Biological effects of exosome derived from Cal27 on normal human gingival fibroblasts

doi:10.7518/hxkq.2021.03.011

Abstract

Abstract: Objective

The proliferation, migration capacity, and expression of activation-related proteins of NHGFs+Cal27-exo were determined by coculturing Cal27 exosome (Cal27-exo) with normal human gingival fibroblasts (NHGFs) to explore the effects of Cal27-exo on the activation and biological behavior of NHGFs.

Methods

Cal27-exo was extracted using supercentrifugation, and exosomes were identified using Western blot, transmission electron microscopy (TEM), and particle size detection. Cal27-exo was cocultured with NHGFs to detect the uptake of Cal27-exo by NHGFs, and the proliferation and migration capacity of NHGFs+Cal27-exo were detected using CCK8 and wound healing tests, respectively. The expression levels of NHGF activation-related proteins, i.e., matrix metalloproteinase-9 (MMP-9), fibroblast-activating protein (FAP), alpha smooth muscle actin (αSMA), and transforming growth factor-β (TGF-β), were detected using real-time quantitative polymerase chain reaction (qRT-PCR).

Results

Cal27-exo was extracted u-sing supercentrifugation, and Western blot showed the positive expression levels of Alix and CD63. TEM showed that Cal27-exo had a circular double-layer vesicle. The particle size was between 30 and 150 nm. Cal27-exo labeled with PKH67 entered NHGFs after the coculture method. The wound healing test showed that the migration capacity of NHGFs+Cal27-exo was stronger after the scratch compared with that of NHGFs. CCK8 results showed that the proliferation activity of NHGFs+Cal27-exo was enhanced. qRT-PCR results showed that the MMP-9 levels of NHGFs+Cal27-exo were upregulated, whereas the TGF-β and αSMA mRNA levels of NHGFs+Cal27-exo were downregulated (P<0.05).

Conclusion

The proliferation and migration ability of NHGFs+Cal27-exo are enhanced, and the mRNA expression of related proteins is changed. Cal27-exo can activate NHGFs, which suggests that Cal27-exo has potential significance in tumor invasion and metastasis.

Key words: oral cancer, exosome, cell proliferation, cell migration, fibroblast

CLC Number:

R 739.8

Jia Mei’e, Li Zhiyong, Xu Kai, Wang Yiheng, Yu Fei, He Xiangyi. Biological effects of exosome derived from Cal27 on normal human gingival fibroblasts[J]. West China Journal of Stomatology, 2021, 39(3): 313-319.

Figures/Tables 6

Fig 1

Fig 2

Fig 3

Fig 4

Fig 5

Fig 6

References 26

1	Ng JH, Iyer NG, Tan MH, et al. Changing epidemiology of oral squamous cell carcinoma of the tongue: a global study[J]. Head Neck, 2017, 39(2): 297-304.
2	Nilendu P, Sarode SC, Jahagirdar D, et al. Mutual concessions and compromises between stromal cells and cancer cells: driving tumor development and drug resistance[J]. Cell Oncol (Dordr), 2018, 41(4): 353-367.
3	Ding L, Ren J, Zhang DY, et al. A novel stromal lncRNA signature reprograms fibroblasts to promote the growth of oral squamous cell carcinoma via LncRNA-CAF/interleukin-33[J]. Carcinogenesis, 2018, 39(3): 397-406.
4	孟琳, 王丹丹, 布文奂, 等. 口腔鳞癌对牙龈成纤维细胞转化为肿瘤相关成纤维细胞的影响[J]. 口腔生物医学, 2017, 8(3): 136-140.
	Meng L, Wang DD, Bu WH, et al. The influence of oral squamous cell carcinomas on normal fibroblasts turning into oral carcinoma associated fibroblasts[J]. Oral Biomed, 2017, 8(3): 136-140.
5	Jiang EH, Xu Z, Wang M, et al. Tumoral microvesicle-activated glycometabolic reprogramming in fibroblasts promotes the progression of oral squamous cell carcinoma[J]. FASEB J, 2019, 33(4): 5690-5703.
6	Pang WJ, Su JJ, Wang YL, et al. Pancreatic cancer-secre-ted miR-155 implicates in the conversion from normal fibroblasts to cancer-associated fibroblasts[J]. Cancer Sci, 2015, 106(10): 1362-1369.
7	Wu XX, Showiheen SA, Sun AR, et al. Exosomes extraction and identification[J]. Methods Mol Biol, 2019, 2054: 81-91.
8	Routray S, Sunkavali A, Bari KA. Carcinoma-associated fibroblasts, its implication in head and neck squamous cell carcinoma: a mini review[J]. Oral Dis, 2014, 20(3): 246-253.
9	Santi A, Caselli A, Ranaldi F, et al. Cancer associated fi-broblasts transfer lipids and proteins to cancer cells through cargo vesicles supporting tumor growth[J]. Biochim Biophys Acta, 2015, 1853(12): 3211-3223.
10	Silva TA, Smuczek B, Valadão IC, et al. AHNAK ena-bles mammary carcinoma cells to produce extracellular vesicles that increase neighboring fibroblast cell motility[J]. Oncotarget, 2016, 7(31): 49998-50016.
11	Prime SS, Cirillo N, Hassona Y, et al. Fibroblast activation and senescence in oral cancer[J]. J Oral Pathol Med, 2017, 46(2): 82-88.
12	Lim KP, Cirillo N, Hassona Y, et al. Fibroblast gene expression profile reflects the stage of tumour progression in oral squamous cell carcinoma[J]. J Pathol, 2011, 223(4): 459-469.
13	Kalluri R, Zeisberg M. Fibroblasts in cancer[J]. Nat Rev Cancer, 2006, 6(5): 392-401.
14	Kessenbrock K, Plaks V, Werb Z. Matrix metalloprotei-nases: regulators of the tumor microenvironment[J]. Cell, 2010, 141(1): 52-67.
15	Ren ZH, Wu K, Yang R, et al. Differential expression of matrix metalloproteinases and miRNAs in the metastasis of oral squamous cell carcinoma[J]. BMC Oral Health, 2020, 20(1): 24.
16	Zhu X, Wang K, Zhang K, et al. Galectin-1 knockdown in carcinoma-associated fibroblasts inhibits migration and invasion of human MDA-MB-231 breast cancer cells by modulating MMP-9 expression[J]. Acta Biochim Biophys Sin (Shanghai), 2016, 48(5): 462-467.
17	Busek P, Balaziova E, Matrasova I, et al. Fibroblast activation protein alpha is expressed by transformed and stromal cells and is associated with mesenchymal features in glioblastoma[J]. Tumour Biol, 2016, 37(10): 13961-13971.
18	Zhou B, Chen WL, Wang YY, et al. A role for cancer-associated fibroblasts in inducing the epithelial-to-mesenchymal transition in human tongue squamous cell carcinoma[J]. J Oral Pathol Med, 2014, 43(8): 585-592.
19	Ba PF, Zhang XJ, Yu M, et al. Cancer associated fibroblasts are distinguishable from peri-tumor fibroblasts by biological characteristics in TSCC[J]. Oncol Lett, 2019, 18(3): 2484-2490.
20	Rønnov-Jessen L, Petersen OW. A function for filamentous alpha-smooth muscle actin: retardation of motility in fibroblasts[J]. J Cell Biol, 1996, 134(1): 67-80.
21	Moinfar F, Man YG, Arnould L, et al. Concurrent and independent genetic alterations in the stromal and epithe-lial cells of mammary carcinoma: implications for tumorigenesis[J]. Cancer Res, 2000, 60(9): 2562-2566.
22	Pang X, Tang YL, Liang XH. Transforming growth factor-β signaling in head and neck squamous cell carcinoma: insights into cellular responses[J]. Oncol Lett, 2018, 16(4): 4799-4806.
23	Li YY, Zhou CX, Gao Y. Interaction between oral squamous cell carcinoma cells and fibroblasts through TGF-β1 mediated by podoplanin[J]. Exp Cell Res, 2018, 369(1): 43-53.
24	Limoge M, Safina A, Beattie A, et al. Tumor-fibroblast interactions stimulate tumor vascularization by enhancing cytokine-driven production of MMP9 by tumor cells[J]. Oncotarget, 2017, 8(22): 35592-35608.
25	Salido-Guadarrama I, Romero-Cordoba S, Peralta-Zaragoza O, et al. MicroRNAs transported by exosomes in body fluids as mediators of intercellular communication in cancer[J]. Onco Targets Ther, 2014, 7: 1327-1338.
26	Vella LJ. The emerging role of exosomes in epithelial-mesenchymal-transition in cancer[J]. Review Front Oncol, 2014, 4: 361.

[1]	Lu Ting, Zhu Jiahao, Yang Shihe, Shen Zhe, Zhong Liangjun. Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment [J]. West China Journal of Stomatology, 2023, 41(3): 269-275.
[2]	Wu Hao, Li Ying, Wang Yuzhuo, Yu Jize, Bao Xingfu, Hu Min. Effect of recombinant human fibroblast growth factor 21 on the mineralization of cementoblasts and its related mechanism [J]. West China Journal of Stomatology, 2023, 41(2): 140-148.
[3]	Hou Yali, Li Hexiang, Song Peng, Yang Yanxiao, Hao Yali, Liu Huijuan. Effect of tumor-stromal fibroblasts on the biological behavior of salivary gland pleomorphic adenoma cells in vitro [J]. West China Journal of Stomatology, 2023, 41(2): 149-156.
[4]	Tan Zhongjuan, Luo Yuanyuan, Yang Li. Basic fibroblast growth factor/chitosan derivatives/collagen composite thermosensitive hydrogel promotes perio-dontal tissue regeneration in rats [J]. West China Journal of Stomatology, 2023, 41(1): 21-28.
[5]	Dai Zhenning, Zheng Weihan, Li Shiyu. Receptor activator of nuclear factor-κB ligand and tumor necrosis factor-α promotes osteoclast differentiation through the exosomes of inflammatory periodontal ligament stem cells [J]. West China Journal of Stomatology, 2022, 40(4): 377-385.
[6]	Chen Jiao, Die Lü, Chen Hongli, Zhang Ping.. Effect of protein kinase D inhibitor CRT0066101 on the cell migration of salivary adenoid cystic carcinoma [J]. West China Journal of Stomatology, 2022, 40(3): 320-327.
[7]	Lu Qian, Guo Liumei, Bi Xiaoqin. Systematic review of risk factors of postoperative dysphagia in patients with oral cancer [J]. West China Journal of Stomatology, 2022, 40(3): 328-334.
[8]	Li Ruohan, Huang Yingzhao, Liao Nailin, Wu Chenzhou, Li Yi.. Oral cancer cells affect pancreatic β-cell function through transmissible endoplasmic reticulum stress [J]. West China Journal of Stomatology, 2022, 40(1): 22-31.
[9]	Zhang Yuwei, He Yuxuan, Ding Yi, Liu Chengcheng.. Porphyromonas gingivalis upregulates calbindin 1 and thus promotes the proliferation of gingival epithelial cells [J]. West China Journal of Stomatology, 2022, 40(1): 93-99.
[10]	Gao Yongqiang, Shi Pengwei, Shi Wenkai, Liu Yiming. Expression and mechanism of long non-coding RNA HCG22 in oral squamous cell carcinoma [J]. West China Journal of Stomatology, 2021, 39(6): 658-666.
[11]	Dai Xiaohua, Wang Guanhua, Lian Xiaoli, Yan Yan, Wang Yue, Zou Huiru, Liu Hao.. Effect of acidic culture conditions on the proliferation, apoptosis, and migration ability of human tongue squamous cell carcinoma cells and its related mechanism [J]. West China Journal of Stomatology, 2021, 39(5): 540-546.
[12]	Jin Xiaolan, Zhang Yanan, Sun Chengrui, Zou Zhaohui. Protective effect of low-level laser irradiation on lipopolysaccharide-mediated inflammatory injury of human periodontal ligament fibroblasts [J]. West China Journal of Stomatology, 2021, 39(3): 260-266.
[13]	Wu Baoqin, Li Chunhui, Zhang Menglian, Nie Minhai. microRNA-1 gene delivery mediated by exosomes suppresses CAL-27 cell proliferation [J]. West China Journal of Stomatology, 2021, 39(2): 136-142.
[14]	Liu Xiang, Kang Wenyan, Shang Lingling, Ge Shaohua. Sitagliptin inhibits lipopolysaccharide-induced inflammatory response in human gingival fibroblasts by blocking nuclear factor-κB signaling pathway [J]. West China Journal of Stomatology, 2021, 39(2): 153-163.
[15]	Xia Xiaoyang, Fang Fei, Liu Yan, Che Chao, Ke Jinjuan, Jiang Shengjun. Expression of cyclophilin A in oral squamous cell carcinoma and its effect on cell proliferation and invasion [J]. West China Journal of Stomatology, 2021, 39(2): 164-169.