TNF-α stimulated exosome derived from fibroblast-like synoviocytes isolated from rheumatoid arthritis patients promotes HUVEC migration, invasion and angiogenesis by targeting the miR-200a-3p/KLF6/VEGFA axis

Figures & data

Figure 1. Content of IL-6 in culture supernatant of FLS and characteristics of exosomes derived from FLS induced by TNF-α or PBS were quantified and characterised by nanoparticle characterization instrument, transmission electron microscope and Western blot. (A) The changes of content of IL-6 in culture supernatant of FLS stimulated by PBS or TNF-α for 48h. (B) Transmission electron microscope pictures of exosome. (C) Particle size distribution of exosomes. (D) The protein of CD9, CD63 and TSG101 in exosome were examined by Western blot. **p < 0.01.

Figure 2. Exosome derived from FLS stimulated by TNF-α promoted invasion and migration of HUVECs. Transwell invasion assay was utilised to assess the invasion ability of HUVECs after different treatment (a); the column presented the relative invasion HUVECs (B); wound healing assay was utilised to assess the migration ability of HUVECs after different treatment (C); the column presented the migration ratio of HUVECs (D). **p < 0.01.

Figure 3. Exosome derived from FLS stimulated by TNF-α promoted angiogenesis of HUVECs. The angiogenesis assays were detected by tube formation assay after different treatment (a); the columns presented the number of total loops (B), tube length (C), and total branching points (D). *p < 0.05.

Table 1. Differentially expressed miRNA in HUVECs and exosome.

	HUVECs			exosome
microRNA_ID	logFC (TNF/PBS)	Pvalue (TNF/PBS)	Qvalue (TNF/PBS)	logFC (TNF/PBS)	Pvalue (TNF/PBS)	Qvalue (TNF/PBS)
hsa-miR-1246	1.857309863	0.001521267	0.068239696	4.968773442	0.008672444	0.089958228
hsa-miR-200a-3p	1.104474994	0.011500491	0.319526725	4.008798516	2.25E-20	1.38E-17
hsa-miR-30a-3p	−0.579654845	0.017988094	0.444300775	−0.78515269	0.030665481	0.21822412
hsa-miR-99b-3p	−1.012746146	0.003937364	0.154541536	−1.153136397	0.018328433	0.151581098

Table 2. Differentially expressed mRNA in HUVECs.

Gene Symbol	PBS Average TPM	TNF Average TPM	PBS Average FPKM	TNF Average FPKM	PBS Average Read Count	TNF Average Read Count	logFC (TNF/PBS)	Pvalue (TNF/PBS)	Qvalue (TNF/PBS)
IL6R	3.98	16.54666667	7.936666667	32.72666667	763	2610.783333	2.194081394	1.43E-283	6.52E-281
EPHA2	100.4766667	417.5533333	200.5566667	825.2433333	20134.16	64885.22333	2.107294855	0	0
KLF6	64.32666667	18.04333333	127.25	36.00333333	10853	3918.666667	−1.888964308	0	0
NAMPT	7.753333333	25.86666667	15.45	51.13666667	1716.666667	4476	1.803161981	3.78E-216	1.02E-213
PRKAB1	5.223333333	15.36	10.42666667	30.35333333	576.04	1294.333333	1.587373242	1.34E-93	1.13E-91

Figure 4. The expression level of miR-200a-3p. The expression level of miR-200a-3p derived from FLS stimulated by PBS or TNF-α were detected by qRT-PCR (a); the expression level of miR-200a-3p in HUVECs after different treatment (B). *p < 0.05, **p < 0.01.

Figure 5. Exosome derived from FLS stimulated by TNF-α decreased the expression of KLF6 and promoted the level of VEGFA in HUVEC. KLF6 (A) and VEGFA (B) were detected by qRT-PCR; Western blot assay was used to detect the proteins expression of KLF6 and VEGFA (C); the columns presented the relative expression of KLF6 (D) and VEGFA (E). *p < 0.05, **p < 0.01.

Figure 6. KLF6 is the target of miR-200a-3p. The 3′UTR of KLF6 mRNA has a base site that binds to miR-200a-3p (a); the dual luciferase activity experiment was used to confirm the targeting relationship between miR-200a-3p and KLF6 in HUVECs. ** p < 0.01.