MicroRNA-339-5p inhibits lipopolysaccharide-induced rat mesangial cells by regulating the Syk/Ras/c-Fos pathway

Chronic glomerulonephritis (CGN) is a disease occurred in glomeruli. The mechanism of CGN is regarded to be involved in a range of inflammatory responses. MicroRNA-339-5p (miR-339-5p) has been reported to be involved in inflammatory responses in many diseases. However, the role of miR-339-5p in CGN remains unclear. The purpose of this study was to investigate the role of miR-339-5p in lipopolysaccharide (LPS)-induced nephritis injury in vitro. The real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and western blot (WB) were used to detect the expression of miR-339-5p and Syk/Ras/c-Fos pathway. Double luciferase was performed to identify targeted binding of miR-339-5p to Syk. Cell counting kit-8 (CCK-8) and flow cytometry were used to observe cell viability and cell cycle. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the concentrations of inflammatory cytokines IL-1β, IL-10, IL-6, and TNF-α. Lipopolysaccharide (LPS) could increase HBZY-1 (rat mesangial cells) cell viability, decrease the G2 phase, and promote cell proliferation and accelerate inflammatory cytokine. However, overexpression of miR-339-5p could inhibit LPS-induced HBZY-1 cell viability, decrease the expression of Syk/Ras/c-Fos signaling pathway, downregulate the expression level of inflammatory cytokines, increase the G2 phase, and inhibit cell proliferation. miR-339-5p could inhibit the proliferation and inflammation of the rat mesangial cells through regulating Syk/Ras/c-Fos signaling pathway.


Introduction
Chronic glomerulonephritis (CGN) is one of the most serious non-communicable diseases in the world. About 10% of world's population suffers from chronic kidney disease (Venuthurupalli et al. 2017). Similarly, the prevalence rate of chronic kidney disease in Chinese is 10.8%, and the incidence of CGN in central China is up to 14.2% (Zhang et al. 2012). The occurrence of CGN is mainly due to glomerular pathological damage caused by the immune-mediated inflammatory response. Patients will have symptoms such as edema, proteinuria, and hypertension . The specific pathogenesis is still not clear, and there is no effective targeted therapy (Jia et al. 2021). If it is not treated immediately, it will develop into end-stage renal disease (ESRD) (Asgari et al. 2016;Zuo et al. 2015). Kidney transplantation will be the only choice (Prionas et al. 2021;Naderi et al. 2020) that will increase the economic burden of the patients and decrease their quality of life. The CGN has become a global public health problem.
MicroRNAs (miRNAs), which usually contain 21-25 nucleotides (Liu et al. 2020;Tzaridis et al. 2020), are a kind of short-stranded non-coding RNA. They regulate gene expressions by targeting mRNAs for translation inhibition or degradation, mainly in post-transcriptional horizontal regulation-related protein expression (Gomez et al. 2020;Ramaswamy et al. 2018). There is increasing evidence that miRNAs play important roles in the regulation of inflammation-mediated disease (Hsu et al. 2012), and participate in a series of important processes such as disease development, inflammation, immune response, and so on Min et al. 2018). By regulating the protein expression of downstream target genes, miR-339-5p can promote cell proliferation, reduce cell inflammation, decrease the production of reactive oxygen species, and inhibit oxidative stress-induced injury (Li et al. 2021). In addition, studies have found that miRNAs can affect the process of cell proliferation and apoptosis ). miR-339-5p has been proved to have an inhibitory effect on the glioblastoma, which can induce the apoptosis of the glioblastoma (Farooq et al. 2019). It has been reported that miR-339 promotes apoptosis of hepatocellular carcinoma cells by inhibiting proliferation and invasion (Zeng et al. 2019). Therefore, that the role of miR-339-5p in CGN may be related to reducing the level of inflammatory factors, regulating immune function, and inhibiting cell viability and proliferation. This study investigated the relationship between miR-339-5p and CGN that will provide a reference for the pathogenesis of CGN.

Cell lines and cell culture
HBZY-1 cells (rat mesangial cells) are a special mesenchyme of glomerular capillary loops, which are composed of mesangial cells and mesangial matrix. These cells, isolated from glomerular tissue, are very active cells in glomeruli. The proliferation of mesangial cells and matrix are the main pathological manifestations of many kinds of glomerulonephritis. Therefore, HBZY-1 cells were selected for the experiment in the following study. In the establishment of the CGN model, an appropriate amount of LPS was added to HBZY-1 cells so that the final concentration of LPS in the medium was 0.5 μg/mL and cultured for 24 h in the incubator of 37 C and 5% CO 2 . The detection of cell proliferation showed that the cell proliferation rate increased significantly, which proved that the cell model of chronic glomerulonephritis was successfully established .
The HBZY-1 cell line was obtained from the Cell Bioscience Inc. (Shanghai, China). After receiving the cells, the cells were placed in an incubator for 3 h to stabilize the cell state. After washing with PBS (Hyclone, Logan, UT, USA), 1 mL trypsin (Beyotime Biotechnology, Shanghai, China) was added for digestion for 2 min. After centrifugation, the suspension was transferred to T-25 culture flask (KIRGEN, Shanghai, China) and cultured in high glucose Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, UT, USA), which contains 10% fetal bovine serum, penicillin (100 U/mL, Invitrogen, Carlsbad, CA, USA), and streptomycin (100 μg/mL, Invitrogen). The HBZY-1 cell line was cultured in a humidified incubator, which contains 5% CO 2 at 37 °C.

Quantitative real-time polymerase reaction
Trizol reagent (Life Technologies, Carlsbad, CA, USA) was used to extract the total RNA in each group. PrimeScript™RT Reagent Kit with gDNA Eraser Kit (TaKaRa, Kyoto, Japan) was used to reverse the transcription into cDNA. SYBR qPCR SuperMix Plus (Novoprotein, Shanghai, China) was used for a real-time quantitative polymerase chain reaction in fluorescent quantitative PCR apparatus (Thermo Fisher Scientific, Waltham, MA, USA). The reaction conditions were pre-denaturated for 1 min at 95 °C at first, and then denatured at 95 °C for the 20 s, annealing at 60 °C for 1 min, for 40 cycles at last. Using β-actin or U6 as control, the relative expression levels of related RNAs were calculated by the 2 −ΔΔCT method. Primer sequences of all the indicators were shown in Table 1.

Western blotting assay
The cell samples were collected and the total cell proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China), after electrophoretic separation and transferring to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Then, the samples were sealed at room temperature for 2 h in a Tris buffer containing 5% skims milk. The primary antibody was incubated overnight. The dilution ratios of the inflammatory factors were as follows: IL-1β (1:500) (Bioss, Beijing, China, bs-0812R), IL-6 (1:1000) (

Cell Counting Kit-8 Proliferation Assay
CCK-8 assay cells in the logarithmic growth phase were all evenly spread in a 96-well plate and cultured for 24 h (5 × 10 4 cells per well). Ten microliters of CCK-8 reagent (Bioss, Beijing, China) was administrated to each well. The absorbance (A) of each hole was determined at the OD value 450 nm of the enzyme labeling instrument (Rayto, Shenzhen, China). Triplicate assays were conducted for each group.

Flow cytometry assay
The cells in all the groups were washed with aseptic PBS, digested with 1 mL trypsin for 2 min, centrifuged at 1000 rpm for 5 min, removed for the supernatant, and fixed with precooled anhydrous ethanol in the refrigerator at − 20 C for 1 h; the fixed cells were washed twice with precooled PBS and added with 20 µL RNase. The resuscitated cells for digested for 30 min in 37 C water baths, and then added with PI staining solution 400 µL, stained for 40 min at 4 C. Flow cytometry (BECKMAN, CA, USA) was used to observe the changes in the cell cycle in each group.

Luciferase reporter assay
The 3'-untranslated region of the Syk gene was amplified from the human genome by polymerase chain reaction. The corresponding sequence of rno-miR-339-5p was amplified from the rat genome and cloned into the 3'end of the pSI-Check2 (Hanbio Biotechnology, Shanghai, China) luciferase vector. Mutated version of each construct was generated by mutating the sequences of rno-miR-339-5p seed sites (psi-CHECK2-wt or mut). HBZY-1 cells, seeded in 96-well plates, were transfected with wt/mut report gene and rno-miR-339-5p/NC mimic with Lipofectamine 2000 (Invitrogen, CA, USA). The dual-luciferase reporter assay system (Promega, Beijing, China) was used to examine the

Statistical analysis
All experiments were repeated three times and data were presented as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS 19.0 statistical software, and the figures were drawn by GraphPad Prism 8.0. One-way ANOVA analysis of variance (ANOVA) was used to calculate the P-values, which was less than 0.05 and considered to be statistically significant. After the ANOVA, LSD is used for multiple comparisons if equivariance is assumed, and TamhaneT2 is used for multiple comparisons if equivariance is not assumed.

miR-339-5p was significantly downregulated in HBZY-1 cells
In this study, we used LPS-induced HBZY-1 cells as CGN experimental model to clarify the relationship between miR-339-5p and CGN. The results of qRT-PCR experiment showed that miR-339-5p expression level was significantly downregulated in the model group when compared with the control group ( Fig. 1) (Supplementary 5, 6, 7).

Overexpression of miR-339-5p reduced the viability of HBZY-1 cells and inhibited cell proliferation
We next utilize the cell transfection experiment to further explore the key role of miR-339-5p in HBZY-1 cells. The results showed that overexpression of miR-339-5p or inhibition of miR-339-5p level would cause changes in the expression level of miR-339-5p RNA ( Fig. 2A) (Supplementary  5, 6, 7). CCK-8 assay showed that overexpression of miR-339-5p could effectively reduce the activity of HBZY-1 cells; however, transfection of miR-339-5p inhibitor had a completely opposite effect on the viability of HBZY-1 cells (Fig. 2B)   expression levels of IL-1β, IL-6, TNF-α, and IL-10 were detected by qRT-PCR. **P < 0.01 vs. control, ## P < 0.01 vs. model, # P < 0.05 vs. model detect the expression levels of inflammatory cytokines to determine whether miR-339-5p affects the level of inflammatory cytokines in HBZY-1 cells. The results showed that overexpression of miR-339-5p significantly reduced the expression levels of inflammatory cytokines IL-1β, IL-6, and TNF-α, and significantly increased the expression level of proinflammatory cytokines IL-10. Inhibition of the miR-339-5p expression had in the opposite result. Therefore, it can be concluded that miR-339-5p reduces the level of inflammatory cytokines in HBZY-1 cells.

Syk was the direct target gene of miR-339-5p
Our team further confirmed that the downstream target gene Syk of miR-339-5p by using Targetscan (https:// www. targe tscan. org/) (Fig. 4A). The results of the double luciferase report showed that the luciferase value of the Syk-WT + miR-339-5p mimics group was significantly lower than that of the Syk-MUT + mimics NC group, and there was no significant change in the Syk-MUT + miR-339-5p mimics group compared with the Syk-MUT + mimics NC group (Fig. 4B) (Supplementary 9), which confirmed that Syk was the direct target gene of miR-339-5p. In addition, the regulatory relationship between miR-339-5p and Syk was studied by western blot (Fig. 4C-D) (Supplementary 3, 4), qRT-PCR (Fig. 4E) (Supplementary 5,6,7), and IF ( Fig. 4F-G) (Supplementary 8, 10, 11) experiments. The results showed that inhibiting the expression of miR-339-5p significantly increased the expression of Syk in HBZY-1 cells, while the expression of overexpressed miR-339-5p was significantly decreased in HBZY-1 cells. It can be concluded that miR-339-5p can negatively regulate the expression of Syk in HBZY-1 cells directly.

Syk downregulation reversed the effects of miR-339-5p on HBZY-1 cells
This study confirmed the inhibitory effect of miR-339-5p on Syk and speculated that Syk was involved in the regulation of miR-339-5p on HBZY-1 cells. Among Syk inhibitors, fostamatinib (prodrug of R406) has been widely proven to have therapeutic effects in a variety of inflammatory diseases (Sun et al. 2019;Koziol-White et al. 2016), and previous studies in our research group also discussed the role of R406 in CGN . The results showed that R406 could downregulate the expression levels of inflammatory cytokines IL-1β, IL-6, and TNF-α and upregulate the expression levels of anti-inflammatory cytokines IL-10 ( Fig. 6A-H) (Supplementary 3, 4, 5, 6, 7). As can be seen from the changes in the cell cycle diagram, R406 can also regulate changes of the cell cycle, stagnating the cell cycle in the G2 phase, reversing the decrease in the G2 phase of HBZY-1 cells caused by miR-339-5p inhibitors, and reducing the cell proliferation ability (Fig. 6J-I) (Supplementary 2). In addition, the CCK-8 assay showed that R406 reduced HBZY-1 cell viability (Fig. 6K) (Supplementary 1). Thus, Syk inhibitor R406 has the opposite effect to miR-339-5p on inflammatory cytokines, cycle and cell viability.  Maity et al. 2018).
In addition, the decrease of GMC biological activity can stimulate glomerular endothelial cell apoptosis and affect renal senescence, which will cause glomerulosclerosis and other pathological conditions (Khan et al. 2011;Feng et al. 2017). After acting on cells, LPS can promote the secretion of various inflammatory cytokines, which is consistent with the pathological manifestation of CGN (Gohda et al. 2001). Therefore, in this paper, we used rat mesangial HBZY-1 cells which were treated with LPS to establish a chronic glomerulonephritis model in vitro.
With the increasing discovery of miRNA, a deeper understanding is gained for their functions. So far, thousands of miRNA have been discovered, which constitute the most abundant class of gene regulatory systems in cells (Siomi and Siomi 2010). Most of the Fig. 6 Syk downregulation reversed the effects of miR-339-5p on HBZY-1 cells. A-D The expression of IL-1β, IL-6, TNF-α, and IL-10 protein was detected by Western blot. E-H The mRNA expression levels of IL-1 β, IL-6, TNF-α, and IL-10 were detected by qRT-PCR. I-J Flow cytometry was used to detect the cycle changes. K CCK-8 was used to detect the viability of HBZY-1 cells. **P < 0.01 vs. control, ## P < 0.01 vs. model, *P < 0.05 vs. control. # P < 0.05 vs. model miRNA expressions are highly tissue-specific and cellspecific, so they can be used in disease diagnosis and treatment, which has become a new research focus (Anelli et al. 2021;Gholizadeh et al. 2020;Ye et al. 2021). The functions for miRNAs are main in the post-transcriptional regulation of protein-coding genes, which binds to the target mRNA and inhibits its translation in a sequence-dependent manner (Navarro 2021;Li and Ni 2017;Guo et al. 2018). Our group screened a series of glomerulonephritis-related miRNA by referring to the related literature. It is predicted that there may be a complementary sequence between miR-339-5p and the central gene Syk of glomerulonephritis through bioinformatics prediction and analysis (Gao et al. 2016), which plays an important role in cell proliferation, apoptosis, inflammation, and so on . In this paper, double luciferase experiments showed that there was a targeted binding relationship between miR-339-5p and Syk, and the overexpression of miR-339-5p could negatively regulate the relative expression of Syk proteins and genes. Studies have shown that the overexpression of miR-339-5p in prostate cancer (Pan et al. 2021) and breast cancer (Feng et al. 2020) decreased cell proliferation activity and caused the cellrelated inflammatory responses. This study showed that the expression level of miR-339-5p in HBZY-1 cells of the model group decreased significantly when compared with the control group. However, overexpression of miR-339-5p can effectively reduce cell viability and the level of inflammatory factors in cells and change the cell cycle. Therefore, the potential molecular mechanism of miR-339-5p in CGN may be related to the inhibition of cell proliferation and inflammation.
As a non-receptor tyrosine kinase, Syk is generally regarded as an important regulator of adaptive immunity. It could phosphorylate substrate proteins, promote the secretion of inflammatory factors, and mediate inflammatory immune responses (Chen et al. 2011;Xia et al. 2017). Syk has been found to be able to activate inflammatory bodies and promote the release of inflammatory factors in diabetic cardiomyopathy and diabetic nephropathy ). In the process of Staphylococcus aureus infection, Syk is phosphorylated rapidly, which reduces the defense function of the immune system and releases pro-inflammatory factors IL-1β and IL-18 ). In addition, Syk is an important part of the middle Syk/ Ras/c-Fos signaling pathway. Through bioinformatics prediction and analysis in the previous study, it is found that miR-339 had the highest binding degree with Syk. We have studied the influence of the Syk/Ras/c-Fos signaling pathway on HBZY-1 cells , while the pathogenesis of CGN at the genetic level has not been discussed.
In this study, it was found that the expressions of proinflammatory factors such as IL-1β, IL-10, and 1L-6 were increased in HBZY-1 cells, while the expression of The targeting combination of miR-339-5p and Syk inhibits the expression level of Syk/ Ras/c-Fos signaling pathway. On the one hand, it inhibits the expression level of inflammatory factors and then inhibits cell proliferation. On the other hand, it inhibits cell proliferation by increasing the G2 phase (red arrows for promotion, green arrows for inhibition) anti-inflammatory factor IL-10 decreased. After the Syk/Ras/ c-Fos signaling pathway was activated, the expression levels of Syk, Ras, MEK1, MEK2, ERK1, ERK2, and c-Fos were significantly increased, and the fluorescence intensity and average optical density in IF were also significantly increased. The overexpression of miR-339-5p could effectively reverse the up-regulated expression of genes and proteins related to Syk/Ras/c-Fos pathway and reduce the level of inflammatory factors in LPS-induced HBZY-1 cells. Then, it was found in the recovery experiment that Syk inhibitor R406 could reverse the increase of cell viability and the decrease of G2 phase caused by miR-339-5p inhibitor, as well as the increase of inflammatory factors. Based on the findings presented herein, a schematic representation of the relationship between miR-339-5p and CGN is proposed (Fig. 7).

Conclusion
In conclusion, this study suggests that the overexpression of miR-339-5p can reduce the viability of LPS-induced HBZY-1 cells and inhibit the expression of inflammatory factors and cell proliferation through regulating the Syk/Ras/c-Fos signaling pathway by targeting Syk. It provides not only a new idea for the further explore of the molecular mechanism of CGN, but also a new target for the clinical treatment of CGN.