Long noncoding RNA MIAT inhibits the progression of diabetic nephropathy and the activation of NF-κB pathway in high glucose-treated renal tubular epithelial cells by the miR-182-5p/GPRC5A axis

Abstract Background Diabetic nephropathy (DN) is a common diabetic complication. Long noncoding RNAs (lncRNAs) have been identified as essential regulators in DN progression. This study is devoted to the research of lncRNA-myocardial infarction-associated transcript (MIAT) in DN. Methods DN cell model was established by high glucose (HG) treatment for human renal tubular epithelial cells (HK-2). Cell viability and colonizing capacity were analyzed by Cell Counting Kit-8 (CCK-8) and colony formation assay. Apoptosis was assessed via caspase-3 detection and flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used for evaluating inflammation. The protein determination was completed using western blot. MIAT, microRNA-182-5p (miR-182-5p), and G protein-coupled receptor class C group 5 member A (GPRC5A) levels were all examined via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Intergenic binding was verified using dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays. Results HG induced the inhibition of cell growth, but accelerated apoptosis and inflammation as well as the activation of nuclear factor kappa B (NF-κB) pathway. MIAT reestablishment prevented the HG-induced cell damages and NF-κB signal activation. Mechanistically, MIAT was proved as a miR-182-5p sponge and regulated the expression of GPRC5A that was a miR-182-5p target. The rescued experiments demonstrated that MIAT downregulation or miR-182-5p upregulation aggravated the HG-induced cell damages and activated the NF-κB pathway via the respective regulation of miR-182-5p or GPRC5A. Conclusion Taken together, MIAT functioned as an inhibitory factor in the pathogenesis to impede the development of DN and inactivate the NF-κB pathway via regulating the miR-182-5p/GPRC5A axis.


Introduction
Diabetic nephropathy (DN), the most familiar microvascular complication in diabetic type 1 and type 2 patients with high incidence rate, is considered as the leading risk factor of chronic kidney failure and end-stage renal diseases [1,2]. The typical pathological feature for DN is the damage of capillaries in the glomeruli, and the inflammation formation has also been involved in the pathological progression of DN [3]. The molecular pathways have become the new insights in the therapeutic paradigms of DN [4], especially long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) [5,6].
lncRNAs (>200 nucleotides) are one common family of regulatory ncRNAs in diverse biological processes of diseases [7], such as osteoarthritis [8], cardiovascular disease [9], and liver disease [10]. lncRNAs have also been regarded as important regulators in the regulation of DN [11,12]. PVT1 silence reduced cell proliferation, migration, and fibrosis in DN cell model [13]; NEAT1 acted as a pro-inflammatory molecule in DN [14]. Myocardial infarction-associated transcript (MIAT) downregulation has been correlated to the renal tubular epithelial injury in DN [12], and MIAT could enhance cell viability in high glucose (HG)-treated renal tubular cells by increasing the expression of Nrf2 [15]. A recent study reported that the antagonistic effect of MIAT on DN progression was related to the sponge function on miR-147a and the expression regulation on E2F3 [16]. It is unknown whether the regulation of MIAT in DN progression is related to other molecular mechanism.
miRNAs are short ncRNAs (∼22 nucleotides) with pivotal effects on the epigenetic modifications of DN [17]. Meta-analysis has identified that miR-126 and miR-770 could be used as noninvasive biomarkers for DN [18], and miR-184 could govern renal fibrosis in DN [19]. lncRNAs can exert the "sponge-like" effects on various miRNAs to limit the miRNA-mediated actions in human diseases [20]. Ming et al. reported that microRNA-182-5p (miR-182-5p) contributed to the pathogenetic development to serve as a risk factor in DN [21]. It remains unclear whether MIAT has a sponge effect on miR-182-5p.
G protein-coupled receptor class C group 5 member A (GPRC5A) is a sub-member of G protein-coupled receptors (GPCRs) superfamily. GPRC5A regulated the progression of human diseases and it was associated with many signaling pathways, such as nuclear factor kappa B (NF-κB) pathway [22]. GPRC5A overexpression could accentuate the HG-induced renal proximal tubule cell injury and it functioned as a target of miR-218 [23]. miRNAs have been implicated in the epigenetic modulation of DN by binding to the 3′-untranslated regions (3′-UTRs) of mRNAs [24]. The target relation between GPRC5A and miR-182-5p will be explored in this study.
Herein the effects of MIAT on other cellular processes of DN were further researched. More importantly, we have disclosed the regulatory network among MIAT, miR-182-5p, and GPRC5A.

Cell culture and HG induction
Human renal tubular epithelial cell line (HK-2) was bought from American Type Culture Collection (ATCC, Manassas, VA, USA) and sustained in Dulbeccoʼs modified Eagleʼs medium (DMEM)/F12 (Gibco, Carlsbad, CA, USA) with the supplement of 10% of fetal bovine serum (FBS; Gibco) and 1% of penicillin/streptomycin solution (Gibco). Cell culture was performed in a 37°C humid incubator containing 5% of CO 2 . DN cell model was established by treating HK-2 cells with HG (25 mM of glucose) for 48 h, using the normal glucose (NG; 0 mM of glucose) and low glucose (LG; 5 mM of glucose) as the control treatment groups.

Colony formation assay
Cell inoculation was conducted at 48 h post-transfection with 500 cells/well in 6-well plates. Cells were cultivated for 2 weeks under the standard culture condition. Subsequently, the colonies were fastened with methanol (Sigma-Aldrich, St. Louis, MO, USA) for 10 min and stained with crystal violet (Sigma-Aldrich) for 15 min. These colonies were photographed by a digital camera and the colonizing number was counted.

Caspase-3 activity detection
Total proteins were obtained via RIPA Lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) and caspase-3 activity was measured via the EnzChek ® Caspase-3 Assay Kit (Invitrogen, Carlsbad, CA, USA) using Z-DEVD-AMC substrate, in compliance with the supplied manual for users.

Flow cytometry
At 72 h post-transfection, apoptosis detection was carried out through the double staining of Annexin V-FITC and propidium iodide (PI) using Apoptosis Detection Kit (Dojindo) as per the operating procedures of the producer. Through the analysis on the flow cytometer (FacsCanto II; BD Biosciences, San Diego, CA, USA), the apoptotic cells were distinguished as the stained cells with Annexin V (+)/PI(−) and Annexin V(+)/PI(+). The common formula: apoptotic cells/total cells × 100% was used to calculate the apoptotic rate.

Western blot
Total proteins were produced by lysing cells in RIPA buffer (Thermo Fisher Scientific), followed by detecting the concentration using BCA Protein Assay Kit (Invitrogen). In this study, an equal amount of proteins (30 µg) were applied to perform the western blot analysis, as reported earlier [25].

Cell transfection
The

Dual-luciferase reporter assay
The miR-182-5p binding sites in the MIAT or 3′-UTR of GPRC5A sequence (wild-type, WT) were mutated and the mutated MIAT or 3′-UTR of GPRC5A sequence was defined as the mutant-type (MUT). These sequences were cloned into the downstream of luciferase reporter gene in the pGL3-control vector (Promega, Madison, WI, USA) to construct the novel luciferase vectors (WT-MIAT, MUT-MIAT, WT-GPRC5A 3′-UTR, and MUT-GPRC5A 3′-UTR). Then, cell transfection of each of the above vectors with miR-182-5p or miR-NC was performed for 48 h. The dualluciferase reporter assay system (Promega) was adopted for the subsequent luciferase activity determination following the user protocols. The relative luciferase activity was exhibited using the normalized firefly activity by renilla luciferase activity.
2.11 RNA immunoprecipitation (RIP) assay RIP assay was conducted by Imprint ® RIP Kit (Sigma-Aldrich). In brief, 1 × 10 6 HG-treated HK-2 cells were lysed in RIP lysis buffer and Protein A magnetic beads were pre-coated with antibodies targeting Argonaute-2 (anti-Ago2) or immunoglobulin G (anti-IgG). Cell lysates were then incubated with the antibody-coated magnetic beads at 4°C overnight. The proteins were removed by proteinase K and total RNA was purified by Trizol, followed by the expression analysis of MIAT and miR-182-5p via RT-qPCR as per the above description.

Biotinylated RNA pull-down assay
Biotinylated miR-182-5p mimic (Bio-miR-182-5p; RIBOBIO) and negative Bio-miR-NC control were respectively transfected into cells for 48 h. Whereafter, cell lysates were incubated with streptavidin magnetic beads (Thermo Fisher Scientific) at 4°C overnight and the combined RNA was isolated from the magnetic beads. Then, RT-qPCR was performed to analyze the enrichment of MIAT.

Statistical analysis
Three independent experiments were conducted with three repetitions each time, and the mean value ± standard deviation (SD) was used for data manifestation. Statistical analysis was completed by SPSS 24.0 (IBM Corp., Armonk, NY, USA) and figures were obtained via Graphpad Prism 7 (GraphPad Inc., La Jolla, CA, USA). Student's t-test for two groups and one-way analysis of variance (ANOVA) followed by Tukey's test for multiple groups were used for the analysis of difference. Statistically, P < 0.05 represented a significant difference.

HG blocked HK-2 cell growth while inducing apoptosis promotion, inflammatory response, and NF-κB activation
To explore the effects of HG on HK-2 cells, we analyzed various biological processes through a series of experiments. In comparison to NG and LG groups, cell viability by CCK-8 ( Figure 1a) and colonizing ability by colony formation assay (Figure 1b) were reduced after HG treatment with significant changes. Differently, the increased caspase-3 activity (Figure 1c) and apoptosis rate (Figure 1d) in HG-treated HK-2 cells indicated that cell apoptosis was notably facilitated by HG. Then, the inflammatory cytokines were determined by ELISA. The results exhibited that IL-6, TNF-α, and IL-1β levels in HG-treated HK-2 cells were evidently higher than those in NG-or LG-treated cells, suggesting that HG treatment resulted in the inflammatory response (Figure 1e-g). Western blot indicated that HG evoked the upregulation of NLRP3 and IL-1β/pro-IL-1β (the characteristic proteins of inflammation) as well as NF-κB p-p65/p65 (Figure 1h). HG treatment could lead to cellular apoptosis and inflammation in HK-2 cells to simulate the DN environment in vitro, and NF-κB pathway was activated by HG.

MIAT reestablishment antagonized the HG-induced cell damages and HGactivated NF-κB pathway in HK-2 cells
MIAT level was determined by RT-qPCR in glucose-treated HK-2 cells. The data revealed that the expression of MIAT was decreased in LG and HG groups relative to NG group, and the inhibitory effect of HG on MIAT was more significant in HG group than that in LG group ( Figure A1a). Furthermore, MIAT vector was constructed for the functional analysis of MIAT in HG-treated HK-2 cells. The RT-qPCR showed that MIAT transfection promoted MIAT expression with 7-fold changes contraposed to vector transfection group in HK-2 cells (Figure 2a) and it also eliminated the HG-induced MIAT downregulation in HK-2 cells (Figure 2b). After MIAT level was upregulated, the HG-mediated inhibitory effects on cell viability (Figure 2c) and colonizing capacity (Figure 2d) together with the stimulative influences on cell apoptosis (Figure 2e-f) and inflammation cytokines levels (Figure 2g-i) were all alleviated at least in part. Also, the inflammation-related NLRP3, IL-1β/pro-IL-1β, and NF-κB-related p-p65/p65 levels were downregulated in HG + MIAT group compared with HG treatment group (Figure 2j). These findings revealed that the reestablishment of MIAT level could inhibit the cell damages and NF-κB signal in HG-treated HK-2 cells.

MIAT directly interacted with miR-182-5p
The online miRcode prediction software exhibited the site binding between the sequences of MIAT (UGCCAAA) and miR-182-5p (ACGGUUU) (Figure 3a), showing the potential of miR-182-5p as a target for MIAT. Compared to MUT-MIAT luciferase reporter plasmid containing the point mutation (CAUUGGG) in miR-182-5p-binding sites, we found that the overexpression of miR-182-5p (Figure 3b) repressed the luciferase intensity of WT-MIAT plasmid in the dual-luciferase reporter assay (Figure 3c). Also, MIAT and miR-182-5p could simultaneously bind to Ago2 protein in RIP assay (Figure 3d). In addition, MIAT was largely captured by Bio-miR-182-5p in comparison to Bio-miR-NC group (Figure 3e). The RT-qPCR manifested that siRNAmediated suppressive effect on the expression of MIAT was successful and si-MIAT-2 was the most efficient ( Figure 3f). MIAT overexpression distinctly reduced the miR-182-5p level, while silence of MIAT induced the opposite influence on miR-182-5p (Figure 3g). All these assays demonstrated the interaction between MIAT and miR-182-5p, and the negative regulation of MIAT on miR-182-5p.

Knockdown of MIAT promoted the HGinduced cell effects and NF-κB signal by upregulating the miR-182-5p level
RT-qPCR demonstrated that miR-182-5p level was increasingly elevated in LG and HG groups in contrast with NG group ( Figure A1b). The transfection of anti-miR-182-5p has reduced the miR-182-5p expression in HK-2 cells compared to anti-NC transfection (Figure 4a). The functional analysis has demonstrated that the inhibition of miR-182-5p could reduce all the HG-triggered toxicity in HK-2 cells ( Figure A2). To explore whether the function of MALAT1 in regulating cell injury was related to miR-182-5p, HG-treated HK-2 cells were transfected with si-NC, si-MIAT-2, si-MIAT-2 + anti-NC, and si-MIAT-2 + anti-miR-182-5p. The RT-qPCR showed that the upregulation of miR-182-5p level caused by si-MIAT-2 was attenuated following the inhibition of miR-182-5p in HG-treated cells (Figure 4b). MIAT downregulation reduced cell viability ( Figure 4c) and colony formation ability (Figure 4d), but promoted cell apoptosis (Figure 4e-f) and inflammatory cytokine release (Figure 4g-i) in HG-treated HK-2 cells, whereas all these effects were abolished by miR-182-5p inhibitor. The protein levels of NLRP3, IL-1β/pro-IL-1β, and p-p65/p65 were all markedly higher in HG + si-MIAT-2 group than those in HG + si-NC group, while the downregulation of miR-182-5p returned the si-MIAT-2-mediated exacerbation of inflammation and activation of NF-κB pathway (Figure 4j). From the above results, we could find that cell damages and NF-κB signal activation induced by HG were enhanced by MIAT downregulation to elevate the level of miR-182-5p.

GPRC5A upregulation suppressed the miR-182-5p-mediated cell damages and NF-κB signal in HG-treated HK-2 cells
The expression analysis for GPRC5A has manifested that the mRNA level of GPRC5A was downregulated after treatment of LG or HG in comparison to the treatment of NG in HK-2 cells, and the HG-induced expression change was more obvious than LG ( Figure A1c). GPRC5A overexpression vector was constructed to analyze the effects of miR-182-5p and GPRC5A on DN. The transfection efficiency of GPRC5A was great in HK-2 cells (Figure 6a and b). HK-2 cells were then treated with HG + miR-NC, HG + miR-182-5p, HG + miR-182-5p + pcDNA, or HG + miR-182-5p + GPRC5A. The introduction of GPRC5A lightened the expression inhibition of GPRC5A mRNA and protein caused by miR-182-5p, also exhibiting the successful overexpression of GPRC5A vector (Figure 6c and d). CCK-8 and colony formation assays manifested that cell viability (Figure 6e) and cloned cells (Figure 6f) were inhibited by miR-182-5p mimic in HK-2 cells treated with HG, while this inhibition was abated after the upregulation of GPRC5A. The same reversal of GPRC5A overexpression was found in miR-182-5p-induced promotion of caspase-3 activity (Figure 6g) and apoptosis rate (Figure 6h) in HG-treated HK-2 cells. The introduction of miR-182-5p also facilitated the IL-6, TNF-α, and IL-1β levels, but the following GPRC5A transfection weakened the inflammation promotion triggered by miR-182-5p (Figure 6i and k). Also, miR-182-5p enhanced the protein expression of NLRP3, IL-1β/pro-IL-1β, and p-p65/p65 to activate cell inflammation and NF-κB pathway via inhibiting the level of GPRC5A in HG-induced DN cell model (Figure 6l). Collectively, miR-182-5p also aggravated the HG-triggered HK-2 cell damages and accelerated the HG-induced NF-κB pathway by downregulating GPRC5A.

Discussion
DN is a progressive disorder with great threat for diabetic patients, and lncRNAs have recently become the research emphasis in DN [26]. In this study, we provided interesting evidence for the involvement and functional mechanism of MIAT in DN progression. Dysfunction of renal tubule is the initial event for DN and renal tubular epithelial cells have crucial functions in DN [27]. In the renal tubular epithelial cells (HK-2), we found that HG treatment generated a suppressive effect on cell growth and a promoting effect on cell apoptosis. By detecting the representative inflammatory cytokines (IL-6, TNF-α, and IL-1β), HG was observed to induce the inflammatory response in HK-2 cells. NLRP3 inflammasome plays a significant role in the initiation of inflammation to evoke the release of inflammatory cytokines [28]. Cellular NF-κB pathway can be activated in the process of inflammatory response of DN [29]. Our western blot results indicated that NLRP3, IL-1β, and NF-κB p-p65 protein levels were all enhanced in HG-treated HK-2 cells, insinuating that HG induced cell inflammation and activated the NF-κB pathway.
Mounting studies have suggested the correlations of lncRNAs with diabetic complications. For instance, MALAT1 enhanced the HG-induced cartilage endplate cell apoptosis by the p38/MAPK signaling pathway [30] and CASC15 promoted diabetes-induced chronic renal failure and podocyte apoptosis [31]. It has been found that MIAT facilitated diabetic retinopathy via activating TGF-β1 signaling [32] and induced cardiomyocyte apoptosis in diabetic cardiomyopathy by targeting the miR-22-3p/DAPK2 axis [33]. lncRNA-Gm4419 knockdown inhibited the NF-κB pathway and inflammatory response in DN [34]. Our analysis exhibited that MIAT impeded the HG-induced cell damages and HG-activated NF-κB pathway in vitro. First, we found that the reestablishment of MIAT expression elevated cell viability in HG-stimulated HK-2 cells and this result was in accordance with the previous study [15]. In conformity to the issued findings of MIAT [16], we furthered affirmed the proliferative effect of MIAT on HG-treated HK-2 cells using colony formation assay. In addition, cell apoptosis and inflammation were repressed after the upregulation of MIAT. Zhang et al. stated that MIAT contributed to the HGinduced apoptosis and inflammation in podocytes to accelerate the DN progression [35]. On the contrary, the current data suggested that MIAT functioned as an inhibitor in the development of DN. lncRNAs can affect gene expression and the downstream signaling pathways by functioning as the sponges of miRNAs [36,37]. Here miR-182-5p was first affirmed as a miRNA target of MIAT and the functional regulation of MIAT on DN or NF-κB pathway was achieved by targeting miR-182-5p in HG-treated HK-2 cells. For miRNAs, miR-451 repressed the NF-κB-mediated inflammatory response in DN by downregulating LMP7 [38] and miR-140-5p lightened HG-induced apoptosis or inflammation via inhibiting the TLR4/NF-κB signaling pathway in HK-2 cells [39]. We have ensured that miR-182-5p targeted GPRC5A to regulate the expression of GPRC5A, negatively. Furthermore, miR-182-5p enhanced the DN progression and NF-κB signal in HG-treated HK-2 cells by targeting GPRC5A. The inhibitory effect of GPRC5A on NF-κB pathway has been discovered in lung inflammation [40]. PVT1 facilitated the HG-induced mesangial cell proliferation and fibrosis via sponging miR-23b-3p to result in the regulation of WT1 [41]. MALAT1 enhanced the DN progression in renal tubular  (a and b) The efficiency of GPRC5A vector was analyzed via RT-qPCR and western blot. (c and d) After HK-2 cells were treated with HG + miR-182-5p, HG + miR-182-5p + GPRC5A, or the relative controls, the RT-qPCR and western blot were conducted to examine the GPRC5A expression. (e-k) CCK-8 for cell viability (e), colony formation assay for clonal ability (f), caspase-3 assay/flow cytometry for cell apoptosis (g and h) and ELISA for inflammation (i-k) were used for the assessment of the different transfections on biological processes. (l) Western blot was administrated to determine the protein levels related to inflammation and NF-κB pathway. *P < 0.05, **P < 0.01, ***P < 0.001. epithelial cells by increasing NLRP3 level through playing the sponge role for miR-30c [42]. Ji et al. have revealed that MIAT served as a miR-147a sponge to regulate E2F3 in DN [16]. MIAT was also involved in the pathogenesis of DN by sponging miR-130a-3p and regulating TLR4 [35]. Herein our data revealed that MIAT could affect the expression of GPRC5A via the sponge effect on miR-182-5p. Thus, this study provided direct evidence for the presence of MIAT/ miR-182-5p/GPRC5A axis in the regulation of DN progression and NF-κB pathway.
However, this study has some limitations. For example, the dose scope of glucose treatment was not wide. Zhou et al. have found that cell viability reduction and MIAT expression downregulation were more significant after 30 or 45 mM of glucose treatment [15]. Exploring whether MIAT or GPRC5A overexpression and miR-182-5p downregulation can antagonize the effects of higher dose of glucose on HK-2 cells is also important. In addition, all experiments were conducted in cell line in vitro. It is necessary to investigate the expression level of MIAT in clinical samples (or primary cells) and the correlation among MIAT, miR-182-3p, and GPRC5A. Moreover, the research of MIAT/miR-182-5p/GPRC5A axis in vivo remains to be performed in future.