Long non‑coding RNA L13Rik promotes high glucose-induced mesangial cell hypertrophy and matrix protein expression by regulating miR-2861/CDKN1B axis

Patients and samples

Human blood specimens were obtained with the approval of the Ethics Committee of Shanghai Jiao Tong University Affiliated Tong Ren Hospital (No. 2019-060) according to the Declaration of Helsinki. In total, 13 DN blood samples were obtained from DN patients aged 53.3 ± 12.6 (seven men and six women), and 18 control blood samples were obtained from healthy volunteers aged 57.2 ± 11.1 (11 men and seven women) in Tong Ren Hospital. DN was diagnosed in accordance with previous criteria (An et al., 2015). Written informed consent was obtained from each donor before sampling. These donors had not received any therapies within 3 months before blood sampling.

Animal model

All procedures were approved by the Animal Ethics Committee of Shanghai Jiao Tong University Affiliated Tong Ren Hospital (No. 2019-060) and performed in accordance with the ARRIVE guidelines. Male C57BL/6J wild-type mice, aged 8 weeks, were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China) and housed in a thermostatic room (25 ± 2 °C, 12/12 light/dark cycle, 50 ± 10% relative humidity). Animals were allowed to access chow and water ad libitum. Mice were randomly divided into a control group (n = 10) and a diabetic group (n = 10). Experimental DN was induced by intraperitoneal injection with streptozotocin (STZ, 50 mg/kg; Aladdin, Shanghai, China) dissolved in sodium citrate buffer (pH = 4.5). Access to 20% glucose water was allowed ad libitum for 18 h to prevent transient hypoglycemia. The blood glucose of each animal was measured from 0 to 35 days after STZ administration. Mice whose blood glucose was greater than 16.7 mmol/L after 3 days of STZ injection and subsequently maintained above this level were considered diabetic (Fig. S1A), as previously described (Fan et al., 2013; Hu et al., 2018; Wang et al., 2020). During the experimental period, any animal suffering from disease or dying was euthanized by cervical dislocation under deep anesthesia using pentobarbital sodium (40 mg/kg).

Cell culture

A murine mesangial cell line, SV40-MES-13, was purchased from the American Type Culture Collection (ATCC, Natick, MA, USA) and maintained in DMEM (Gibco, New York, CA, USA) containing 10% FBS (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C under 5% CO₂. To explore the function of glucose concentration on L13Rik expression, MCs were challenged with 30 mM mannitol (Ma) or glucose at concentrations of 5 (normal glucose, NG), 15, and 30 mM (high glucose, HG). After exposure to glucose, cells were harvested for qRT-PCR and western blot assays.

Overexpression and RNA interference (RNAi)

The recombinant lentivirus harboring full-length L13Rik (Lv-L13Rik) and negative control lentivirus were purchased from Sangon (Shanghai, China). L13Rik siRNA (siL13Rik) and miR-2861 mimics were synthesized by Genewiz (Jiangsu, China) and transfected into MCs with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s introduction. The sequence for RNAi and miRNA was shown in Table S1.

Fluorescence in situ hybridization (FISH)

The L13Rik probe was synthesized by Sangon (Shanghai, China). After culturing in 12-well plates in the presence or absence of HG for 48 h, SV40-MES-13 cells were washed by PBS and fixed by 4% PFA for 15 min at room temperature, following treatment with proteinase K, glycine and acetic anhydride. Pre-hybridization was performed at 37 °C for 1 h, followed by hybridization of L13Rik against a 250 ng/mL L13Rik probe. Nuclei were labeled with DAPI, and the L13Rik signal was captured as mentioned earlier.

qRT-PCR

Total RNA in renal tissues and SV40-MES-13 cells was extracted with the RNA Simple Total RNA Kit (Tiangen, Beijing, China). cDNA was synthesized with the TIANScript IIRT Kit (Tiangen, Beijing, China) at 42 °C for 60 min. qRT-PCR was performed in triplicate with FastFire qPCR PreMix (SYBR Green) (Tiangen, Beijing, China) on a ViPlex Fluor real-time PCR system (Vivantis, Selangor, Malaysia). Thermocycling conditions were set as follows: 95 °C for 60 s, followed by 30 cycles of 95 °C for 15 s and 67 °C for 30 s. Gene expressions were calculated by the 2^−ΔΔCT method. Expressions of fibronectin (FN), collagen IV (Col-IV), N-cadherin (N-cad), CDKN1B, and L13Rik were normalized by GAPDH, and expression of miR-2861 was normalized by U6. All primer sequences were listed in Table S1.

Western blot

SV40-MES-13 cells were lysed with the xTractor Buffer Kit (Clontech Laboratories, Inc., Mountain View, CA, USA). Protein specimens were quantified through the BCA assay kit (Beyotime, Shanghai, China) and electrophoresed through 10% SDS-PAGE gels. After transferring onto PVDF membranes, interested proteins were masked with 2% non-fat milk at 37 °C for 30 min, then incubated with primary antibodies against FN (1:700, SAB5700724; Sigma-Aldrich, St. Louis, MO, USA), Col-IV (1:500, ab52235; Abcam, Fremont, CA, USA), CDKN1B (1:1,000, ab32034; Abcam, Fremont, CA, USA), N-cad (1:8,000, ab76011; Abcam, Fremont, CA, USA), Bax (1:3,000, ab32503; Abcam, Fremont, CA, USA), Bcl-2 (1:1,500, ab196495; Abcam, Fremont, CA, USA), and β-actin (1:20,000, AF7018; Affinity, Jiangsu, China) at 4 °C for 20 h. The membranes were rinsed three times with PBST, then immersed in HRP-conjugated anti-rabbit IgG secondary antibody solution (1:5,000, S0001; Affinity, Jiangsu, China) at 37 °C for 60 min. Interesting proteins were visualized using a commercial ECL kit (Glpbio, Montclair, CA, USA). Fluorescence intensities were quantified using Fiji software.

RNA pull-down assay

Biotin-labeled miR-2861 was purchased from Genewiz (Jiangsu, China). Biotin-labeled miR-2861 (3 μg) was incubated with streptavidin-coated beads (434341; Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C overnight. Then, streptavidin-coated beads were incubated with SV40-MES-13 cell lysate at 4 °C for 12 h. RNAs bound by miR-2861 were purified with Trizol reagent (Takara, Shiga, Japan) and assessed through qRT-PCR.

RNA immunoprecipitation (RIP) assay

After miR-2861 knockdown, the RIP assay was performed as per the instructions of the Immunoprecipitation Kit with Protein A+G Magnetic Beads (Beyotime, Shanghai, China). At first, 1 × 10⁶ SV40-MES-13 cells were lysed with 200 μL of RIPA buffer (Beyotime, Shanghai, China). Lysates were incubated with 20 μL of magnetic beads labeled with normal mouse IgG (A7028; Beyotime, Shanghai, China) or rabbit anti-Ago2 antibody (DF12246; Affinity, Jiangsu, China) at 4 °C for 16 h. Immunoprecipitated RNA was obtained by digesting proteins with Proteinase-K, and quantified on a microplate reader. Lastly, immunoprecipitated L13Rik and miR-2861 were assessed through qRT-PCR.

Dual-luciferase reporter assay

The wild-type or mutant predictive binding site of miR-2861 on L13Rik was cloned into the PGL3 vector (Fenghui Biotechnology, Hunan, China) to construct pGL3-L13Rik-WT or pGL3-L13Rik-Mut plasmids. The 3′-UTR of CDKN1B or its mutant was inserted into the PGL3 vector to construct pGL3-CDKN1B-3′UTR-WT or pGL3-CDKN1B-3′UTR-Mut plasmids. HEK293 cells (1 × 10⁶) were plated into 24-well plates. pGL3-L13Rik-WT (20 ng), pGL3-L13Rik-Mut (20 ng), pGL3-CDKN1B-3′UTR-WT (20 ng), or pGL3-CDKN1B-3′UTR-Mut (20 ng) plasmids were co-transfected with 40 nM of miR-2861 and 2 ng of pRL-TK (Beyotime, Shanghai, China) into HEK293 cells with Lipofectamine™ 3,000 according to the manufacturer’s protocol. After incubation at 37 °C for 48 h, luciferase activities were assessed using the Dual-Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China) in accordance with the manufacturer’s protocol. Renilla luciferase was used for internal control.

Protein synthesis

After treatment with HG, SV40-MES-13 cells were incubated with [³⁵S]-methionine and then protein synthesis was assessed as described previously (Mahimainathan et al., 2006).

Cell hypertrophy

SV40-MES-13 cells were treated with HG and then lysed with the xTractor Buffer Kit. Protein content was quantified through the BCA assay kit, and cell hypertrophy was evaluated by testing the ratio of total protein to cell number (Mahimainathan et al., 2006).

Flow cytometry

SV40-MES-13 cells were treated with HG and dyed with propidium iodide (PI) for 1 h. the cell cycle was measured using an Attune N×T flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA).

Statistical analysis

Data were presented as mean ± standard deviation (SD) from three independent experiments and analyzed using SPSS 22.0 software (IBM, Armonk, NY, USA). The difference among groups was compared with the student’s t-test or one-way analysis of variance (ANOVA) followed by the Scheffé test. A p-value less than 0.05 was defined as statistically significant.

L13Rik was increased in DN patients, diabetic mice, and HG-treated MCs

Given that L13Rik expression is up-regulated in renal tissues of mice with type two diabetes compared with normal mice (Figs. S1B and S1C), we next investigated whether L13Rik is correlated with DN progression through regulating MC hypertrophy. To this end, L13Rik levels were first assessed in DN patients and diabetic mice. As shown in Fig. 1A, L13Rik levels were significantly up-regulated in the peripheral blood of DN patients (n = 13) compared with healthy controls (n = 18). Similarly, L13Rik expression was also increased in the renal tissues of diabetic mice compared with control mice (Fig. 1B). Consistently, L13Rik expression was remarkably up-regulated in HG-cultured SV40-MES-13 cells (Fig. 1C). The relationships between treatment time and glucose concentration on L13Rik expression were also explored. As shown in Figs. 1D and 1E, HG treatment resulted in a significant increase in the L13Rik level in a time- and dose-dependent manner. Furthermore, the results from the FISH assay verified that L13Rik was up-regulated in HG-cultured SV40-MES-13 cells and that L13Rik was mainly located in the cytoplasm of SV40-MES-13 cells (Fig. 1F).

L13Rik depletion alleviated HG-induced MC hypertrophy and ECM accumulation

Cell cycle arrest in the G1 phase and subsequent hypertrophy are critical pathological characteristics of MC injury (Rojas-Canales et al., 2019; Sun, 2019). To investigate the biological effect of L13Rik on regulating MC injury, SV40-MES-13 cells were treated with HG alone or combined with siRNA against L13Rik (siL13Rik), and then the cell cycle was assessed through flow cytometry. Figures 2A and 2B showed that HG treatment resulted in the arrest of cells at the G1 phase, whereas the changes were reversed by L13Rik knockdown. The protein levels were next examined in SV40-MES-13 cells after treatment with HG and siL13Rik. HG enhanced the amount of newly synthesized proteins, as evidenced by ³⁵S-methionine incorporation (Fig. 2C). HG further resulted in cell hypertrophy, as suggested by elevated total protein content in SV40-MES-13 cells (Fig. 2D). These effects were blocked by L13Rik knockdown (Figs. 2C and 2D).

To identify the effect of L13Rik on ECM accumulation in SV40-MES-13 cells, FN, Col-IV, and N-cad expression were assessed by qRT-PCR and western blot analysis after treatment with HG in the presence or absence of siL13Rik. Figures 2E–2G showed that the mRNA and protein levels of FN, Col-IV, and N-cad were markedly increased after treatment with HG, whereas these effects were blocked by L13Rik depletion.

L13Rik acted as a ceRNA to sponge miR-2861

LncRNA, located in the cytoplasm, commonly acts as ceRNA to control gene expression via sponging specific miRNAs (Thomson & Dinger, 2016). Given that L13Rik mainly locates in the cytoplasm in SV40-MES-13 cells (Fig. 1F), we speculated that L13Rik might exert its biological effect in this way. To prove that, a bioinformatics tool, miRDB (http://mirdb.org/cgi-bin/search.cgi) (Chen & Wang, 2020), was applied to predict miRNAs sponged by L13Rik, and 63 candidate miRNAs were identified (Table S2). A previous study demonstrated that 86 miRNAs were dysexpressed in DN progression (Table S3) (Chen et al., 2020). Venn diagram analysis revealed that miR-2861 was the only miRNA that could be sponged by L13Rik and dysregulated in DN patients (Fig. S3).

The miR-2861 level was next assessed in DN patients and diabetic mice. As shown in Fig. 3A, miR-2861 expression was obviously decreased in the peripheral blood of DN patients (n = 13) compared to healthy controls (n = 18). miR-2861 was also down-regulated in the renal tissues of diabetic mice compared to control mice (Fig. 3B). In addition, HG treatment resulted in a significant decrease in miR-2861 in a dose-dependent manner (Fig. 3C). To verify the direct combination of miR-2861 with L13Rik, the binding sites between L13Rik and miR-2861 were analysed, and recombinant plasmids containing the binding sites (pGL3-L13Rik-Wt and pGL3-L13Rik-Mut) were constructed (Fig. 3D). pGL3-L13Rik-Wt or pGL3-L13Rik-Mut was co-transfected with miR-2861 into HEK293 cells, and then relative luciferase activity was measured using the dual luciferase reporter assay. As shown in Fig. 3E, miR-2861 significantly decreased the luciferase activity of pGL3-L13Rik-Wt, but did not affect the luciferase activity of pGL3-L13Rik-Mut. Furthermore, the miR-2861 mutant lost the inhibitor effect on the luciferase activity of pGL3-L13Rik-Wt (Fig. 3F), indicating that the combination of miR-2861 with L13Rik was sequence-specific. The results from the RNA pull-down assay further demonstrated that L13Rik was more enriched in the biotin-labeled miR-2861 precipitates than in the biotin-labeled miR-cont precipitates (Fig. 3G). Argonaute 2 (Ago2) is the core component of the “miRNA-induced silencing complex (miRISC)”, a multi-protein complex that incorporates miRNA and its target mRNA (or lncRNA) (Chendrimada et al., 2005; Liu et al., 2004). The results from RIP with Ago2 antibody showed that miRNA-2861 and L13Rik were concurrently enriched in SV40-MES-13 cells, and miRNA-2861 inhibitor significantly reduced miRNA-2861 level and increased L13Rik enrichment in Ago2 precipitates (Figs. 3H and 3I). These results demonstrate that L13Rik functions as a ceRNA to sponge miR-2861 in a sequence-specific manner.

MiR-2861 suppressed HG-induced MC hypertrophy and ECM accumulation

The roles of miR-2861 in regulating MC hypertrophy and ECM accumulation were next investigated. As shown in Figs. 4A and 4B, miR-2861 overexpression alleviated HG-induced arrest of cells at the G1 phase. miR-2861 further reduced HG-induced new protein synthesis (Fig. 4C) and cell hypertrophy (Fig. 4D), as evidenced by decreased ³⁵S-methionine incorporation and total protein content, respectively. To identify the effect of miR-2861 on ECM accumulation in SV40-MES-13 cells, FN, Col-IV, and N-cad expression were assessed by qRT-PCR and western blot analysis after treatment with HG in the presence or absence of miR-2861. Figure 4E showed that miR-2861 significantly repressed the HG-induced increase in FN, Col-IV, and N-cad mRNA levels. miR-2861 also significantly suppressed the HG-induced increase in FN, Col-IV, and N-cad protein levels (Figs. 4F and 4G).

MiR-2861 antagonized the effect of L13Rik on MC hypertrophy

To explore whether L13Rik accelerated MC hypertrophy through sponging miR-2861, SV40-MES-13 cells were overexpressed by L13Rik in the presence or absence of miR-2861 mimic, and cell hypertrophy and ECM accumulation were assessed. L13Rik overexpression resulted in the arrest of cells at the G1 phase (Fig. 5A), enhanced the amount of newly synthesized proteins (Fig. 5B), and caused cell hypertrophy (Fig. 5C). All these effects were reversed by miR-2861 (Figs. 5A–5C). Moreover, L13Rik increased the mRNA and protein expression of FN, Col-IV, and N-cad, whereas miR-2861 overexpression reversed these changes (Figs. 5D–5F).

L13Rik increased CDKN1B expression by sponging miR-2861

The potential target genes of miR-2861 were predicted using the TargetScan tool (http://www.targetscan.org/vert_71/). A total of 4,407 genes were predicted to be potential targets of miR-2861. Among these genes, CDKN1B was reported to be associated with cell cycle and MC hypertrophy (Awazu et al., 2003; Liao et al., 2020). To reveal the regulatory role of miR-2861 in CDKN1B expression, recombinant plasmids of pGL3-CDKN1B-3′UTR-Wt and pGL3-CDKN1B-3′UTR-Mut were constructed and co-transfected with miR-2861 (Fig. 6A). As shown in Fig. 6B, miR-2861 significantly decreased the luciferase activity of pGL3-CDKN1B-3′UTR-Wt but had no impact on the luciferase activity of pGL3-CDKN1B-3′UTR-Mut. Moreover, CDKN1B protein expression was further assessed in SV40-MES-13 cells after treatment with the miR-2861 mimic. Figures 6C and 6D showed that CDKN1B expression was obviously decreased after miR-2861 overexpression. More importantly, L13Rik increased CDKN1B expression in SV40-MES-13 cells, whereas miR-2861 overexpression reversed the effect (Figs. 6E and 6F).