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LncRNA GAS5 reduces blood glucose levels and alleviates renal fibrosis in diabetic nephropathy by regulating the miR-542-3p/ERBB4 axis

Diabetology & Metabolic Syndrome volume 17, Article number: 30 (2025) Cite this article

Abstract

Objective

The present study was implemented to unravel the effect of lncRNA GAS5 on renal fibrosis induced by diabetic nephropathy (DN) by regulating the miR-542-3p/ERBB4 axis.

Methods

db/db mice were injected with lncRNA GAS5 high expression or miR-542-3p low expression related vectors. Biochemical experiments were performed to assess blood glucose level and urine protein concentration. HE, TUNEL and Masson stainings were employed to observe the cellular morphology, apoptosis, and fibrosis of renal tissues, respectively. ELISA was executed to examine the levels of IL-1β, IL-6, and TNF-α; and the superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) activities were evaluated. Bioinformatics analysis, dual-luciferase and RIP assays were performed to verify the relationship between lncRNA GAS5 and miR-542-3p, and miR-542-3p and ERBB4.

Results

LncRNA GAS5 and ERBB4 were lowly expressed and miR-542-3p was highly expressed in the renal tissues of DN mice. Overexpression of lncRNA GAS5 or low-expression of miR-542-3p diminished DN-induced renal fibrosis. LncRNA GAS5 could bind to miR-542-3p and miR-542-3p further modulating ERBB4 expression. Up-regulation of miR-542-3p neutralized the suppressive effect of lncRNA GAS5 overexpression and down-regulation of ERBB4 also counteracted the inhibitory impact of down-regulation of miR-542-3p on renal fibrosis in DN mice.

Conclusion

Up-regulation of lncRNA GAS5 alleviates renal fibrosis in DN mice via down-regulation of miR-542-3p and up-regulation of ERBB4.

Introduction

Diabetes nephropathy (DN) is the primary reason for end-stage renal disease (ESRD) worldwide. Its main initiating mechanism is hyperglycemia-evoked vascular dysfunction, while its progression resultes from different pathological mechanisms, consisting of oxidative stress, inflammation, as well as fibrosis [1]. Nowadays, the first-line medicines for treating DN mainly include angiotensin receptor blockers or renin-angiotensin system inhibitors, as well as the latest approved aldosterone receptor antagonist finerenone. These treatments can delay the progression of DN to ESRD, while the treatment efficacy is still not ideal [2]. Therefore, there is an urgent demand to create new therapies that are both safe and effective. Renal fibrosis is a complicated and irreversible process in DN’s late stage, further exacerbating the disease progression [3]. Fibrosis is regarded as a harmful condition, but this process has a protective effect as it helps maintain crosstalk with damaged proximal renal tubular cells, supporting their regeneration [4]. Therefore, evaluating the existence and degree of renal fibrosis in DN patients and forecasting their long-term outcomes are of great importance.

Recently, multiple lncRNAs have been investigated in the therapy of DN [5, 6]. People are increasingly concerned about lncRNA GAS5 as a potential biomarker for the pathogenesis and development of various diseases [7, 8]. The lncRNA GAS5 is able to mediate cell growth, and proliferation, along with survival. LncRNAGAS5 downregulation is associated with the occurrence of diabetes [9]. As previously reported, lentivirus-mediated GAS5 overexpression can alleviate streptozocin-induced renal interstitial fibrosis together with inflammatory reaction in DN rats [10]. miRNAs participate in the modulation of many cellular biological processes, consisting of proliferation, differentiation, and apoptosis [11]. Due to the stability and detectability of miRNA in human biological fluids, correct evaluation of miRNA profiles in biological fluids is attractive in clinical translational research, as early diagnosis of DN can effectively prevent renal failure [12]. Existing data has indicated that miR-542-3p is reduced in diverse types of cancers, which is involved in multiple cancer-related behaviors, such as glycolysis, metastasis, apoptosis, as well as proliferation [13]. In particular, miR-542-3p is a potent biomarker of corneal neuropathy in diabetes. Suppressing miR-542-3p enhances autophagy and augments corneal debridement healing, supplying a novel therapeutic option for clinical therapy [14]. ERBB4 is a vital developmental protein expressed in developing nephrons, playing a part in epithelial cell proliferation and tubular formation, and is found in podocytes [15]. ERBB4 may function in glucose homeostasis and fat production, and its deficiency has been concerned with obesity and adipose tissue inflammation, contributing to the development of metabolic syndrome [16]. Competing endogenous RNA (ceRNA) is a pivotal modulatory mechanism between lncRNAs and miRNAs, and has been regarded as a novel mechanism for DN [17]. Herein, the present study was implemented to unravel the effect of lncRNA GAS5 on renal fibrosis induced by DN by regulating the miR-542-3p/ERBB4 axis.

Materials and methods

Ethics statement

This study was conducted following the protocols approved by the Animal Ethics Committee of West China Hospital of Sichuan University.

Animals and treatment

Eight-week-old male db/m mice and db/db mice (provided by the Animal Research Center of Sichuan University) were housed in a standard animal room at a temperature of 20–25 °C with a 12/12 h light/dark cycle. The db/db mice were divided into the following groups: lncRNA GAS5 overexpression negative control group (oe-NC), lncRNA GAS5 overexpression group (oe-lncRNA GAS5), miR-542-3p low-expression negative control group (anti-NC), miR-542-3p low-expression group (anti-miR-542-3p), lncRNA GAS5 overexpression and miR-542-3p high-expression negative control group (oe-lncRNA GAS5 + miR-NC), lncRNA GAS5 overexpression and miR-542-3p high-expression group (oe-lncRNA GAS5 + miR-542-3p), miR-542-3p low-expression and ERBB4 low-expression blank control group (anti-miR-542-3p + si-NC), as well as miR-542-3p low-expression and ERBB4 low-expression group (anti-miR-542-3p + si-ERBB4) (n = 6). Meanwhile, db/m mice were set as a normal control group (control). At the age of 10 weeks, db/db mice were injected every 2 weeks via tail vein [18] with the related vectors of oe-lncRNA GAS5, anti-miR-542-3p, miR-542-3p, or si-ERBB4. Mice were sacrificed at 20 weeks of age [19].

Biochemical analysis

Fasting blood glucose was measured by Glucose LiquiColor Test (Stanbio Laboratory, Boerne, TX). Urine samples were collected from mice (24 h) using metabolic cages and urinary albumin was determined by competitive ELISA as per the manufacturer’s instructions [20].

HE staining

Kidney tissues of mice in each group were subjected to fixation with 4% paraformaldehyde, 1-h washing with distilled water, dehydration in gradient ethanol at 70%, 80%, 90%, and 100% (each 1 min), and permeabilization with xylene twice, each time for 5 min, followed by paraffin-embedding and slicing into 5-µm-thick slices. The sections were then spread on slides, baked at 60 °C for 1 h, and stored at ambient temperature. After routine dewaxing with xylene, the sections were subjected to 10-min staining with hematoxylin, treatment with 1% hydrochloric acid aqueous solution, and 10-min staining with eosin. After routine dehydration, cleaning, and neutral resin blocking, the pathology of kidney tissue was observed under a light microscope. Images were acquired using ImageJ software [21].

TUNEL staining

TUNEL kit (In Situ Cell Death Detection Kit, Fluorescein, Roche) was implemented to detect apoptosis in kidney tissues. Briefly, mouse kidney tissue sections were deparaffinized and hydrated, and then treated with Proteinase K (Roche) at 37 °C for 15–30 min. After that, the sections were incubated with TUNEL reaction solution (50 µl) for 60 min at 37 °C. The nuclei were stained with DAPI for 10 min. Fluorescence microscopy was used to observe the TUNEL-positive cells (Olympus, Tokyo, Japan), and positive cells were counted using Image J [22].

Masson staining

After 5-min Reguad staining and color separation by picric acid ethanol solution, the sections were rinsed once with double-distilled water. After 5-min staining with Ponceau S, the sections were rinsed once with double-distilled water, and then sequentially immersed in 1% phosphoaluminate for 5 min, aniline acetate blue solution for 7 min, and 1% acetic acid for 1 min, followed by dehydration with 95% ethanol and anhydrous ethanol, permeabilization by xylene, and blocking with neutral gum. Digital images of kidney tissues were acquired using a light microscope (Olympus) equipped with image analysis software (Image-Pro Plus version 6.0; Media Cybernetics, Bethesda, MD, USA). Ten fields of view were randomly selected in each section and images were captured with a microscope. Image acquisition and analysis were performed using ImageJ software [21].

Measurement of inflammatory response and oxidative stress indices

Kidney tissue was homogenized and the supernatant was acquired by centrifugation at 1,000 ×g for 20 min, and then assayed immediately. The levels of TNF-α, IL-1β and IL-6 were evaluated by ELISA kits. The levels of malondialdehyde (MDA) were measured by MDA assay kit (TBA method), Superoxide Dismutase (SOD) levels by SOD assay kit (WST-1 method), and catalase (CAT) levels by CAT assay kit (Ammonium Molybdate method). The above kits were available from Nanjing JianCheng Bioengineering Institute (Nanjing, China).

Dual-luciferase assay

Wild and mutant reporter plasmids for GAS5 (lncRNA GAS5-WT and lncRNA GAS5-MUT) and ERBB4 (ERBB4-WT and ERBB4-MUT) containing miR-542-3p binding sites were synthesized by GenePharma (Shanghai, China). For reporter gene detection, the reporter plasmids (lncRNA GAS5-WT or lncRNA GAS5-MUT, ERBB4-WT or ERBB4-MUT) were co-transfected with miR-542-3p mimic or anti-NC into HEK293T cells using Lipofectamine 2000. Upon 48-h transfection, luciferase activity was tested using a dual luciferase reporter gene assay system (Promega, Madison WI, USA), and standardized by measuring Renilla luciferase activity [23].

RNA immunoprecipitation (RIP) assay

RIP experiments were implemented using the EZ-Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Millipore, Beford, MA, USA). Briefly, cells were lysed in RIP lysis buffer as per the manufacturer’s recommendations. Subsequently, pre-incubated magnetic beads coated with the indicated antibodies were immunoprecipitated with the supernatant of cell lysates for 6 h at 4 °C. After that, purified RNA was examined by RT-qPCR [24].

RT-qPCR

Total RNA content was extracted using Trizol kit (Invitrogen, Car, USA). LncRNA and mRNA were detected using FastQuant RT kit including gDNase (Tiangen, China) and SuperReal Premix Plus-SYBR Green (Tiangen). miRNAs were examined using miScript reverse transcription kit (QIAGEN, Germany) and miScript SYBR Green PCR kit (QIAGEN). RT-qPCR was performed on the LightCycler96 qPCR system. Relative expression was calculated using the 2−ΔΔCt method. Primer sequences are shown in Supplementary Table 1. GAPDH and U6 were used as endogenous controls for lncRNA, mRNA and miRNA normalization, respectively.

Western blot

Total protein was extracted using RIPA lysis buffer (Elabscinece, Pudong, Shanghai, China). Protein concentration was assessed by BCA (Thermo Fisher Scientific, USA) method. Extracted proteins were subjected to SDS-PAGE (10%) and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). After blocking with 5% bovine serum albumin, the membranes were incubated with the appropriate primary antibodies overnight at 4 °C and then with secondary antibodies ((ab6721, 1:2000, Abcam) for 1 h at ambient temperature. Primary antibodies: ERBB4 (4795, 1:1000, Cell Signaling Technology, USA) and GAPDH (2118, 1:1000, Cell Signaling Technology). After that, the ECL kit (34096, ThermoFisher Co, San Francisco, California, USA) was then used for the detection of protein blotting [25].

Statistical methods

All data were processed using SPSS 21.0 statistical software (IBM, Armonk, NY, USA). Data were expressed as mean ± standard deviation and compared by the t-test or one-way analysis of variance (ANOVA) and Tukey’s post-hoc test. P < 0.05 was considered statistically significant.

Results

Establishment of DN mouse models

The blood glucose levels of the mice were measured using a glucometer, while their urine albumin concentrations were determined through the Bradford method. Figure 1A-B showed that DN mice had significantly higher blood glucose and urine protein excretion rates. ELISA assay suggested that the inflammatory factors IL-1β, IL-6 and TNF-α had significantly elevated levels in kidney tissues in DN mice (Fig. 1C).TUNEL staining revealed that apoptosis was more pronounced in kidney tissues of DN mice (Fig. 1D). Oxidative stress measurements indicated that DN mice exhibited elevated MDA content and reduced SOD and CAT activities in kidney tissues (Fig. 1E-G). Based on the observations of HE staining, we found that control mice had an intact renal base without glomeruli, fibrous tissue hyperplasia, or interstitial hyperplasia, whereas DN mice had hyperplasia of mesangial cells, increased extracellular matrix of the mesangial membranes, thickening of the basement membranes, increased inflammatory cells in the interstitium, and infiltration of fibrous tissue hyperplasia (Fig. 1H). Masson staining showed a large number of fibroblasts proliferating in the kidney tissues and forming collagen fibers (blue) in the renal interstitium in the DN mice with diffuse inflammatory cell infiltration as compared with the control mice (Fig. 1I). Additionally, the mRNA levels of fibrosis-related factors in the renal tissue of DN mice were newly increased (Fig. 1J). These results indicated that the DN mouse model was established successfully.

Fig. 1
figure 1

DN mouse model establishment. A-B: Biochemical experiments were conducted to detect blood glucose level and urine protein concentration. C. ELISA was utilized to test the levels of three inflammatory factors (IL-1β, IL-6 and TNF-α). D. TUNEL staining was employed to assess apoptosis in kidney tissues. E-G: Oxidative stress measurement was adopted to measure MDA content and SOD and CAT activities. H. HE staining was used to observe the cellular morphology of kidney tissues (left: 100×; right: 400×). I. Masson staining was implemented to observe the fibrosis of kidney tissues (400×). J: qRT-PCR were used to detect the mRNA levels of fibrosis-related factors in the renal tissue of DN mice. # P < 0.05 vs. control group

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Up-regulation of lncRNA GAS5 suppresses renal fibrosis caused by DN

Relevant studies have demonstrated that lncRNA GAS5 is down-regulated in DN, inhibits the development of DN in vivo, and is negatively correlated with the severity of DN-related complications [17]. RT-qPCR was executed to compare lncRNA GAS5 expression in kidney tissues of DN mice and control mice. LncRNA GAS5 expression in the kidney tissues of DN mice was found to be down-regulated in DN mice (Fig. 2A). To further unravel the role of lncRNA GAS5 on renal fibrosis in DN mice, we up-regulated lncRNA GAS5 in kidney tissues of DN mice (Fig. 2B). Biochemical analysis showed that blood glucose and urinary protein excretion rates were markedly reduced in DN mice overexpressing lncRNA GAS5 (Fig. 2C-D). ELISA assay indicated that the levels of IL-1β, IL-6 and TNF-α were notably reduced in DN mice overexpressing lncRNA GAS5 (Fig. 2E). TUNEL staining unveiled that the cell apoptosis was significantly reduced in kidney tissues of DN mice overexpressing lncRNA GAS5 (Fig. 2F). Oxidative stress measurements showed that up-regulation of lncRNA GAS5 suppressed the MDA content and promoted the activities of SOD and CAT in the kidney tissues of DN mice (Fig. 2G-I). HE and Masson stainings presented that glomerular mesangial cell proliferation, glomerular and interstitial hyperplasia, and fibrosis were alleviated in DN mice overexpressing lncRNA GAS5 (Fig. 2J), and collagen fibers (blue) were significantly reduced (Fig. 2K). Furthermore, after overexpressing lncRNA GAS5, the mRNA levels of fibrosis-related factors in the renal tissue of DN mice were reduced (Fig. 2L). These experimental results revealed that lncRNA GAS5 was lowly expressed in kiney tissues of DN mice, and up-regulation of lncRNA GAS5 had an inhibitory effect on DN-induced renal fibrosis.

Fig. 2
figure 2

Up-regulation of lncRNA GAS5 inhibits renal fibrosis caused by diabetic nephropathy. A-B. RT-qPCR was performed to detect the expression level of lncRNA GAS5. C-D. Biochemical experiments were conducted to detect blood glucose levels and urine protein concentration after oe-lncRNA GAS5 treatment. E. ELISA was utilized to test the levels of three inflammatory factors (IL-1β, IL-6 and TNF-α) after oe-lncRNA GAS5 treatment. F. TUNEL staining was employed to assess apoptosis in kidney tissues after oe-lncRNA GAS5 treatment. G-I: Oxidative stress measurement was adopted to measure MDA content and SOD and CAT activities after oe-lncRNA GAS5 treatment. J. HE staining was used to observe the cellular morphology of kidney tissues after oe-lncRNA GAS5 treatment (left: 100×; right: 400×). K. Masson staining was implemented to observe the fibrosis of kidney tissues after oe-lncRNA GAS5 treatment (400×). L. qRT-PCR were used to detect the mRNA levels of fibrosis-related factors in the renal tissue of DN mice. # P < 0.05 vs. control group; * P < 0.05 vs. oe-NC group

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LncRNA GAS5 binds mir-542-3p and modulates its expression

Down-regulation of miR-542-3p prevents liver fibrosis in vitro and in vivo [26]. To test whether there is a binding relationship between lncRNA GAS5 and miR-542-3p, the binding site of lncRNA GAS5 to miR-542-3p was predicted using the bioinformatics website DIANA (Fig. 3A). The results were verified by dual-luciferase and RIP assays, which showed that miR-542-3p overexpression reduced the luciferase activity of lncRNA GAS5-WT, and both lncRNA GAS5 and miR-542-3p were more enriched in Ago2, demonstrating that lncRNA GAS5 could bind miR-542-3p (Fig. 3B-C).

Fig. 3
figure 3

LncRNA GAS5 can bind miR-542-3p and regulate its expression. (A) The existence of binding site of lncRNA GAS5 and miR-542-3p. (B) Dual-luciferase reporter gene assay was conducted to verify the binding of lncRNA GAS5 to miR-542-3p. (C) RIP assay was utilized to detect the enrichment of lncRNA GAS5 with miR-542-3p. D-E. RT-qPCR was employed to evaluate the miR-542-3p expression level. # P < 0.05 vs. control group; * P < 0.05 vs. oe-NC group

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miR-542-3p expression in kidney tissues of DN mice and control mice was tested by RT-qPCR, and the findings demonstrated that miR-542-3p was up-regulated in kidney tissues of DN mice (Fig. 3D). To investigate the regulatory relationship between lncRNA GAS5 and miR-542-3p in kidney tissues of DN mice, miR-542-3p expression in DN mice after overexpression of lncRNA GAS5 was examined by RT-qPCR analysis, and oe-lncRNA GAS5 remarkably down-regulated miR-542-3p expression (Fig. 3E). It was suggested that lncRNA GAS5 could bind miR-542-3p and modulate its expression.

Down-regulation of mir-542-3p restricts renal fibrosis induced by DN

To verify the mechanism of miR-542-3p in kidney tissues of DN mice, we down-regulated miR-542-3p expression in kidney tissues of DN mice (Fig. 4A). Biochemical analyses showed that blood glucose and urinary protein excretion rate were reduced in DN mice with down-regulated miR-542-3p (Fig. 4B-C). Meanwhile, in DN mice with down-regulated miR-542-3p, there showed a reduction in IL-1β, IL-6 and TNF-α levels (Fig. 4D), a significant reduction in apoptosis (Fig. 4E), a reduction in the MDA content, and an elevation in the activities of SOD and CAT in the kidney tissues (Fig. 4F-H). HE and Masson stainings showed that glomerular mesangial cell proliferation, glomerular and interstitial hyperplasia, and fibrosis were alleviated in DN mice after down-regulation of miR-542-3p (Fig. 4I), and collagen fibers (blue) were notably reduced (Fig. 4J). Additionally, after downregulating miR-542-3p, the mRNA levels of fibrosis-related factors in the renal tissue of DN mice were reduced (Fig. 4K). It was summarized that down-regulation of miR-542-3p had an inhibitory effect on DN-induced renal fibrosis.

Fig. 4
figure 4

Down-regulation of miR-542-3p inhibits renal fibrosis caused by diabetic nephropathy. A. RT-qPCR was performed to detect the expression level of miR-542-3p. B-C. Biochemical experiments were conducted to detect blood glucose levels and urine protein concentration after down-regulated miR-542-3p treatment. D. ELISA was utilized to test the levels of three inflammatory factors (IL-1β, IL-6 and TNF-α) after down-regulated miR-542-3p treatment. E. TUNEL staining was employed to assess apoptosis in kidney tissues after down-regulated miR-542-3p treatment. F-H: Oxidative stress measurement was adopted to measure MDA content and SOD and CAT activities after down-regulated miR-542-3p treatment. I. HE staining was used to observe the cellular morphology of kidney tissues after down-regulated miR-542-3p treatment (left: 100×). J. Masson staining was implemented to observe the fibrosis of kidney tissues after down-regulated miR-542-3p treatment (400×). K: qRT-PCR were used to detect the mRNA expression levels of fibrosis-related factors in the renal tissue of DN mice. # P < 0.05 vs. anti-NC group

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Mir-542-3p directly targets ERBB4

As reported, ERBB4 deficiency accelerates the development and progression of renal fibrosis in obstructive nephropathy [27]. We predicted the possible targets of miR-542-3p with ERBB4 by bioinformatics website RNA22 (Fig. 5A). To confirm whether ERBB4 is a direct target of miR-542-3p, we inserted WT or MUT containing the binding sequences into luciferase reporter vectors and co-incorporated them into HEK293T cells with mimic NC or miR-542-3p mimic, respectively, and the experimental results demonstrated that overexpression of miR-542-3p resulted in decreased luciferase activity in ERBB4-WT, but no significant change in luciferase activity in ERBB4-MUT (Fig. 5B). RIP experiments show elevated enrichment of miR-542-3p and ERBB4 in Ago2 (Fig. 5C). ERBB4 was significantly down-regulated in kidney tissues of DN mice (Fig. 5D-E). ERBB4 expression in DN mice was also examined after overexpression of lncRNA GAS5 or down-regulation of miR-542-3p, which showed that both overexpression of lncRNA GAS5 or down-regulation of miR-542-3p resulted in a significant up-regulation of the expression levels of ERBB4 in the kidney tissues of DN mice (Fig. 5F-G). The above experimental results revealed that miR-542-3p directly targeted ERBB4 in kidney tissues of DN mice.

Fig. 5
figure 5

miR-542-3p directly targets ERBB4. (A) Bioinformatics website RNA22 was utilized to predict the presence of the target binding site of miR-542-3p with ERBB4. (B) Dual-luciferase reporter gene assay was employed to verify the binding of miR-542-3p with ERBB4. (C) RIP assay was conducted to test the enrichment of miR-542-3p with ERBB4. D-E. RT-qPCR and western blot were carried out to determine ERBB4 expression levels in kidney tissues of DN mice. F-G. RT-qPCR and Western blot were performed to determine ERBB4 expression levels in kidney tissues of DN mice with lncRNA GAS5 overexpression or miR-542-3p down-regulation. ^ P < 0.05 vs. mimic NC group; * P < 0.05 vs. control group; & P < 0.05 vs. oe-NC group; # P < 0.05 vs. anti-NC group

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LncRNA GAS5 promotes DN-induced renal fibrosis in mice via the miR-542-3p/ERBB4 axis

To unveil the impact of lncRNA GAS5 on renal fibrosis induced by DN in mice by mediating the miR-542-3p/ERBB4 axis, oe-lncRNA GAS5 + miR-NC, oe-lncRNA GAS5 + miR-542-3p, anti-miR-542-3p + si-NC, and anti-miR-542-3p + si-ERBB4 were injected into DN mice, and successful injection was verified by RT-qPCR and western blot (Fig. 6A-B). The results of biochemical analysis, ELISA assay, TUNEL staining, oxidative stress measurement, HE staining, and Masson staining indicated that up-regulation of miR-542-3p neutralized the suppressive effect of lncRNA GAS5 overexpression and down-regulation of ERBB4 also counteracted the inhibitory impact of down-regulation of miR-542-3p on renal fibrosis in DN mice (Fig. 6C-L).

Fig. 6
figure 6

LncRNA GAS5 promotes DN-induced renal fibrosis in mice via the miR-542-3p/ERBB4 axis. A-B. RT-qPCR and western blot were performed to detect the expression levels of ERBB4. C-D. Biochemical experiments were conducted to detect blood glucose level and urine protein concentrations in the rescue experiment. E. ELISA was utilized to test the levels of three inflammatory factors (IL-1β, IL-6 and TNF-α) in the rescue experiment. F. TUNEL staining was employed to assess apoptosis in kidney tissues in the rescue experiment. G-I: Oxidative stress measurement was adopted to measure MDA content and SOD and CAT activities in the rescue experiment. J. HE staining was used to observe the cellular morphology of kidney tissues in the rescue experiment. K. Masson staining was implemented to observe the fibrosis of kidney tissues in the rescue experiment. L. qRT-PCR were used to detect the mRNA levels of fibrosis-related factors in the renal tissue of DN mice. # P < 0.05 vs. oe-lncRNA GAS5 + miR-NC group; * P < 0.05 vs. anti-miR-542-3p + si-NC group

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Discussion

The pathogenesis of DN is complex and remains to be fully elucidated. The traditional view is that DN pathogenesis involves hemodynamic effects, genetic factors, blood glucose levels, and lipid metabolism disorders [28]. With the development of multiple pharmacological and non-pharmacological interventions, significant progress has been achieved in DN therapy. This is due to people’s increasing recognition of the molecular mechanisms of this disease, such as the effects of oxidative stress, inflammation, and epigenetic modifications [29]. Herein, the present study was implemented to unravel the effect of lncRNA GAS5 on renal fibrosis induced by DN by regulating the miR-542-3p/ERBB4 axis.

Several lncRNAs have been identified as modulators of diabetes-related complications. The up-regulation or down-regulation of lncRNA expression depends on the target genes, disease background, as well as regulatory partners [30]. Relevant research has demonstrated that lncRNA GAS5 is down-regulated in DN, inhibits the development of DN in vivo, and is negatively correlated with the severity of DN-related complications [17]. Similarly, in this study, we observed that lncRNA GAS5 was lowly expressed in kiney tissues of DN mice, and up-regulation of lncRNA GAS5 had an inhibitory effect on DN-induced renal fibrosis. This confirms the significant role of lncRNA GAS5 in the process of DN renal fibrosis. It is also suggested that lncRNA GAS5 levels are linked with the prevalence of diabetes. Evaluation of GAS5 in the serum offers greater accuracy in the identification of individuals experiencing diabetes [31]. Another article has unveiled that lncRNA GAS5 is decreased in serum of diabetes and DN subjects. GAS5 is an independent protective index for fasting blood glucose, implying that depletion of GAS5 may lead to hyperglycemia [32]. Our study also indicated that the blood glucose levels of DN mice with overexpressed lncRNA GAS5 were significantly reduced.

Furthermore, GAS5 overexpression has been revealed to restrict the inflammation and oxidative stress of high glucose-induced HK-2 cells through reducing miR-452-5p, offering a new sight for DN therapy [33], this leads to the role of miRNA in the response of GAS5 to DN. As verified by dual-luciferase and RIP assays, miR-542-3p overexpression reduced the luciferase activity of lncRNA GAS5-WT, and both lncRNA GAS5 and miR-542-3p were more enriched in Ago2, demonstrating that lncRNA GAS5 can bind miR-542-3p, and lncRNA GAS5 may participate in the process of DN renal fibrosis by regulating the expression of miR-542-3p. Changes in miRNA expression profiles are detected in both diabetes and diabetes-related complications. Therefore, these transcripts have remarkable potential and novelty as markers for therapy [34]. miR-542-5p overexpression decreases lipid accumulation, blood glucose, liver weight, plasma insulin, as well as hepatic and plasma triglyceride contents in diabetic mice livers [35]. Guo et al. have stated that miR-542-5p is down-regulated in diabetic retinopathy and in high glucose-induced retinal pigment epithelial cells. Meanwhile, miR-542-5p overexpression suppresses apoptosis in high glucose-induced retinal pigment epithelial cells [36]. Similarly, miR-542-3p is highly expressed in renal fibrosis, and overexpression of miR-542-3p deteriorates renal fibrosis [37]. In our study, it was summarized that DN mice with downregulated miR-542-3p exhibited decreased blood glucose levels and urinary protein excretion rates, and the downregulation of miR-542-3p inhibited DN-induced renal fibrosis.

Targeting tumor miRs offers potential benefits by influencing the expression of diverse mRNAs that serve as their targets, thereby potentially modulating various signaling pathways [38]. Our results also revealed that miR-542-3p directly targeted ERBB4 in kidney tissues of DN mice. Both the overexpression of lncRNA GAS5 and the downregulation of miR-542-3p resulted in an upregulation of ERBB4 expression levels in the renal tissue of DN mice. This indicates that ERBB4 may be involved in the regulation of DN renal fibrosis by lncRNA GAS5 and miR-542-3p. Recently, it is reported that in type 1 diabetes mice, the ERBB4 ligand NRG1 slows down the development of kidney disease by activating the ERBB4 pathway, thus preventing the formation of glomerular matrix and glomerulosclerosis [39]. Zheng et al. have found that ERBB4 deficiency accelerates the development of renal fibrosis after renal injury. The elevated ERBB4 in chronic kidney injury may reflect a compensatory impact in preventing renal tubulointerstitial injury development [27]. Our study further found that upregulating miR-542-3p could reverse the inhibitory effect of lncRNA GAS5 overexpression on DN renal fibrosis, and downregulating ERBB4 could reverse the inhibitory effect of miR-542-3p downregulation on DN renal fibrosis. This suggests that the inhibitory effect of lncRNA GAS5 on DN renal fibrosis is mediated through the regulation of the miR-542-3p/ERBB4 axis.

In summary, we observe that lncRNA GAS5 reduces blood glucose levels and alleviates DN renal fibrosis in DN mice by regulating the miR-542-3p/ERBB4 axis. This lncRNA GAS5/miR-542-3p/ERBB4 network may shed light on DN development and may result in therapeutic targeting for DN, thereby providing new ideas and methods for the treatment of DN. However, this study still has some limitations, such as the limited sample size. Moreover, the interaction network between lncRNA, miRNA, and mRNA is extremely complex, involving multiple regulatory mechanisms and signaling pathways. The current study may have only uncovered a portion of these mechanisms, and there are still many unknown areas that need to be explored. Therefore, future research needs to delve deeper into the mechanisms, expand the sample size, and consider individual differences to promote the development of this field and provide stronger support for its clinical application.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

We thank the associate editor and the reviewers for their useful feedback that improved this paper.

Funding

This study was supported by the Science and Technology Department of Sichuan Province of China (Grants number: 2022YFS0330).

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Authors and Affiliations

  1. Division of Nephrology, Kidney Research Institute, West China Hospital of Sichuan University, Chengdu, 610047, Sichuan, China

    Qinghua Yin

  2. Occupational Disease and Toxicosis/Nephrology, West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu, 610000, Sichuan, China

    Na Guo

  3. Division of Nephrology, West China Hospital of Sichuan University, No.37 Guoxue Lane, Wuhou District, Chengdu, 610047, Sichuan, China

    Ruoxi Liao

Authors
  1. Qinghua Yin
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  2. Na Guo
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Contributions

Y.Q.H. contributed to study design; G.N. contributed to manuscript editing; L.R.X. and Y.Q.H. contributed to experimental studies; G.N. and Y.Q.H. contributed to data analysis. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ruoxi Liao.

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This study was conducted following the approved protocols by the Animal Ethics Committee of West China Hospital of Sichuan University (approval number: 20210417).

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The authors declare no competing interests.

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Yin, Q., Guo, N. & Liao, R. LncRNA GAS5 reduces blood glucose levels and alleviates renal fibrosis in diabetic nephropathy by regulating the miR-542-3p/ERBB4 axis. Diabetol Metab Syndr 17, 30 (2025). https://doi.org/10.1186/s13098-025-01593-z

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  • DOI: https://doi.org/10.1186/s13098-025-01593-z

Keywords

Diabetology & Metabolic Syndrome

ISSN: 1758-5996

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