miR-99a-3p Targeting EIF4EBP1 Affects B Lymphocytes Function Through Autophagy and Aggravates SLE Disease Progression

Background:Overactivation of immune cells plays a key role in the pathogenesis of systemic lupus erythematosus (SLE). The regulation of immune cells by miRNA is a research hotspot.In this study, the second-generation high-throughput sequencing found that the expression of miR-99a-3p in SLE decreased, but the specic mechanism is still unclear.The purpose of this study is to explore the potential target genes, target cells of miR-99a-3p and their potential mechanisms affecting the progression of SLE. Methods: Isolate PBMC from healthy individulas and SLE patients, transfect Ball-1, Jurkat, THP-1 and K562 cells with miR-99a-3p agomir and antagomir,detect miR-99a-3p, predict target genes and autophagy pathway mRNA and protein expression by RT-qPCR and Western blotting.CCK-8 method detects cell proliferation, PI method detects cell cycle, ow cytometry detects cell apoptosis, and luciferase reporter gene experiment determines miR-99a-3p target genes.With C57BL/6J mice as control,construct miR-99a-3p overexpression and interference model based on MRL/lpr mice,ELISA detects plasma ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS.Pathological analysis of HE staining and C3 immunouorescence(IF) deposition in mouse kidney tissue,Immunohistochemistry(IHC) detects changes in target genes and pathway proteins in kidney tissue,isolate B cells to verify the differential expression of miR-99a-3p, target genes, pathway mRNA and protein. Results: detection proved that miR-99a-3p directly targets EIF4EBP1. Rescue experiments conrmed the interaction model between miR-99a-3p and EIF4EBP1. Clinical, in vitro, and in vivo experiments further conrmed that miR-99a-3p agomir can reduce the expression of EIF4EBP1, LC3-(cid:0), and LAMP-2A, while miR-99a-3p antagomir had the opposite effect.In vivo experiment antagomir group mice serum ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS were higher than those in the MRL/lpr group, EIF4EBP1, LC3-(cid:0), LAMP-2A mRNA, protein and IHC levels also increased, and the urinary protein and C3 IF deposition of mice in the antagomir group were increased, and the related indexes of mice in the agomir group were lower than those in the MRL/lpr group. SLE blue method to detect the changes of 8w, 10w, 12w, 13w urine protein after intervention of MRL/lpr and C57 mice(cid:0)C(cid:0)HE staining of glomerular area after intervention of MRL/lpr and C57 mice(cid:0)D(cid:0)HE staining of MRL/lpr and C57 mice after intervention (×2.5,×400)(cid:0)E(cid:0)The C3 IF of MRL/lpr and C57 mice after intervention (×400, scale 50um). Green uorescence was C3 positive staining, blue uorescence was DAPI nuclear staining, and Merge uorescence was the image after fusion of C3 and DAPI(cid:0)F(cid:0)C3 IF after intervention of MRL/lpr and C57 mice(cid:0)G(cid:0)ELISA detected ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS expression after intervention in MRL/lpr and C57 signicant(cid:0)


Introduction
Systemic lupus erythematosus (SLE) is a type of autoimmune-mediated diffuse connective tissue disease that involves multiple systems throughout the body, characterized by pathological in ammation [1]. Studies had shown that genetic factors in uence the clinical phenotype and progression of SLE, while environmental factors trigger the occurrence of SLE [2], and ultraviolet rays play an important role in stimulating SLE [3]. The age of onset of SLE in high altitude areas is lower, the course of disease is shorter. The proportion of patients with Anti-Sm positive, anemia, thrombocytopenia and elevated serum creatinine is signi cantly higher than that in low altitude areas [4].
The main ethnic group in the Diqing Tibetan Autonomous Prefecture in Yunnan Province is the Tibetans, they live in a high-altitude, low-oxygen, high-cold, dry and strong ultraviolet environment, relatively isolated, lack of gene exchange with the outside world. The epidemiological and transcriptomics characteristics of Tibetan SLE patients deserve in-depth and systematic research.
MicroRNAs (miRNAs) are a class of highly conserved endogenous non-coding single-stranded small RNAs with a length of about 21-25 nucleotides. More and more miRNAs had been found to play an important role in the pathogenesis of SLE [5]. Our research group found that UVB may be involved in the pathogenesis of SLE by decreasing miR-125b-5p of SLE and increasing the expression of target gene UVRAG and cell autophagy [6].
We collected venous blood from 10 Tibetan SLE patients and 10 healthy Tibetans. The miRNA was differentiated by high throughput sequencing of the second generation after RNA extraction (Fig. 1a). The sequencing results were veri ed by RT-qPCR, and it was found that miR-99a-3p in Tibetan SLE patients decreased signi cantly ( Fig 1B).
As a member of the miR-99 family, miR-99a-3p is transcribed from the 21 region of the long arm of chromosome 21. miR-99 is expressed at low levels in a variety of human malignant tumors. It participates in the occurrence and development of a variety of urinary tumors [7], head and neck squamous cell carcinoma [8], liver cancer [9] and ovarian cancer [10], and has certain signi cance in the early diagnosis and staging of tumors [11].
Pradhan and Tomankova were the rst to nd that the expression of miR-99 in SLE decreased [12]. Jin et al found that the expression of miR-99a in South Korean SLE patients was down-regulated by miRNA PCR chip detection [13]. Frangou et al used cDNA microarrays to compare the gene expression in the effector cells and target tissues of SLE patients and control individuals, and found that miR-99a expression in SLE PBMC decreased, and it was related to the regulation of type I IFN pathway [14].There is no report on the expression of miR-99a-3p in SLE in the Chinese population.
Therefore, this study explores the role of miR-99a-3p in the pathogenesis of SLE. This study provides help to clarify the complex mechanisms involved in the pathogenesis of SLE and new targets for SLE. Take the frozen Ball-1, Jurkat, THP-1 and K562 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) from the liquid nitrogen tank and dissolve them in a 37℃water bath, centrifuge to discard the supernatant, and add 1640 complete medium (containing 1% Double antibody +10%% FBS, Thermo, Germany) to resuspend and culture.
PI method to detect cell cycle Take 250ul (1x10 6 cells) cell suspension, add 750ul of pre-cooled absolute ethanol, seal and place at -20℃for xation overnight; add 500ul PI staining solution (Becton, Dickinson and Company, USA) to a nal concentration of 65ug/ml, Incubate at 37℃for 30 minutes, and immediately perform ow cytometric detection (Thermo, Germany

Dual-Luciferase Reporter Assay
The miR-99a-3p target genes were predicted, and EIF4EBP1, NCAPG, IKBKB, PRKCB were selected for luciferase detection. The related target gene 3'UTR Wild/Mutant type luciferase vector pmirGLO Vector was constructed by Wuhan GeneCreate Company. Take miRNA and plasmid 2ug incubated for 5min, and add 2ul lipo2000 transfection reagent to culture medium for incubation. When the cell fusion degree reaches 80%, add 500ul of the above transfection complex, add the extracted protein to 100ul of re y luciferase substrate, measure its re y luciferin value, add Renilla luciferase substrate, and measure the sea level. Kidney uorescence value. The renilla uorescence value of each well divided by the re y uorescein value was the uorescence value of the reporter plasmid in each well.

Western-blotting
Add 250ul RIPA lysis buffer (containing protease inhibitor, Thermo, Germany) to each group of cells to be tested, and BCA protein quanti cation kit (P0010, Beyotime, China) to determine the protein concentration. Take 30ug of total protein for SDS-PAGE electrophoresis, and block after transfer to membrane. Add the primary antibodies EIF4EBP1 (1:1000, GTX133182, GeneTex, USA), LAMP-2A (1:1000, ab24170, Abcam, USA), LC3B (1:1000, ABS1513, Millipore, USA), β-Actin (LMAI Bio,Shanghai, China), shake overnight at 4℃, rewarm for one hour the next day, add secondary antibody (Peroxidase labeled goat anti-rabbit IgG, 1:5000, Sigma, USA), incubate for 30min, exposure instrument (Monad, Suzhou, China), add ECL color developing solution (Thermo, Germany) to take pictures,use Image-Pro Plus 6.0 software to analyze the optical density of the stripe, the optical density ratio of the target protein and the light density ratio of the endoprotein β-actin represents the relative content of the target protein to compare the difference in protein expression.
SiRNA and antagomir jointly intervene in Ball-1 cells for Rescue experiment siRNA EIF4EBP1 was synthesized by Guangzhou Ruibo Biological Company, and transfected Ball-1 cells with siEIF4EBP1-1, siEIF4EBP1-2, and siEIF4EBP1-3. After 48 hours, RT-qPCR and Western-blotting were detected. It was found the expression of EIF4EBP1 in the siEIF4EBP1-1 group was lower than siNC, the difference was statistically signi cant (sequence and screening see supplementary material Table3, Fig  2). Collect Ball-1 cell count and plate, add siRNA EIF4EBP1 and NC respectively, and then add lipo2000 transfection reagent. After 24h, half of the sample was separated from the siRNA tube and added miR-99a-3p antagomir for further culture. After incubation at 37℃for 48 hours, proliferation, cycle, apoptosis and Western blotting were detected.
Eyeball venous blood was collected, and B lymphocytes were separated by immunomagnetic bead method, followed by Western-blotting, RT-qPCR , and plasma retention for ELISA . Mice were sacri ced by neck breaking method, and the kidneys were removed in layers and placed in 4% paraformaldehyde (P0099, Beyotime, China) for xation. The experimental protocol was approved by the Animal Research Committee of Kunming Medical University (kmmu2021724).
Coomassie brilliant blue method for quantitation of total protein in the urine Take appropriate amount of standard dilution, and take appropriate amount of urine to be measured, PBS equal multiple dilution, add 5 ml dilution of the Coomassie brilliant blue solution (Xinfan Biological Biological Technology Co., Shanghai, China), the color changes from red to blue , the absorbance was determined at 595 nm.
ELISA detects ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS Take out the ELISA kit (JL12477-96T, Jiang Lai Bio, China) slats, add 100μL of HRP-labeled antibody, incubate at 37℃for 60min, wash the plate; add substrate A and B 50μl to each well , then incubate at 37℃in dark incubation and add the stop solution. Measure the OD value of each well at 450nm wavelength, and calculate the sample concentration by the absorbance value of the sample and the standard curve.

HE staining
Take the longitudinally sectioned kidney tissues of each group and x them in paraformaldehyde for 24 hours, and then put them in a low-to-high concentration ethanol solution for dehydration, transparency, wax immersion, embedding, and cut into slices with a thickness of about 4um, and bake at 64℃for 30 minutes. After dewaxing with xylene, put the slides in the ethanol solution of high concentration to low concentration to gradually hydrate, counterstain with hematoxylin (C0105-1, Beyotime, China) for 4 minutes, rinse with distilled water, and put it into alcohol-hydrochloric acid solution for differentiation, return to blue in tap water for 20 minutes, stain with eosin (C0109, Beyotime, China) for 10 seconds, and wash with distilled water. Finally, use ethanol dehydration, transparent xylene and neutral gum to mount the slides, take pictures with a microscope (Lab.A1, ZEISS, Germany) and analyze the staining results.

Immunohistochemical staining
Prepare slices, bake slices, and dewax.After hydration with gradient alcohol, add 0.01M citric acid buffer and boil for 15 minutes to restore the antigen, block with 5% BSA (LMAI Bio, Shanghai, China) at room temperature for 30 minutes, add primary antibody EIF4EBP1 (1:100, GTX133182, GeneTex, USA), LAMP-2A (1:100, ab24170, Abcam, USA), LC3B (1:100, ABS1513, Millipore, USA) overnight at 4℃, dropwise reaction Incubate with the enhancement solution, add the secondary antibody (Sheep anti-mouse, A21235, Invitrogen) and incubate at 37℃for 30 minutes, add the DAB (ZLI-9019, Zhongshan Golden Bridge, China) dye solution dropwise to the tissue block for 5 minutes, and place the slide on the Stained in hematoxylin for 4 minutes, and the tap water turned blue for 20 minutes; dehydrated, transparent, and photographed after mounting, scanning the positive area and calculating the positive rate.First score according to the staining intensity: 0 is divided into colorless, 1 is divided into light yellow, 2 is divided into brown and 3 is divided into brown, and the staining intensity needs to be compared with the background coloring;Then score according to the percentage of positive cells: 0 is negative, 1 is positive cells≤25%, 2 is 25%-50%, and 3 is >50%.
Tissue and cell immuno uorescence staining Prepare slices, bake slices, dewax, and put them in the repair solution after hydration with gradient alcohol; draw circles with oily brushes, add diluted serum, Anti-C3 antibody (1:100, ab11887, Abcam, USA) incubate overnight and then add secondary antibody Goat Anti-Rabbit IgG H&L (AmyJet Scienti c, Wuhan, China), drop DIPA (Weifang Bincheng Chemical Industry) dyeing solution.Put it under a uorescence microscope (Mshot, Guangzhou, China), and take photos of a total of 5 elds of view in the center and surrounding areas of each lm, and calculate the positive rate.

Statistical methods
The data were expressed as Mean±SD, analyzed by ANOVA and LSD-t test, and statistically analyzed by SPSS 23.0 software. Two tailed P<0.05 was statistically signi cant. The correlation analysis used Spearman's rank correlation analysis, the count data used the χ 2 test. GraphPad Prism 6.0 is used for statistical analysis of histograms.

Results
Differential veri cation of miR-99a-3p in SLE The venous blood of Tibetan SLE patients and healthy individulas was collected, and RNA was extracted and then subjected to second-generation high-throughput sequencing. RT-qPCR veri ed the sequencing results which shows miR-99a-3p in Tibetan SLE patients had decreased signi cantly (Fig 1A, B).
RT-qPCR detected miR-99a-3p in PBMCs of 10 Tibetan&Han SLE patients and 10 Tibetan&Han healthy individulas. The miR-99a-3p in both Tibetan and Han SLE patients was decreased compared with the healthy controls, with statistical signi cance (Fig 1C and D).
After Ball-1, Jurkat, THP-1 were transfected with miR-99a-3p agomir, the apoptosis rate was signi cantly higher than that of NC group; Ball-1, Jurkat were transfected with miR-99a-3p antagomir, the apoptosis rate was signi cantly lower than that of NC group, and the difference was statistically signi cant.After K562 transfection,the apoptosis rate of agomir and antagomir groups did not change signi cantly compared with NC group (Fig 2C).
After Ball-1 and THP-1 transfected miR-99a-3p agomir, the number of cells in G0/G1 phase was higher than that of NC group; Ball-1 and THP-1 transfected miR-99a-3p antagomir , the number of cells in G0/G1 phase was lower than that of NC group, and the difference was statistically signi cant.
After Ball-1 transfected miR-99a-3p agomir, the number of cells in G2/M and S phase decreased compared with NC group;after transfected miR-99a-3p antagomir, the number of cells in G2/M and S phase was higher . After Jurkat transfected miR-99a-3p antagomir, the number of cells in G2/M phase was lower than that in NC group (P<0.05). After K562 transfected, the agomir and antagomir groups had no signi cant changes in each cycle compared with the NC group ( Fig 2D). The cell proliferation, apoptosis and cycle changes of the Ball-1 line were relatively stable after transfected miR-99a-3p agomir, antagomir and NC.
Target gene con rmation The target genes of miR-99a-3p were predicted by database TarBase RT-qPCR detected the expression of EIF4EBP1, NCAPG, IKBKB, and PRKCB in the PBMC of Han SLE patients and Han healthy individulas. The expression of related genes was up-regulated in the PBMC of SLE patients, and the difference was statistically signi cant ( Fig 3A).
Ball-1 cells were transfected with miR-99a-3p agomir, antagomir, and NC, and the target gene expression was detected by RT-qPCR 48h after transfection. Compared with NC group, the target genes all decreased after miR-99a-3p agomir transfection.The target genes all increased after miR-99a-3p antagomir transfection, and the difference was statistically signi cant ( Fig 3B).
Western blotting further veri ed the expression of target genes in SLE and healthy individulas. The expression of EIF4EBP1 and NCAPG in SLE increased, and the difference was statistically signi cant ( Fig   3E).
miR-99a-3p participates in autophagy signaling pathway through target genes RT-qPCR detected the expression of LC3-and LAMP-2A in the PBMC of SLE and healthy individulas. SLE was higher than that of healthy control group, and the difference was statistically signi cant ( Fig 4A). Western blotting analyzed SLE and healthy individual PBMC autophagy pathway marker protein LC3and LAMP-2A.The ratio of LC3-and LAMP-2A in SLE was higher than that of healthy individulas (P=0.031, Fig 4B).
Fluorescence microscope was used to observe the IF of LC3-after transfection. The IF of LC3-in the miR-99a-3p agomir group in Ball-1 and B cells was lower than that in NC group. The LC3-II IF of the miR-99a-3p antagomir group was higher than that of NC group (Fig 4F).

Ball-1 function changes after Rescue experiment
Western blotting was used to detect the expression of EIF4EBP1, LC3-, LAMP-2A after siNC, siEIF4EBP1, siEIF4EBP1+antagomir transfected Ball-1. After transfection with siEIF4EBP1, Ball-1's EIF4EBP1, LC3-, LAMP-2A protein expressions were all lower than that of the siNC group. After transfection with siEIF4EBP1+antagomir, the protein expressions of EIF4EBP1, LC3-, and LAMP-2A in Ball-1 were higher than those in the siEIF4EBP1 group, and the difference was statistically signi cant (Fig 5A).
Flow cytometry was used to detect the difference in cell apoptosis after transfection of Ball-1. After Ball-1 was transfected with siEIF4EBP1, the apoptotic rate was signi cantly higher than that of the siNC group; after transfection with siEIF4EBP1+antagomir,the apoptosis rate was signi cantly lower than that of the siEIF4EBP1 group, and the difference was statistically signi cant (Fig 5B).
After Ball-1 transfection, the cell proliferation of the siEIF4EBP1 group at 1, 2, 3, and 4 days was lower than that of the siNC group; the cell proliferation of the siEIF4EBP1+antagomir group at 2, 3, and 4 days was higher than that of the siEIF4EBP1 group.The difference was statistically signi cant ( Fig 5C).
After transfection with siEIF4EBP1, the number of cells in the G0/G1, G2/M phase was higher than that in siNC group, and the number of cells in the S phase was less than that in siNC group. After transfection with siEIF4EBP1+antagomir, the number of cells in the G0/G1 phase decreased compared with the siEIF4EBP1 group, and the number of cells in the G2/M and S phase increased and the cell proliferation recovered (Fig 5D). Fluorescence microscope was used to observe the LC3-II IF of siNC, siEIF4EBP1, siEIF4EBP1+antagomir transfected with Ball-1. It was found that the LC3-II IF of siEIF4EBP1 group was lower than that of siNC group, and the LC3-II IF of siEIF4EBP1+antagomir group was higher than that of siEIF4EBP1 group. The difference was statistically signi cant (Fig 5E).

Disease progression of MRL/lpr lupus mice after experimental intervention of miR-99a-3p in vivo
The body weight of the four groups of mice increased with the increase in the number of feeding weeks. One mouse in the agomir group died at 12w, and the body weight of the MRL/Lpr group was higher than that of C57 at 10w, 12w, and 13w. However, miR-99a-3p intervention did not signi cantly change the body weight (Fig 6A).At 13w, the hair around the nose and eyes of the MRL/lpr group decreased,and there was fewer and slower behavioral activities;the antagomir group mice had alopecia area around the nose, eyes and forehead, and the behavioral activity was obviously slow.
There was no difference in urine protein between the four groups at 8 weeks (P>0.05). However, at 10w, 12w, and 13w, the urine protein in the MRL/Lpr, agomir, and antagomir group increased signi cantly. The urine protein of the MRL/Lpr group was higher than that of the C57 group. The urine protein in the antagomir group was higher than that of the C57 group; the urine protein in the agomir group was lower than that of the C57 group, and the difference was statistically signi cant (Fig 6B). CaseViewer 3.3 counted the glomerular area,the glomerular area of the MRL/lpr group was higher than that of the C57 group (P=0.0184), suggesting that the mice in the MRL/lpr group had some glomerular edema. The glomerular area of the agomir group was lower than that of the MRL/lpr group, but the difference was not statistically signi cant (P=0.2098). The glomerular area of the antagomir group was higher than that of the MRL/lpr group, but the difference was not statistically signi cant (P=0.5888) ( Fig  6C, D).
The C3 IF deposition of MRL/lpr mice and C57 mice after intervention was calculated, and it was found that the C3 IF deposition of the MRL/lpr group was higher than that of the C57 group (P<0.0001). C3 deposition in the agomir group was lower than that in the MRL/lpr group (P=0.0002). C3 deposition in the antagomir group was higher than that in the MRL/lpr group (P=0.0008, Fig 6E, F).
ELISA detection found that the levels of ANA, dsDNA, IgE, IgM, IL-6, IL-10, and BLyS in the MRL/lpr group were higher than those in the C57 group.The levels of ANA, dsDNA, IgE, IgM, IL-6, IL-10 and BLyS in the agomir group were lower than those in the MRL/lpr group. The levels of ANA, dsDNA, IgE, IgM, IL-10, and BLyS in the antagomir group were higher than those in the MRL/lpr group, and the difference was statistically signi cant (Fig 6G).
Changes of target genes and pathway proteins in MRL/lpr lupus mice after experimental intervention of miR-99a-3p in vivo RT-qPCR detected the expression of miR-99a-3p, EIF4EBP1, LC3-, and LAMP-2A after tail vein injection.
The expression of miR-99a-3p in the MRL/lpr group was lower than that in the C57 group (P=0.0293). The expression of miR-99a-3p in agomir group was higher than that in MRL/lpr group (P=0.0013). The expression of miR-99a-3p in the antagomir group was lower than that in the MRL/lpr group (P=0.0272).
The expression of EIF4EBP1, LC3-and LAMP-2A in the MRL/lpr group was higher than that in the C57 group. The mRNA expression of EIF4EBP1, LC3-and LAMP-2A in agomir group was lower than that in MRL/lpr group. The expression levels of EIF4EBP1, LC3-, and LAMP-2A mRNA in the antagomir group were higher than those in the MRL/lpr group, and the difference was statistically signi cant ( Fig 7A).
Western blotting was used to detect the protein expression of EIF4EBP1, LC3-and LAMP-2A after intervention in MRL/lpr and C57 mice. The expression levels of EIF4EBP1, LC3-, and LAMP-2A in the MRL/lpr group were higher than those in the C57 group (P=0.0293). The expression of EIF4EBP1, LC3and LAMP-2A in agomir group was lower than that in MRL/lpr group (P=0.0013). The expression of EIF4EBP1, LC3-and LAMP-2A in the antagomir group was higher than that in the MRL/lpr group (P=0.0272) (Fig 7B).
IHC staining showed that the expression of EIF4EBP1, LC3-, LAMP-2A in the kidney of C57 mice was weak, while the MRL/lpr group increased, the antagomir group increased much more signi cantly, and in the agomir group signi cantly weakened (Fig 6C). EIF4EBP1, LC3-, LAMP-2A in the MRL/lpr group were lower than the C57 and agomir groups at 0 points, and higher than the antagomir group, the difference was statistically signi cant. As the score increased, the intensity of IHC staining and the percentage of positive cells in the C57 and agomir groups gradually decreased, while the trend in the antagomir group was opposite (Fig 7C).

Discussion
SLE is an immune system disease caused by over-activation of immune cells and massive secretion of autoantibodies. Extremely active B cells are involved in almost all the pathogenesis of SLE. Although the treatment of SLE has made great progress, there are still patients with ineffective treatment or relapse.
miRNA is a type of endogenous non-coding RNA widely distributed in the human body, which plays an important role in regulating cell proliferation, apoptosis and disease progression. MiRNAs are involved in immune disorders and organ damage in SLE. MiRNA-based biomarkers and treatment methods may become viable options for the treatment of SLE [5].
Zhang et al provide a novel insight into the role of the circRNA-miRNA-mRNA regulation network in the SLE and 29 DECs (2 up and 27 down) of SLE were found [17]. Latini et al found that miR-155, miR-499a and miR-142 are involved in the pathogenesis and clinical phenotype of SLE [18]. Tao et al found that miR-152-3p promotes TLR-mediated CD4+T cell in ammatory response by regulating the DNMT1/MyD88 signaling pathway, which may become a new target for SLE treatment [19].
This study found that miR-99a-3p decreased signi cantly in Tibetan and Han SLE patients, which was consistent with Pradhan's report on the Indian population [12], Jin's report on the Korean population [13], and Frangou's report on the European population [14].There was no report on the expression of miR-99a-3p in SLE in Chinese population. This study aimed to explore the functional mechanism of miR-99a-3p.
MiR-99a-3p decreased in both Tibetan and Han SLE patients. The Han SLE patients who are more convenient to obtain were selected as further research objects.
In order to clarify the function of miR-99a-3p in different immune cells, this study selected Ball-1, Jurkat, THP-1, K562 cell lines for functional veri cation. After Ball-1 transfection of miR-99a-3p agomir, antagomir, NC, cell proliferation, apoptosis and cycle changes were relatively stable. In this experiment, Ball-1 was selected as the research object. In the pathogenesis of SLE, B cells not only produce autoantibodies, but also regulate the activation of T cells through various cytokines and antigen presentation processes, which also aggravates the progress of SLE [20]. Therefore, it is particularly important to study how B cells play a role in SLE and how to produce pathogenic auto antibodies through signal transmission between cells.
In order to further study the speci c molecular mechanism of miR-99a-3p functioning, it was predicted that EIF4EBP1 was the target gene of miR-99a-3p through bioinformatics methods. In this study, 293T cells were transfected to construct the luciferase reporter gene vector of the 3'UTR wild-type/mutant region of EIF4EBP1, which con rmed that miR-99a-3p can combine with the 3'UTR complementary sequence of EIF4EBP1 and down-regulate EIF4EBP1.
EIF4EBP1 is a translation initiation inhibitor, which regulates its activity by preventing the assembly of eIF4E into the eIF4F complex: hypophosphorylated form competes with EIF4G1/EIF4G3 and strongly binds to EIF4E, leading to repress translation. In contrast, hyperphosphorylated form dissociates from EIF4E, leading to initiation of translation [21]. The pathogenesis of EIF4EBP1 and SLE had not been reported.
Autophagy is a process of engul ng one's own cytoplasmic proteins or organelles, coating them into vesicles, and then fusing with lysosomes to degrade the contents of the package. A large number of studies had con rmed that miRNA can regulate autophagy [22,23]. Abnormal autophagy function leads to the accumulation of apoptosis and induces the production of autoantibodies, thereby inducing and aggravating the condition of SLE.EIF4EBP1 can regulate mTORC1 to induce autophagy [24], microtubulassociated protein 1 light chain 3 (LC3) is a key protein involved in autophagy [25]. The lysossomal associated protein 2a (LAMP-2A) is a key regulatory protein in the chaperonmediated autophagy(CMA) pathway. Inhibiting the LAMP-2A protein can speci cally block the CMA pathway [26].
This study further con rmed that transfection of miR-99a-3p agomir reduced the expression of autophagy-related genes LC3-and LAMP-2A in clinical, in vitro cells (Ball-1, B cells), and in vivo experiments (MRL/lpr mouse B cells).Transfection of miR-99a-3p antagomir had the opposite effect, suggesting that miR-99a-3p had a negative regulatory effect on autophagy, and the change trend of EIF4EBP1 is consistent with the change trend of autophagy level, suggesting that EIF4EBP1 had a positive regulatory effect on autophagy.
In this study, rescue experiment was carried out to con rm the interaction model of miR-99a-3p and EIF4EBP1, miR-99a-3p affects cell proliferation and apoptosis through target genes.
B lymphocyte stimulator (BLyS) is a new member of the tumor necrosis factor family, involved in the regulation of B cell proliferation, differentiation and antibody production. Transgenic mice overexpressing BLyS increased the number of B cells, increased serum ANA and dsDNA, and deposited immunoglobulin in the kidney [27]. Benlysta was the rst inhibitor to act on BLyS, which binds to soluble BLyS with high a nity and inhibits its activity to achieve disease control [28].
In this study, an in vivo experimental model of MRL/Lpr was constructed by injecting miR-99a-3p agomir, antagomir, and NC into the tail vein. The ANA, dsDNA, IgE, IgM, IL-6, IL-10, and BLyS of mice in the antagomir group were signi cantly increased, SLE disease activity was stronger, and B cell proliferation and differentiation actively produced more antibodies. In the antagomir group, the urinary protein and C3 IF deposition in the kidneys were increased, and the kidney damage was more serious, consistent with reports in the literature [29]. They all re ected that miR-99a-3p inhibition increased the progression of SLE disease. However, the IgM level of mice in the antagomir group was higher than that of the MRL/lpr group, and the IgM level was not negatively correlated with the severity of SLE disease [30]. This may be related to the 13-week-old when the mice were sacri ced in this study, and they were still in the early stage of negative feedback.

Conclusions
This study rst discovered that miR-99a-3p targeting EIF4EBP1 participates in the autophagy signaling pathway and affects the function of B cells, thereby aggravating the progression of SLE. This study had deepened the understanding of the molecular mechanisms of miRNA regulatory networks related to the pathogenesis of SLE.The abnormally expressed miR-99a-3p and its target gene EIF4EBP1 found in the study are expected to become potential targets of SLE.

Limitations
There are some limitations in this study. First, most of the experiments in this study were done on Ball-1 and B cells, but there are still some differences between Ball-1, B cells of healthy individulas and B cells of SLE patients. The results of the B cells in this study were not ideal, and the research was not in-depth.
Second, the rescue experiment did not use gene knockout mice and lacked relevant functional veri cation experiments. Third, due to various restrictions,Tibetan patients were not directly involved in the research.
The number of clinical cases was small, and the correlation analysis between clinical indicators and laboratory indicators was lacking.   cells (×400, scale 50um). The green uorescence was the positive staining of LC3-, the blue uorescence was the nuclear staining of DAPI, and the Merge uorescence was the image of the fusion of LC3-and DAPI.

Figure 5
Ball-1 function changes after Rescue experiment. A Western blotting was used to detect the expression of EIF4EBP1, LC3-, LAMP-2A after siNC, siEIF4EBP1, siEIF4EBP1+antagomir transfected Ball-1 B After siNC, siEIF4EBP1, siEIF4EBP1+antagomir were transfected with Ball-1, ow cytometry was used to analyze the changes in apoptosis of each cell line and the histogram to analyze the differences in the proportion of apoptosis in each group(C1 necrosis, C2 late apoptosis, C3 normal, C4 early apoptosis) C CCK-8 method to detect the difference in cell proliferation of siNC, siEIF4EBP1, siEIF4EBP1+antagomir transfected with Ball-1 at 1, 2, 3, and 4 days D After siNC, siEIF4EBP1, siEIF4EBP1+antagomir were transfected into Ball-1, ow cytometry was used to analyze the differences in the cycle changes of each cell and the histogram to compare the differences in the proportion of cells in each group. E The IF of Page 24/26 LC3-after siNC, siEIF4EBP1, siEIF4EBP1+antagomir transfected Ball-1 (×400, scale 50um). Green uorescence was the positive staining of LC3-, blue uorescence was the nuclear staining of DAPI, and