Treg cells promote the proliferation, migration and invasion of esophageal cancer cells by up-regulating the expression of TGF-β1 and B7-H3

doi:10.3877/cma.j.issn.2095-1221.2023.02.001

Abstract

Abstract:

Objective

To investigate the effect and molecular mechanism of regulatory T (Treg) cells on the proliferation, migration and invasion of esophageal cancer cells.

Methods

Treg cells in the peripheral blood of patients were isolated by flow cytometry; transforming growth factor β1 (TGF-β1), TGF-β1/Smad inhibitor (SB431542), TGF-β1 overexpression (TGF-β1 OE) and B7 homolog 3 (B7-H3) inhibition lentiviral vector (sh-B7-H3) were used to regulate the expression of TGF-β1 and B7-H3 in esophageal cancer cells; qRT-PCR and western blot were used to detect the expression levels of TGF-β1 and B7-H3; CCK-8 and Transwell were used to detect the proliferation, migration and invasion of esophageal cancer cells; the TGF-β1/B7-H3 axis was verified in xenograft nude mice by HE staining and immunohistochemistry. An independent sample t-test was used to compare two groups, a one-way analysis of variance was used to compare multiple groups and an LSD-t test was used for duo comparison between various groups.

Results

Compared with PBS, the co-culture of Treg cells promoted the proliferation (0.93 ± 0.18, 0.99 ± 0.18 vs 0.63 ± 0.12, 0.63 ± 0.14) and migration (74.67 ± 4.16, 61.00 ± 2.65 vs 55.67 ± 5.13, 37.33 ± 2.08), invasiveness (105.70 ± 2.13, 63.67 ± 3.51 vs 77.33 ± 3.22, 55.33 ± 2.52) and expression of TGF-β1 (2.17 ± 0.02, 2.82 ± 0.08 vs 1.00 ± 0.02, 1.00 ± 0.05) of Eca-109 and TE-1 esophageal cancer cells; SB431542 effectively inhibited TGF-β1 induced cell proliferation (0.96 ± 0.14 vs 1.46 ± 0.22), migration (84.67 ± 5.03, 62.67 ± 5.69 vs 120.30 ± 4.51, 119.30 ± 7.02) and invasion (45.33 ± 4.04, 47.33 ± 2.52 vs 59.33 ± 2.52, 58.00 ± 2.00) ; Compared with Het-1a, B7-H3 mRNA levels were increased in Eca-109 and TE-1 (2.65 ± 0.04, 3.30 ± 0.27 vs 1.00 ± 0.03). Compared with the control group, TGF-β1 further induced an increase in B7-H3 protein level in esophageal cancer cells (1.57 ± 0.10, 1.41 ± 0.02 vs 1.00 ± 0.02, 1.00 ± 0.06), and the knockdown of B7-H3 reversed the carcinogenic effect induced by TGF-β1. In addition, similar results were observed in a nude mouse model.

Conclusion

Treg cells promote esophageal cancer cell proliferation, migration and invasion by up-regulating the expression of TGF-β1 and B7-H3.

Key words: Treg cells, TGF-β1, B7-H3, Esophageal cancer, Proliferation, Migration and invasion

Woda Shi, Yajun Zhang, Zhan Shi, Jixiang Wu, Huiwen Chang, Zhongquan Yi, Xiaodong Liang, Jingjing Zhou, Jianxiang Song. Treg cells promote the proliferation, migration and invasion of esophageal cancer cells by up-regulating the expression of TGF-β1 and B7-H3[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2023, 13(02): 65-75.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhxbygxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-1221.2023.02.001

https://zhxbygxbzz.cma-cmc.com.cn/EN/Y2023/V13/I02/65

Figures/Tables 19

References 27

1	Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018, 68(6):394-424.
2	Lin Y, Totsuka Y, Shan B, et al. Esophageal cancer in high-risk areas of China: research progress and challenges[J]. Ann Epidemiol, 2017, 27(3):215-221.
3	Allemani C, Matsuda T, Di Carlo V, et al. Global surveillance of trends in cancer survival 2000-14 (CONCORD-3): analysis of individual records for 37 513 025 patients diagnosed with one of 18 cancers from 322 population-based registries in 71 countries[J]. Lancet, 2018, 391(10125):1023-1075.
4	Zheng Y, Chen Z, Han Y. Immune suppressive landscape in the human esophageal squamous cell carcinoma microenvironment[J]. Nat Commun, 2020, 11(1):6268. doi: 10.1038/s41467-020-20019-0.
5	Wang Y, Xu G, Wang J, et al. Relationship of Th17/Treg cells and radiation pneumonia in Locally Advanced Esophageal Carcinoma[J]. Anticancer Res, 2017, 37(8):4643-4647.
6	Perez LG, Kempski J, McGee HM. TGF-β signaling in Th17 cells promotes IL-22 production and colitis-associated colon cancer[J]. Nat Commun, 2020, 11(1):2608. doi: 10.1038/s41467-020-16363-w.
7	周彦彤, 李春晓, 王劲松, 等. 单细胞转录组测序分析结肠癌微环境中免疫细胞亚群与癌症进程的关系[J]. 解放军医学杂志, 2021, 46(7):692-701.
8	臧超然, 张永宏. 免疫抑制性细胞在肝细胞癌发生发展中的作用[J]. 临床肝胆病杂志, 2018, 34(7):1560-1564.
9	冯雪, 王利群, 卢美松. 调节性T细胞与卵巢癌肿瘤免疫的相关研究进展[J]. 国际妇产科学杂志, 2020, 47(4):429-432.
10	Bai F, Zhang P, Fu Y, et al. Targeting ANXA1 abrogates Treg-mediated immune suppression in triple-negative breast cancer[J]. J Immunother Cancer, 2020, 8(1):e000169. doi: 10.1136/jitc-2019-000169.
11	Ji L, Qian W, Gui L,et al. Blockade of β-catenin-induced CCL28 suppresses gastric cancer progression via inhibition of treg cell infiltration[J]. Cancer Res, 2020, 80(10):2004-2016.
12	郑爱华. 肺肿瘤小鼠MDSC, Treg及传统T细胞变化研究[J]. 天津医药, 2016, 44(8):996 -1000.
13	Landskron G, De la Fuente M, Thuwajit P, et al. Chronic inflammation and cytokines in the tumor microenvironment[J]. J Immunol Res, 2014, 2014:149185. doi: 10.1155/2014/149185.
14	Tomova R, Pomakov J, Jacobs JJ, et al. Changes in cytokine profile during local IL-2 therapy in cancer patients[J]. Anticancer Res, 2006, 26(3A):2037-2047.
15	Das S, Surve V, Marathe S, et al. IL-9 abrogates the metastatic potential of breast cancer by controlling extracellular matrix remodeling and cellular contractility[J]. J Immunol, 2021, 206(11):2740-2752.
16	Shi J, Song X, Traub B, et al. Involvement of IL-4, IL-13 and their receptors in pancreatic cancer[J]. Int J Mol Sci, 2021, 22(6):2998. doi: 10.3390/ijms22062998.
17	Prabhala RH, Pelluru D, Fulciniti M, et al. Elevated IL-17 produced by TH17 cells promotes myeloma cell growth and inhibits immune function in multiple myeloma[J]. Blood, 2010, 115(26):5385-5392.
18	Streel GD, Bertrand C, Chalon N, et al. Selective inhibition of TGF-β1 produced by GARP-expressing Tregs overcomes resistance to PD-1/PD-L1 blockade in cancer[J]. Nat Commun, 2020, 11(1):4545. doi: 10.1038/s41467-020-17811-3.
19	Shrestha R, Bridle KR, Crawford DHG, et al. Immune checkpoint molecules are regulated by transforming growth factor (TGF)-β1-induced epithelial-to-mesenchymal transition in hepatocellular carcinoma[J]. Int J Med Sci, 2021, 18(12):2466-2479.
20	Liu C, Zhao Y, Wang J, et al. FoxO3 reverses 5-fluorouracil resistance in human colorectal cancer cells by inhibiting the Nrf2/TR1 signaling pathway[J]. Cancer Lett, 2020, 470:29-42.
21	Picarda E, Ohaegbulam KC, Zang X. Molecular pathways:targeting B7-H3 (CD276) for human cancer immunotherapy[J]. Clin Cancer Res, 2016, 22(14):3425-3431.
22	Yang M, Tang X, Zhang Z, et al. Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors[J]. Theranostics, 2020, 10(17):7622-7634.
23	Loos M, Hedderich DM, Ottenhausen M, et al. Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer[J]. BMC Cancer, 2009, 9:463.doi: 10.1186/1471-2407-9-463.
24	Zhou X, Mao Y, Zhu J, et al. TGF-β1 promotes colorectal cancer immune escape by elevating B7-H3 and B7-H4 via the miR-155/miR-143 axis[J]. Oncotarget, 2016, 7(41):67196-67211.
25	Chen L, Xie Q, Wang Z, et al. Assessment of combined expression of B7-H3 and B7-H4 as prognostic marker in esophageal cancer patients[J]. Oncotarget, 2016, 7(47):77237-77243.
26	Zhao X, Li DC, Zhu XG, et al. B7-H3 overexpression in pancreatic cancer promotes tumor progression[J]. Int J Mol Med, 2013, 31(2):283-291.
27	Yang X, Feng KX, Li H, et al. MicroRNA-199a inhibits cell proliferation, migration, and invasion and activates AKT/mTOR signaling pathway by targeting B7-H3 in cervical cancer[J]. Technol Cancer Res Treat, 2020, 19:1533033820942245. doi: 10.1177/1533033820942245.

基因	序列（5'-3'）	预期扩增长度（bp）
TGF-β1	上游GGATACCAACTATTGCTTCAGCTCC	318
	下游AGGCTCCAAATATAGGGGCAGGGTC
B7-H3	上游TGCACCACCAACTGCTTAGC	328
	下游GGCATGGACTGTGGTCATGAG
GAPDH	上游TGTGGGCATCAATGGATTTGG	300
	下游ACACCATGTATTCCGGGTCAAT

基因	序列（5'-3'）	预期扩增长度（bp）
TGF-β1	上游GGATACCAACTATTGCTTCAGCTCC	318
	下游AGGCTCCAAATATAGGGGCAGGGTC
B7-H3	上游TGCACCACCAACTGCTTAGC	328
	下游GGCATGGACTGTGGTCATGAG
GAPDH	上游TGTGGGCATCAATGGATTTGG	300
	下游ACACCATGTATTCCGGGTCAAT

分组	实验次数	细胞迁移数目（个）	侵袭细胞数目（个）
PBS+Eca-109	3	55.67 ± 5.13	77.33 ± 3.22
Treg+Eca-109	3	74.67 ± 4.16	105.70 ± 2.13
t值		4.980	12.180
P值		0.008	0.003
PBS+TE-1	3	37.33 ± 2.08	55.33 ± 2.52
Treg+TE-1	3	61.00 ± 2.65	63.67 ± 3.51
t值		8.104	3.341
P值		0.001	0.029

分组	实验次数	细胞迁移数目（个）	侵袭细胞数目（个）
PBS+Eca-109	3	55.67 ± 5.13	77.33 ± 3.22
Treg+Eca-109	3	74.67 ± 4.16	105.70 ± 2.13
t值		4.980	12.180
P值		0.008	0.003
PBS+TE-1	3	37.33 ± 2.08	55.33 ± 2.52
Treg+TE-1	3	61.00 ± 2.65	63.67 ± 3.51
t值		8.104	3.341
P值		0.001	0.029

分组	实验次数	Eca-109细胞迁移数（个）	Eca-109侵袭细胞数（个）	TE-1细胞迁移数（个）	TE-1侵袭细胞数（个）
对照	3	60.00 ± 3.61	30.00 ± 2.89	59.67 ± 1.52	28.33 ± 1.53
TGF-β1	3	120.30 ± 4.51^a	59.33 ± 2.52^a	119.30 ± 7.02^a	58.00 ± 2.00^a
TGF-β1+SB431542	3	84.67 ± 5.03^ab	45.33 ± 4.04^ab	62.67 ± 5.69^ab	47.33 ± 2.52^ab
F值		141.214	40.643	121.116	160.427
P值		< 0.001	< 0.001	< 0.001	< 0.001

Please choose a citation manager

Content to export