (IL1B), interleukin 1 receptor type 1 (IL1R1), and interleukin 1 receptor type 1 (CSF1). Conclusion ·Down-regulation of TCF3 in HESCs inhibits decidualization maybe through regulating the expression of cytokines IL-6, IL1B, IL1R1 and CSF1.

Key words: human endometrial stromal cell (HESC), transcription factor 3 (TCF3), decidualization, cytokine

CLC Number:

R711.6

WEI Xiaowei, TIAN Fuju, LIU Xiaorui, ZENG Weihong, CHEN Cailian, LIN Yi. TCF3 knockdown inhibits the decidualization of human endometrial stromal cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(9): 1247-1257.

Figures/Tables 7

TrendMD

References 25

1	LIU H, HUANG X B, MOR G, et al. Epigenetic modifications working in the decidualization and endometrial receptivity[J]. Cell Mol Life Sci, 2020, 77(11): 2091-2101.
2	BRAVERMAN M B, BAGNI A, DE ZIEGLER D, et al. Isolation of prolactin-producing cells from first and second trimester decidua[J]. J Clin Endocrinol Metab, 1984, 58(3): 521-525.
3	WATHEN N C, CAMPBELL D J, PATEL B, et al. Dynamics of prolactin in amniotic fluid and extraembryonic coelomic fluid in early human pregnancy[J]. Early Hum Dev, 1993, 35(3): 167-172.
4	LI J X, QI J, YAO G X, et al. Deficiency of sirtuin 1 impedes endometrial decidualization in recurrent implantation failure patients[J]. Front Cell Dev Biol, 2021, 9: 598364.
5	PLANTE B J, KANNAN A, BAGCHI M K, et al. Cyclic regulation of transcription factor C/EBP β in human endometrium[J]. Reprod Biol Endocrinol, 2009, 7: 15.
6	MOLENAAR M, VAN DE WETERING M, OOSTERWEGEL M, et al. XTcf-3 transcription factor mediates β-catenin-induced axis formation in Xenopus embryos[J]. Cell, 1996, 86(3): 391-399.
7	GRUMOLATO L, LIU G Z, HAREMAKI T, et al. β-Catenin-independent activation of TCF1/LEF1 in human hematopoietic tumor cells through interaction with ATF2 transcription factors[J]. PLoS Genet, 2013, 9(8): e1003603.
8	MERRILL B J, PASOLLI H A, POLAK L, et al. Tcf3: a transcriptional regulator of axis induction in the early embryo[J]. Development, 2004, 131(2): 263-274.
9	MARTELLO G, SUGIMOTO T, DIAMANTI E, et al. Esrrb is a pivotal target of the Gsk3/Tcf3 axis regulating embryonic stem cell self-renewal[J]. Cell Stem Cell, 2012, 11(4): 491-504.
10	WEI X W, LIU X Q, ZHANG Y C, et al. TCF3 regulates human endometrial stromal cell proliferation and migration in RPL[J]. Reproduction, 2022, 163(5): 281-291.
11	IHNATOVYCH I, LIVAK M, REED J, et al. Manipulating actin dynamics affects human in vitro decidualization[J]. Biol Reprod, 2009, 81(1): 222-230.
12	ZHOU Q, YAN G J, DING L J, et al. EHD1 impairs decidualization by regulating the Wnt4/β-catenin signaling pathway in recurrent implantation failure[J]. EBioMedicine, 2019, 50: 343-354.
13	SALKER M, TEKLENBURG G, MOLOKHIA M, et al. Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss[J]. PLoS One, 2010, 5(4): e10287.
14	HAI T, HARTMAN M G. The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis[J]. Gene, 2001, 273(1): 1-11.
15	JAMIL M, WANG W Y, XU M Y, et al. Exploring the roles of basal transcription factor 3 in eukaryotic growth and development[J]. Biotechnol Genet Eng Rev, 2015, 31(1/2): 21-45.
16	WANG Z L, LIU Y, LIU J Y, et al. ATF3 deficiency impairs the proliferative-secretory phase transition and decidualization in RIF patients[J]. Cell Death Dis, 2021, 12(4): 387.
17	DE POOTER R F, KEE B L. E proteins and the regulation of early lymphocyte development[J]. Immunol Rev, 2010, 238(1): 93-109.
18	DE JAIME-SOGUERO A, ABREU DE OLIVEIRA W A, LLUIS F. The pleiotropic effects of the canonical Wnt pathway in early development and pluripotency[J]. Genes, 2018, 9(2): 93.
19	SHUYA L L, MENKHORST E M, YAP J, et al. Leukemia inhibitory factor enhances endometrial stromal cell decidualization in humans and mice[J]. PLoS One, 2011, 6(9): e25288.
20	STAHL N, BOULTON T G, FARRUGGELLA T, et al. Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 β receptor components[J]. Science, 1994, 263(5143): 92-95.
21	THOMA B, BIRD T A, FRIEND D J, et al. Oncostatin M and leukemia inhibitory factor trigger overlapping and different signals through partially shared receptor complexes[J]. J Biol Chem, 1994, 269(8): 6215-6222.
22	FAHMI A, SMART N, PUNN A, et al. p42/p44-MAPK and PI3K are sufficient for IL-6 family cytokines/gp130 to signal to hypertrophy and survival in cardiomyocytes in the absence of JAK/STAT activation[J]. Cell Signal, 2013, 25(4): 898-909.
23	ZHOU M J, XU H H, ZHANG D, et al. Decreased PIBF1/IL6/p-STAT3 during the mid-secretory phase inhibits human endometrial stromal cell proliferation and decidualization[J]. J Adv Res, 2020, 30: 15-25.
24	GEISERT R, FAZLEABAS A, LUCY M, et al. Interaction of the conceptus and endometrium to establish pregnancy in mammals: role of interleukin 1β[J]. Cell Tissue Res, 2012, 349(3): 825-838.
25	FAZLEABAS A T, KIM J J, STRAKOVA Z. Implantation: embryonic signals and the modulation of the uterine environment: a review[J]. Placenta, 2004, 25(Suppl A): S26-S31.

siRNA	Sense （5'→ 3'）	Antisense （5'→ 3'）
siTCF3	CCGGAUCACUCAAGCAAUATT	UAUUGCUUGAGUGAUCCGGTT
NC	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT

siRNA	Sense （5'→ 3'）	Antisense （5'→ 3'）
siTCF3	CCGGAUCACUCAAGCAAUATT	UAUUGCUUGAGUGAUCCGGTT
NC	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT

Primer	Forward （5'→3'）	Reverse （5'→3'）
TCF3	ACGAGCGTATGGGCTACCA	GTTATTGCTTGAGTGATCCGGG
IGFBP1	TCCTTTGGGACGCCATCAGTAC	GATGTCTCCTGTGCCTTGGCTA
PRL	GAGGAGCAAACCAAACGGCTTC	AAGGCGAGACTCTTCATCAGCC
β-actin	GTCATTCCAAATATGAGATGCGT	GCTATCACCTCCCCTGTGTG
GAPDH	GTCTCCTCTGACTTCAACAGCG	ACCACCCTGTTGCTGTAGCCAA
IL-6	AGACAGCCACTCACCTCTTCAG	TTCTGCCAGTGCCTCTTTGCTG
LIF	AGATCAGGAGCCAACTGGCACA	GCCACATAGCTTGTCCAGGTTG
LIFR	CACCTTCCAAAATAGCGAGTATGG	ATGGTTCCGACCGAGACGAGTT
IL1B	CCACAGACCTTCCAGGAGAATG	GTGCAGTTCAGTGATCGTACAGG
IL1R1	GTGCTTTGGTACAGGGATTCCTG	CACAGTCAGAGGTAGACCCTTC
CXCL12	CTCAACACTCCAAACTGTGCCC	CTCCAGGTACTCCTGAATCCAC
CSF1	TGAGACACCTCTCCAGTTGCTG	GCAATCAGGCTTGGTCACCACA

Primer	Forward （5'→3'）	Reverse （5'→3'）
TCF3	ACGAGCGTATGGGCTACCA	GTTATTGCTTGAGTGATCCGGG
IGFBP1	TCCTTTGGGACGCCATCAGTAC	GATGTCTCCTGTGCCTTGGCTA
PRL	GAGGAGCAAACCAAACGGCTTC	AAGGCGAGACTCTTCATCAGCC
β-actin	GTCATTCCAAATATGAGATGCGT	GCTATCACCTCCCCTGTGTG
GAPDH	GTCTCCTCTGACTTCAACAGCG	ACCACCCTGTTGCTGTAGCCAA
IL-6	AGACAGCCACTCACCTCTTCAG	TTCTGCCAGTGCCTCTTTGCTG
LIF	AGATCAGGAGCCAACTGGCACA	GCCACATAGCTTGTCCAGGTTG
LIFR	CACCTTCCAAAATAGCGAGTATGG	ATGGTTCCGACCGAGACGAGTT
IL1B	CCACAGACCTTCCAGGAGAATG	GTGCAGTTCAGTGATCGTACAGG
IL1R1	GTGCTTTGGTACAGGGATTCCTG	CACAGTCAGAGGTAGACCCTTC
CXCL12	CTCAACACTCCAAACTGTGCCC	CTCCAGGTACTCCTGAATCCAC
CSF1	TGAGACACCTCTCCAGTTGCTG	GCAATCAGGCTTGGTCACCACA

[1]	YANG Wenqian, CHEN Chiqi, ZHAO Lu, CAO Liyuan, XIA Yiqiu, LU Zhigang, ZHENG Junke. Immune inhibitory receptor LILRB2 enhances SARS-CoV-2 spike protein-mediated immune inflammation [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(9): 1188-1196.
[2]	LI Xinyu, ZUO Bin, WANG Wen, NIU Xiaoyin, WENG Zhen, HE Yang. Levels of adiponectin in the peripheral blood of patients with immune thrombocytopenia and its effect on the differentiation of megakaryocyte cell line [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(7): 866-874.
[3]	LEI Haitao, TIAN Xuemei, JIN Fangquan. Advances in the correlation between cytokine signal transduction inhibitors and rheumatoid arthritis [J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(7): 945-951.
[4]	Xiaowen ZHANG, Yi WANG, Chan ZHANG, Di ZHANG, Hang YUN, Di HUANG. Effects of Pcsk9 gene interference on high fat-induced nonalcoholic fatty liver disease with atherosclerosis in rats [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2022, 42(2): 150-157.
[5]	Rui CHEN, Yun ZHAO, Xiao-xia ZHAO, Dong MA, Yi-jiang HAN, Deng-ming LAI, Wei-zhong GU, Jin-fa TOU. Expression characteristics of silent information regulator transcript 1 in intestinal tissues of neonatal necrotizing enterocolitis [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(9): 1154-1161.
[6]	Chao CHEN, Cong-cong LI, Feng GUO, Qiao-hong WANG, Ai-min ZHAO. Different expression of prolactin in unexplained recurrent pregnancy loss and normal early pregnancy [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(4): 489-496.
[7]	DAI Fei, WEI Jin-jin, TANG Xin-yue, CHEN Zheng, LIN Lin. Effect of sublingual immunotherapy on functions of effector T cells and regulatory T cells [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2020, 40(5): 626-632.
[8]	GAO Han, ZHANG Ping. Research progress on endometriosis-related immune events [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2020, 40(11): 1544-1549.
[9]	WANG Ling-xiao, LIU Ting-ting, YANG Xiao-hui, YAO Zhi-qing, CAI Hui-zhen. Effect of Lycium barbarum polysaccharides on inflammatory cytokines in type 2 diabetes mellitus model mice without myeloid differentiation factor 88 gene [J]. , 2019, 39(2): 136-.
[10]	LIN Hui, CHEN Xi, LIU Xin-mei, HUANG He-feng. Intrauterine high-nutrition induced immunoreaction abnormality in monocytes and nature killer cells in offspring [J]. , 2018, 38(7): 753-.
[11]	LIU Yang-chun, YE Hai-feng, LI Xiao-yan, HU Chuan, HUANG Jian, ZHENG Yue-hui. Regulatory factors and modulation mechanisms of functions of ovarian germline stem cell niche [J]. , 2018, 38(6): 708-.
[12]	ZHOU Ju-mei1, YUAN Ke-yong2, LIN Wen-zhen2, HU Xu-chen2, JIN Qiao-qiao2, NIU Chen-guang2. Influence of 3,3’-diindolylmethane on expression of inflammatory cytokines in periodontal ligament cells induced by lipopolysaccharide [J]. , 2018, 38(2): 138-.
[13]	XING Meng-juan, PENG Dai-hui. Research progress of NLRP3 inflammasome in the pathogenesis of depression disorder [J]. , 2018, 38(2): 217-.
[14]	FANG Xin-yu, ZHANG Chen. Research progress of correlation between interleukin-17 and schizophrenia#br# [J]. , 2017, 37(9): 1266-.
[15]	DI Fang-fang, LIU Jian-sheng, LI Shang, DU Yan-zhi . Progress on roles and mechanisms of CCAAT/enhancer binding protein β in endometrium decidualization [J]. , 2017, 37(6): 865-.