MiR-339-3p aggravates rat vascular inflammation induced by AT1R autoantibodies by down-regulating BKα protein expression

The abnormality of large-conductance calcium-activated potassium channels (BK channels) is an important factor in inducing vascular inflammation. BK channel agonists can readily recover BK channel function and improve vascular inflammation. However, it is not clear how to improve BK dysfunction caused by downregulation of BK channel protein expression. This study found that angiotensin II-1 receptor autoantibodies (AT1-AA), which are widely present in the body of various types of cardiovascular diseases, can down-regulate the expression of BK channel protein and induce vascular inflammation. Further research found that the elevated neural precursor cells expressed developmentally downregulated 4-like (NEDD4L) protein level is involved in the down-regulation of BK channel α subunit (BKα) protein level by AT1-AA. Bioinformatics analysis and experiments have confirmed that miR-339-3p plays an irreplaceable role in the high expression of NEDD4L and the low expression of BKα, and aggravates the vascular inflammation induced by AT1-AA. Overall, AT1-AA increased miR-339-3p expression (targeting BKα via the miR-339-3p/NEDD4L axis or miR-339-3p alone), reduced BKα protein expression in VSMCs, and induced vascular inflammation. The results of the study indicate that miR-339-3p may become a new target for reversing vascular inflammation in AT1-AA-positive patients.

The results of each sample were tested three times.  To verify the effective combination of miR-339-3p and the NEDD4L 5'UTR and evaluate the effect 206 14 of miR-339-3p on NEDD4L expression, we constructed a psi-check 2-NEDD4L-5'UTR plasmid 207 (Suppl. Figure 4B), and it was cotransfected with miR-339-3p mimic into HEK293A cells. The results 208 of the dual luciferase reporter assay showed that compared with psi-check 2, miR-339-3p mimics and 209 psi-check 2-NEDD4L-5'UTR cotransfection significantly increased the luciferase activity ( Figure.  suggesting that miR-339-3p was an important mechanism of AT1-AA-induced changes in NEDD4L 216 protein levels. 217 To verify the effective binding of miR-339-3p to the BKα 3'UTR and evaluate whether miR-339-218 3p has a direct effect on the expression of BKα, we also constructed a psi-check 2-BKα-3'UTR 219 plasmid (Suppl. Figure 4C) and cotransfected the plasmid with miR-339-3p mimic into HEK293A 220 cells. The results showed that miR-339-3p mimic and psi-check 2-BKα-3'UTR cotransfection 221 The effective binding of miR-339-3p and the BKα 3'UTR was proven by the luciferase reporter gene 244 method, n=6. (K) Western blot was used to detect the changes in BKα protein levels after 245 overexpression/inhibition of miR-339-3p, and (L) the inhibition of miR-339-3p before treatment with 246 AT1-AA was used to detect BKα protein levels, n=6. The results of each sample were tested three 247 times. 248

Inhibition of miR-339-3p can reverse vascular inflammation induced by AT1-AA in vivo 249
We tried to use antagomir-339-3p to reverse the decrease in BKα protein levels and vascular 250 17 inflammation in AT1-AA-positive rats. First, to prove the effectiveness of antagomir-339-3p, VSMCs 251 were transfected with antagomir-339-3p, and miR-339-3p expression in VSMCs was significantly 252 reduced (Suppl. Figure 5A). Similarly, antagomir-339-3p successfully inhibited the expression of 253 miR-339-3p in SD rats through tail vein injection ( Figure.   that after the AT1-AA-positive rats were injected with antagomir-339-3p, the thickness of the thoracic 261 aortic wall of the rats was significantly improved compared with the rats in the AT1-AA group (Suppl. 262 Figure 5B). In addition, from recorded arterial blood pressure data of these model rats, we found that 263 antagomir-339-3p can alleviate the increase in systolic and diastolic arterial blood pressure caused by 264 AT1-AA (Suppl. Figure 5C). The above results suggested that inhibiting miR-339-3p can significantly 265 reverse the vascular inflammation induced by AT1-AA. The results of each sample were tested three times. 277

Discussion 278
In general, VSMCs play an important role in the inflammatory process of blood vessel walls. The 279 role and mechanism of VSMCs in the inflammatory process of the aortic wall have not been fully 280 elucidated. Once the body has an inflammatory response, blood vessels serve as conduits through 281 various tissues and organs, which can cause more extensive pathological changes when inflamed, and 282 deliver the produced inflammatory factors to various parts of the body [19]. In this study, we 283 investigated the mechanism of AT1-AA-induced inflammation in the vascular wall, mainly in VSMCs,  small conductance (SKCa) types. Among these channels, BKCa is expressed mainly in VSMCs [32]. 317 BKCa channels (BK channels) are composed mainly of α subunits that form pores and auxiliary β 318 subunits that regulate channel Ca 2+ sensitivity, activity and structure, and they play an important role 319 in regulating physiological processes, including smooth muscle tension and neuronal excitability [33]. 320 Studies have shown that blocking BK channel function can promote the occurrence and development 321 of vascular inflammation, and the vascular inflammation caused by ischaemia-reperfusion is partially 322 reversed after activation of the BK channel induced by NS1619 [5]. Our group previously found that 323 AT1-AA can downregulate the function of BK channels and damage blood vessels [34]. In this 324 experiment, we pretreated VSMCs with the BK channel agonist NS1619 to upregulate the function of 325 the BK channel and then treated them with AT1-AA. We found that NS1619 indeed upregulated the 326 activity of the BK channel (Suppl. Figure 2B) and partially reversed the vascular inflammation 327 induced by AT1-AA (Suppl. Figure 2C decreasing BKα, we established a negative IgG group, an unrelated antibody group, namely, the β1 334 adrenergic receptor autoantibody (β1-AA) group, and a positive group (Ang II), and found that only 335 22 AT1-AA can lower BKα protein levels (Suppl. Figure 2D). 336 To confirm the role of BKα in AT1-AA-induced vascular inflammation, we observed a significant 337 increase in inflammatory cell infiltration in the thoracic aorta of BKα-knockout rats. However, we 338 observed that the changes in systolic and diastolic arterial blood pressure in 5-month-old BKα-339 knockout rats were not obvious (Suppl. Figure 2E). Although vessels without BK channels may 340 increase contractility due to calcium flow in VSMCs, we also found that the heart function of BKα-341 knockout rats was damaged, leading to cardiac contractility and ejection dysfunction. We speculate 342 that this may be the reason why there was no significant change in arterial blood pressure in BKα-343 knockout rats. However, after BKα overexpressing adenovirus was injected into the tail vein of AT1-344 AA-positive rats, it was detected that the thoracic aortic wall thickness and arterial blood pressure of 345 AT1-AA-positive rats had a reversal effect (Suppl. Figure 2L  Suppl. Figure 3B), and therefore the degradation of the BK channel ubiquitination pathway was the 366 focus of the present study. 367 The ubiquitin process is a three-enzyme cascade catalytic process that consists of E1-ubiquitin 368 activating enzyme, E2-ubiquitin-binding enzyme and E3-ubiquitin ligase. The interaction between the 369 E3-ubiquitin ligase and target protein is the core step of ubiquitin-mediated protein degradation [41]. 370 In the whole process, the main function of ubiquitin is to mark proteins that need to be decomposed so 371 that they can be hydrolysed. In addition to labelling proteins present in the cytoplasm, ubiquitin can 372 also label transmembrane proteins and remove them from the cell membrane [42]. In this experiment, 373 we screened and identified different ubiquitin-related proteases directly connected to BKα through 374 protein profiling methods on the vehicle group and the VSMCs treated with AT1-AA. The data were 375 analysed by GO, and 32 proteins were found in the "protein binding" category after classification 376 according to molecular function. The biological effects of these proteins were queried by the UniProt 377 database (https://www.UniProt.org/), and only one of them was related to protein degradation (Suppl. 378

Establishment of model animals 416
Two-hundred-gram 8-week-old male Sprague-Dawley rats were used in the experiment. AT1R-417 ECII (0.4 μg/g) was injected subcutaneously into the back neck every two weeks to complete active 418 immunization, which lasted for three months. Ad-BKα-GFP (1×10 10 pfu/kg) and antagomir-339-3p 419 (20 nmol/rat) injection started in the 6 th week of active immunization, and intravenous injection into 420 the rat tail was administered every two weeks until the end of active immunization. All animals used 421 in the experiment were approved by the Animal Protection Ethics Committee of Capital Medical 422

Dual-luciferase reporter assay 500
The test was performed according to the operating instructions of the dual luciferase activity test kit 501 (Vazyme Biotech, DL101-01, USA). The reagents in the kit were diluted and prepared in advance. 502 After washing the cells with PBS, 100 μl diluted lysis buffer was added to each well, and the culture 503 plate was shaken with a shaker for 15 min at room temperature. The lysate was centrifuged, 20 μl 504 supernatant was added to each well of the test plate, 100 μl Luciferase Assay Buffer II (LAR II) was 505 added to detect firefly luciferase activity, and 100 μl stop solution was added to detect Renilla 506 luciferase activity. The ratio of the fluorescence intensity of fireflies to the fluorescence intensity of 507 Renilla reflects the relative fluorescence value of each group. 508

Selection of AT1-AA monoclonal antibody cell line 509
The hybridoma cells were made from the human AT1R extracellular second loop sequence. The 510 30 hybridoma cells in good condition and able to bind to the extracellular second loop of AT1R were 511 injected into the abdomen of mice to produce ascites. After AT1-AA extraction, the purity and activity 512 of the resulting ascites were detected, including the detection of antibody light and heavy chain (Suppl. 513 Figure 1D), increased beating of neonatal rat cardiomyocytes (Suppl. Figure 1E) and vascular ring 514 detection of vasoconstriction (Suppl. Figure 1F). Finally, select the cell line that allows mice to 515 produce active AT1-AA for follow-up research. 516

Preparation of monoclonal AT1-AA 517
The selected hybridoma cells are cultured, and monoclonal AT1-AA is obtained from the culture 518 supernatant. Hybridoma cells were grown in 1640 RPMI medium containing 8% fetal bovine serum. 519 Select hybridoma cells with good growth status and suitable growth rate, and collect the culture 520 supernatant by centrifugation. The culture supernatant of hybridoma cells was filtered with 0.45 μm 521 filters, and the IgG in the culture supernatant of hybridoma cells was purified with a protein G affinity 522 chromatography column, which is the AT1-AA required for our experiment. 523

Isolation and identification of circulating extracellular vesicles in rats 524
Ultracentrifugation is the most commonly used method for purification of extracellular vesicles. 525 Use low-speed centrifugation and high-speed centrifugation to alternately separate vesicles of similar 526 size. The whole process includes centrifugation at 300 g for 10 min, centrifugation at 2000 g for 10 527 min, and centrifugation at 10,000 g for 30 min. So far, the supernatant has been retained at each step. 528 Finally, the supernatant was collected, centrifuged at 100,000 g for 70 min, and centrifuged twice to 529 collect the precipitate. The precipitate was an extracellular vesicle. The extracted extracellular vesicles 530 were observed under electron microscope, the particle diameter was detected by Nanoparticle 531 Tracking Analysis (NTA), and the surface markers CD9, CD81 and calnexin of the extracellular 532 31 vesicles were detected to identify the isolated particles as extracellular vesicles. 533

Statistical Analysis 534
We used GraphPad Prism 8 and SPSS 26 software to draw and analyse the data. All the data are 535 presented as the mean ± standard error (SEM). The differences in normally distributed data were 536 analysed using independent sample t-tests (two groups) or one-way ANOVA (> 2 groups). Pearson test 537 is used for correlation analysis. A value of p < 0.05 was considered statistically significant. 538