Circ-FBXW12 aggravates the development of diabetic nephropathy by binding to miR-31-5p to induce LIN28B

Background The involvement of circular RNAs (circRNAs) in diabetic nephropathy (DN) has been gradually identified. In this study, we aimed to explore the functions of circRNA F-box/WD repeat-containing protein 12 (circ-FBXW12) in DN development. Methods Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay was performed for the levels of circ-FBXW12, FBXW12 mRNA, microRNA-31-5p (miR-31-5p) and Lin-28 homolog B (LIN28B) mRNA. RNase R assay was used to analyze the stability of circ-FBXW12. Cell Counting Kit-8 (CCK-8) assay, flow cytometry analysis and 5-ethynyl-2′- deoxyuridine (EdU) assay were employed to evaluate cell viability, cell cycle and proliferation, respectively. Enzyme linked immunosorbent assay (ELISA) was done to measure the concentrations of inflammatory cytokines. Western blot assay was conducted for protein levels. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were examined with commercial kits. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the relationships among circ-FBXW12, miR-31-5p and LIN28B. Results Circ-FBXW12 level was increased in DN patients’ serums and high glucose (HG)-induced human mesangial cells (HMCs). Circ-FBXW12 knockdown suppressed cell proliferation, arrested cell cycle, reduced extracellular matrix (ECM) production and oxidative stress in HG-induced HMCs. Circ-FBXW12 was identified as the sponge for miR-31-5p, which then directly targeted LIN28B. MiR-31-5p inhibition reversed circ-FBXW12 knockdown-mediated effects on cell proliferation, cell cycle process, ECM production and oxidative in HG-triggered HMCs. Moreover, miR-31-5p overexpression showed similar results with circ-FBXW12 knockdown in HG-stimulated HMC progression, while LIN28B elevation reversed the effects. Conclusion Circ-FBXW12 knockdown suppressed HG-induced HMC growth, inflammation, ECM accumulation and oxidative stress by regulating miR-31-5p/LIN28B axis.


Introduction
Diabetes mellitus is a common metabolic disease that can cause chronic renal impairment and lead to diabetic nephropathy (DN) [1,2]. At present, DN has been one of the major reasons for end-stage renal disease around the world [3]. The clinical features of DN include mesangial cell (MC) hyperplasia, proteinuria, extracellular matrix (ECM) accumulation, and renal fibrosis [4,5]. Oxidative stress and inflammation caused by elevated blood glucose are considered to be inseparable factors in the occurrence of DN [6]. Hence, it is crucial to explore the mechanism of MC injury under high glucose (HG) for understanding DN development.
The small ncRNAs, miRNAs have been demonstrated to be implicated in DN pathogenesis [13]. For example, miR-325-3p restrained renal inflammatory response and fibrosis by interacting with C-C motif chemokine ligand 19 (CCL19) [14]. MiR-15b-5p alleviated HG-induced inflammatory damage and oxidative damage in podocytes by binding to semaphorin 3A (Sema3A) [15]. Moreover, miR-31 level was declined in T2D patients with DN and negatively related to the secretion of inflammatory factors [16].
With the assistance of bioinformatics tools circinteractome and starBase V2.0, miR-31-5p was found to contain the binding sequences of circ-FBXW12 and LIN28B, thus, we explored their functions and relationships in regulating DN development.

Clinical sample acquisition
A total of 23 healthy volunteers who underwent routine health checks (Normal group), 23 type-2 diabetes patients with DN (DN group) and 14 diabetic patients without DN (DM group) at Zibo First Hospital were enrolled in the study. The diabetic patients were diagnosed according to urinary albumin excretion and divided into two groups: diabetes with normoalbuminuria (DM group) (urinary albumin excretion rate (UAER) < 30 mg/24 h and serum creatinine (Scr) < 133 μmol/L), diabetes with albuminuria (DN group) (UAER > 30 mg/24 h). The DN patients were then subdivided into two groups: microalbuminuria group (30 mg/24 h < UAER < 300 mg/24 h and macroalbuminuria group (UAER > 300 mg/24 h). The patients were excluded this study if they had a history of cardiovascular disease, morbified obesity, organic or inflammatory disease, infectious, autoimmune, hematologic disease, malignancy, fever and diabetic neuropathy. The blood samples were acquired after the research was approved by the Ethics Committee of Zibo First Hospital and written informed consents were provided by the participants. The serums were acquired through centrifugation. The clinical characteristics of the participants were exhibited in Table 1.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay
The RNA was obtained utilizing TRIzol (Invitrogen) and cDNAs was generated via All-in-  Table 2. The abundance was estimated with the 2 −ΔΔCt method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for the internal reference for circ-FBXW12, FBXW12 and LIN28B, while U6 was used for the internal reference for miR-31-5p. For RNase R assay, total RNA was exposed to RNase R (Epicentre, Madison, WI, USA) for 15 min at 37 °C. Then the expression levels of circ-FBXW12 and FBXW12 were quantified by RT-qPCR assay.

Subcellular fraction assay
The PARIS Kit (Invitrogen) was adopted to separate the cytoplasm and nucleus in HMCs according to the manufacturers' instructions. The level of circ-FBXW12 in the cytoplasm and nucleus was quantified.

Cell counting kit-8 (CCK-8) assay
To test cell viability, CCK-8 assay kit (Sigma-Aldrich) was used. In short, the transfected HMCs were plated into 96-well plates and then CCK-8 was supplemented into each well for further 2 h of incubation. The absorption was measured at 450 nm via a microplate reader (Bio-Rad, Hercules, CA, USA).

Enzyme linked immunosorbent assay (ELISA)
The concentrations of interleukin-6 (IL-6) and tumour necrosis factor α (TNF-α) in the culture medium of HMCs were examined by using relevant ELISA kits (ab178013; ab181421; Abcam, Cambridge, MA, USA) referring to the manufacturers' instructions.

Flow cytometry analysis
To assess cell cycle process, HMCs with relevant transfection were collected, rinsed with phosphate buffer saline (PBS; Sigma-Aldrich) and fixed for 8 h with 75% ethanol at 4 °C. Then the cells were resuspended in PBS (Sigma-Aldrich) and stained with propidium iodide (PI; Beyotime, Shanghai, China) supplemented with RNase A (Sigma-Aldrich) for 15 min in darkness. Cell proportion in each stage was analyzed through FACScan ® flow cytometry (BD Biosciences, San Jose, CA, USA).

5-ethynyl-2′-deoxyuridine (EdU) assay
Cell proliferation was assessed by EdU incorporation kit (RiboBio, Guangzhou, China). In short, HMCs were seeded into 24-well plates and EdU was added into the plates for 2 h. Next, the cells were fixed using 4% paraformaldehyde (Sigma-Aldrich) for 0.5 h, permeabilized for 10 min using 0.3% Triton X-100, and rinsed in PBS. Thereafter, the cells were incubated with Aollo fluorescent staining solution for 0.5 h in darkness. Next, the cells were dyed with Hoechst 33342 solution. The positive cells were counted under a fluorescence microscope (Olympus, Tokyo, Japan).

Measurement of superoxide dismutase (SOD) activity and malondialdehyde (MDA) level
The activity of SOD and the content of MDA in HMCs were examined with SOD assay kit (Sigma-Aldrich) and MDA assay kit (Sigma-Aldrich) strictly according to the guidelines.

RNA immunoprecipitation (RIP) assay
HMCs were lysed in RIP buffer and cell extracts were cultivated with magnetic beads conjugated with IgG (Abcam) or Ago2 (Abcam). Thereafter, the samples were maintained with proteinase K (Sigma-Aldrich) for 0.5 h to separate the RNA-protein complexes from beads followed by RT-qPCR assay for the abundance of circ-FBXW12, miR-31-5p and LIN28B.

Statistical analysis
The sample size was evaluated by G*Power. The experiments were performed in triple times and the data were estimated by GraphPad Prism 7 and exhibited as mean ± SD. The data were normally distributed. The differences of two sets and three sets were analyzed by Student's t-test or one-way analysis of variance followed by Tukey's test. It was considered as significant when P < 0.05.

Circ-FBXW12 was highly expressed in DN patients and HG-induced HMCs
To explore the function of circ-FBXW12 in DN progression, the expression of circ-FBXW12 in the serums of DN patients, diabetic patients without DN (DM group) and healthy volunteers was determined by RT-qPCR assay. The results showed that circ-FBXW12 level was apparently increased in DN patients and DM patients in comparison with normal controls (Fig. 1A). Moreover, circ-FBXW12 was markedly increased in HG-treated HMCs compared to control groups (Fig. 1B). RNase R assay indicated that circ-FBXW12 was resistant to RNase R treatment, while linear FBXW12 was digested by RNase R treatment (Fig. 1C). Moreover, our results exhibited that circ-FBXW12 was mainly enriched in the cytoplasm of HMCs (Fig. 1D). These results suggested the potential role of circ-FBXW12 in DN development.

Silencing of circ-FBXW12 suppressed HG-induced cell proliferation, inflammation, cell cycle, ECM production and oxidative stress in HMCs
To explore the exact roles of circ-FBXW12 in DN development, HMGs were transfected with si-circ-FBXW12 to knock down circ-FBXW12 expression in HMGs. As a result, the upregulation of circ-FBXW12 in HMGs caused by HG treatment was reversed by the transfection of si-circFBXW12 ( Fig. 2A). CCK-8 assay indicated that HG treatment led to a distinct promotion in the viability of HMCs compared to control group, while circ-FBXW12 silencing reversed the effect (Fig. 2B). ELISA showed that the concentrations of IL-6 and TNF-α were increased in HMCs treated with HG, whereas the effects were abated by downregulating circ-FBXW12 (Fig. 2C). As illustrated by flow cytometry analysis, the percentage of HMCs in G0/G1 phases was reduced and the percentage of HMCs in S phase was increased after HG exposure, while circ-FBXW12 knockdown ameliorated the effects (Fig. 2D). EdU assay indicated that HG treatment promoted HMC proliferation, with circ-FBXW12 deficiency rescued the impact (Fig. 2E). HG treatment increased CyclinD1 protein level and decreased P21 level in HMCs, with circ-FBXW12 silencing rescued the effects (Fig. 2F). Moreover, we found that HG treatment increased the protein levels of ECM markers (collagen I and collagen IV) in HMCs, while the effects were overturned by decreasing circ-FBXW12 (Fig. 2G). TGF-β1 is closely related to the production of ECM [22]. Thus, we detected the protein level of TGF-β1 in HG-treated HMCs. Our results showed that TGF-β1 was elevated in HG-treated HMCs, but circ-FBXW12 interference reversed the effect (Fig. 2H). Besides, it was found that the activity of SOD was inhibited and the level of MDA was increased in HG-treated HMCs, while circ-FBXW12 silencing rescued the effects ( Fig. 2I and J). Taken together, HG treatment promoted cell proliferation, inflammation, cell cycle process, ECM production and oxidative stress in HMCs, with circFBXW12 silencing abrogated the impacts.

LIN28B knockdown suppressed cell proliferation, inflammation, cell cycle process, ECM production and oxidative stress in HG-treated HMCs
Subsequently, the functional roles of LIN28B in HGinduced HMCs progression were investigated. As shown in Fig. 6A, si-LIN28B transfection led to a distinct reduction in LIN28B protein level in HG-treated HMCs. CCK-8 assay showed that LIN28B silencing suppressed HG-induced HMC cell viability compared to si-con control groups (Fig. 6B). HG-induced elevation of IL-6 and TNF-α in HMCs was reversed by silencing LIN28B (Fig. 6C). Flow cytometry analysis showed that the promotional effect of HG treatment on cell cycle process in HMCs was abated by LIN28B knockdown (Fig. 6D). LIN28B silencing inhibited the ability of HG-treated HMCs to proliferate compared to si-con groups (Fig. 6E). Moreover, LIN28B interference reduced the protein levels of CyclinD1, collagen I, collagen IV and TGF-β1 and elevated the protein level of P21 in HG-triggered HMCs (Fig. 6F-H). In addition, we found that LIN28B knockdown enhanced SOD activity and reduced MDA level in HG-stimulated HMCs in comparison with si-con groups ( Fig. 6I and J). Collectively, LIN28B knockdown suppressed HGinduced HMCs development.

MiR-31-5p overexpression repressed cell proliferation, inflammation, cell cycle, ECM production and oxidative stress in HG-stimulated HMCs by targeting LIN28B
The transfection of LIN28B overexpression vector elevated LIN28B protein level in HMCs compared to pcDNA control groups (Fig. 7A). MiR-31-5p transfection reduced LIN28B protein level in HG-treated HMCs, while LIN28B transfection reversed the effect (Fig. 7B). CCK-8 assay presented that miR-31-5p overexpression restrained the viability of HG-treated HMCs, while LLIN28B elevation rescued the effect (Fig. 7C). Overexpression of miR-31-5p caused a distinct reduction in IL-6 and TNF-α concentrations in HG-treated HMCs, whereas the impacts were abolished by increasing LIN28B (Fig. 7D). As demonstrated by flow cytometry analysis and EdU assay, miR-31-5p overexpression arrested cell cycle and suppressed cell proliferation in HG-stimulated HMCs, while LIN28B upregulation overturned the impacts (Fig. 7E and F). The protein levels of CyclinD1, collagen I, collagen IV and TGF-β1 were reduced and the protein level of P21 was increased in HG-triggered HMCs after miR-31-5p transfection, while LIN28B overexpression weakened the effects (Fig. 7G-I). Additionally, miR-31-5p overexpression enhanced SOD activity and declined MDA level in HG-treated HMCs, with LIN28B elevation ameliorated the effects (Fig. 7J and K). All these findings suggested that miR-31-5p overexpression suppressed the progression of HG-treated HMCs by targeting LIN28B.

Circ-FBXW12 knockdown suppressed LIN28B expression by sponging miR-31-5p
At last, the relationships of circ-FBXW12, miR-31-5p and LIN28B were analyzed. It was found that circ-FBXW12 silencing resulted in a marked reduction in LIN28B mRNA and protein levels in HG-treated HMCs, while these effects were rescued by the inhibition of miR-31-5p ( Fig. 8A and B). Overexpression of miR-31-5p directly targeted LIN28B to repress the development of HG-stimulated HMCs. A The protein level of LIN28B in HMCs transfected with pcDNA or LIN28B was measured via western blot assay. B-I HMCs were transfected with miR-NC, miR-31-5p, miR-31-5p + pcDNA or miR-31-5p + LIN28B in HG condition. B The protein level of LIN28B in HMCs was measured using western blot assay. C HMC viability was assessed by CCK-8 assay. D The concentrations of IL-6 and TNF-α in HMCs were examined with ELISA kits. E Cell cycle in HMCs was analyzed by flow cytometry analysis. F HMC proliferation was evaluated by EdU assay. G-I The protein levels of CyclinD1, P21, collagen I, collagen IV and TGF-β1 in HMCs were measured by western blot assay. J and K The activity of SOD and the level of MDA in HMCs were examined with commercial kits. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Discussion
In the last decade, circRNAs have been gradually identified and discovered to play crucial regulatory roles in a wide range of human diseases [23]. Moreover, the involvement of circRNAs in DN was reported. For example, circ_0000491 level was elevated in DN mice and HG-induced MMCs and promoted ECM production through the mediation of miR-101b and TGFβRI [24]. Circ_0037128 silencing repressed MC growth and fibrosis by reducing AKT Serine/Threonine Kinase 3 (AKT3) through adsorbing miR-17-3p [25]. Circ_WBSCR17 contributed to HG-induced HK2 cell inflammation and fibrosis by miR-185-5p/SRY-box transcription factor 6 (SOX6) axis [26]. In this study, circ-FBXW12 level was abnormally increased in DN patients' serums. HG treatment also activated circ-FBXW12 expression in HMCs. Thus, we speculated that circ-FBXW12 might exert a function in DN. Proliferation, inflammation, and fibrosis of MCs are important pathophysiological features of DN [27,28]. Herein, our results showed that HG promoted cell proliferation and inflammatory factors release in HMCs, while circ-FBXW12 knockdown reversed the impacts. In addition, the enhancement of oxidative stress caused by hyperglycemia can aggravate DN progression [29]. In this work, HG-triggered oxidative injury in HMCs was abolished by decreasing circ-FBXW12. As DN is a metabolic disorder characterized by ECM accumulation, which can be induced by some pathological conditions such as oxidative stress and autophagy [30,31]. Thus, we explored the effect of circ-FBXW12 on ECM by detecting the ECM related markers, including collagen I, collagen IV and TGF-β1 [32,33]. Our results demonstrated that circ-FBXW12 knockdown reduced the levels of collagen I, collagen IV and TGF-β1 in HG-triggered HMCs, suggesting the suppression of ECM production. All these findings indicated that circ-FBXW12 knockdown was able to alleviate HG-stimulated HMC damage.