LncRNA KCNQ1OT1 affects cell proliferation, apoptosis and fibrosis through regulating miR-18b-5p/SORBS2 axis and NF-ĸB pathway in diabetic nephropathy

Blood samples were collected from 30 patients with DN (DN group) and 30 healthy volunteers (normal group). DN patients were diagnosed in First People’s Hospital of Jingzhou, Hubei Province. Serum samples were separated from blood and stored at -80℃. All patients signed informed consents. This study had acquired approval from the Ethics Committee of First People’s Hospital of Jingzhou, Hubei Province.

Human glomerular mesangial cells (HGMCs) and human renal glomerular endothelial cells (HRGECs) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA). 293 T cells were bought from BeNa Culture Collection (Beijing, China). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (Invitrogen) at 37℃ with 5% CO₂. HGMCs and HRGECs were stimulated with 5.5 mM glucose (normal group) or 30 mM glucose (high glucose group). High glucose-induced HGMCs and HRGECs were served as models of DN cells.

Total RNAs were isolated using TRIzol Reagent (Invitrogen) under the instructions. Subsequently, reverse-transcription was performed by PrimeScript RT Reagent Kit (TaKaRa, Dalian, China) and microRNA First-Strand cDNA Synthesis Kit (Sangon Biotech, China). SYBR Green Master Mix (TaKaRa) was used for gene amplification. Gene expressions were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 and calculated by 2^−∆∆Ct method. The primers were listed in Table 1.

RIPA solution (Thermo Fisher Scientific, Waltham, MA, USA) was applied to extract total proteins. Then equal amounts of proteins were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and shifted onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocked with 5% nonfat milk, the membrane was incubated with the primary antibodies overnight at 4℃. Next, the secondary antibody was used to incubate the membrane for 1.5 h at room temperature. The protein bands were showed by enhanced chemiluminescence reagents (Millipore). The primary antibodies against SORBS2, Col-4, FN, TGF-β1, Twist, NF-κB, STAT3 and GAPDH were bought from Abcam (Cambridge, United Kingdom).

The viability of HGMCs was determined by cell counting kit-8 (CCK-8; Beyotime, Shanghai, China). Cells were placed into a 96-well plate with 0, 10, 20 or 30 mM glucose. After cultured for 48 h, the cells were added with CCK-8 reagent and incubated for 2 h. Finally, a microplate reader (Thermo Fisher Scientific) was used to measure the absorbance at 450 nm.

Small interfering RNA (siRNA) of KCNQ1OT1 and SORBS2 (si-KCNQ1OT1 and si-SORBS2), miR-18b-5p mimic (miR-18b-5p), miR-18b-5p inhibitor and their control fragments (si-NC and miR-NC) were acquired from GenePharma (Shanghai, China). The full-length sequences of KCNQ1OT1 and SORBS2 were respectively inserted into pcDNA3.1 (Invitrogen) for the overexpression of KCNQ1OT1 and SORBS2. High glucose-induced HGMCs and HRGECs were transfected with si-RNA (si-NC, si-KCNQ1OT1 or si-SORBS2), miRNA (miR-NC, miR-18b-5p or miR-18b-5p inhibitor) or vector (pcDNA, pcDNA-KCNQ1OT1 or pcDNA-SORBS2) by Lipofectamine 2000 reagent (Invitrogen).

Cell proliferation was tested by MTT assay. Transfected cells were cultured in a 96-well plate for 24 h and then added 20 µL of MTT (5 mg/mL). After incubation for 4 h, the supernatants were removed. Subsequently, the formazan crystals was dissolved with 150 µL of dimethyl sulfoxide. The proliferation was assessed via the absorbance at 490 nm wavelength using a microplate reader (Thermo Fisher Scientific).

Apoptosis was detected using annexin V-fluorescein isothiocyanate (V-FITC)/ propidium iodide (PI) apoptosis kit (BD Bioscience, San Diego, CA, USA), cells transfected for 48 h were collected and added annexin V-FITC and PI. After incubation for 20 min in a dark condition, cell apoptosis was tested by flow cytometry (BD Bioscience).

The putative binding sequences between KCNQ1OT1 and miR-18b-5p, miR-18b-5p and SORBS2 were predicted by starBase 3.0. The wild type sequences of KCNQ1OT1 (WT-KCNQ1OT1) and 3′-UTR of SORBS2 (WT-SORBS2 3′UTR) contain the predicted binding sites of miR-18b-5p, the mutant sequences of KCNQ1OT1 (MUT-KCNQ1OT1) and mutant 3′-UTR of SORBS2 (MUT-SORBS2 3′UTR) were cloned into pGL3 reporter vectors (Promega, Madison, WI, USA), respectively. The reporter vectors with miR-18b-5p or miR-NC were co-transfected into 293 T cells for 48 h. The activities of firefly and renilla luciferases were detected with a Dual-Luciferase reporter system (Promega).

Each experiment was done with at least three biological replicates. Statistical analysis was conduct by SPSS 22.0. Data were expressed as mean ± standard deviation (SD). Student’s t-test or one-way analysis of variance (ANOVA) with Tukey test was used to compare the differences of two groups or multiple groups. P-value < 0.05 was considered statistically significant.

Firstly, KCNQ1OT1 level in the serum of DN patients was detected by qRT-PCR, and the result showed that the level of KCNQ1OT1 was significantly increased in DN patients compared with that in normal group (Fig. 1a). Next, the level of SORBS2 was measured by qRT-PCR and western blot, and the data revealed that SORBS2 was upregulated in DN patients (Fig. 1b, c). Besides, the levels of KCNQ1OT1 and SORBS2 mRNA had obviously positive correlation (Fig. 1d). Subsequently, we measured the viability of HGMCs treated with glucose. The cell viability was gradually enhanced with the increasing of glucose concentration (Fig. 1e). So 30 mM glucose was used for high glucose treatment to simulate the condition of DN. Additionally, KCNQ1OT1 and SORBS2 levels in HGMCs and HRGECs were detected. The results indicated that KCNQ1OT1 and SORBS2 were both elevated in high glucose group compared with normal group in both HGMCs and HRGECs (Fig. 1f, h). In addition, the cell viability was markedly increased and cell apoptosis rate was remarkably decreased in high glucose group compared with normal group (Additional file 1: Fig. S1). These data implied that KCNQ1OT1 and SORBS2 played vital roles in DN.

To investigate the role of KCNQ1OT1 in DN cells, HGMCs and HRGECs were treated with high glucose and transfected with si-KCNQ1OT1. The qRT-PCR result revealed that KCNQ1OT1 level was obviously reduced in HGMCs and HRGECs transfected with si-KCNQ1OT1 (Fig. 2a). Subsequently, proliferation and apoptosis of HGMCs and HRGECs was assessed by MTT and Flow cytometry assay. MTT assay showed that KCNQ1OT1 knockdown suppressed cell proliferation in both HGMCs and HRGECs (Fig. 2b, c). On the contrary, apoptosis was promoted in HGMCs and HRGECs transfected with si-KCNQ1OT1 (Fig. 2d). In addition, knock-down of KCNQ1OT1 down-regulated the expression levels of FN, Col-4 and TGF-β1 in HFMC and HRGEC cells (Fig. 2e). These results proved that KCNQ1OT1 played a promoting effect on DN cells.

To explore the effects of SORBS2 in DN in vitro, high glucose-induced HGMCs and HRGECs were transfected with si-SORBS2. Then the expression of SORBS2 was measured to evaluate the silencing efficiency. The mRNA level of SORBS2 was significantly downregulated in HGMCs and HRGECs transfected with si-SORBS2 (Fig. 3a). And the protein level of SORBS2 in HGMCs and HRGECs consisted with the mRNA level (Fig. 3b). Subsequently, MTT assay implied that proliferation was inhibited in HGMCs and HRGECs transfected with si-SORBS2 (Fig. 3c, d). In addition, silencing of SORBS2 facilitated apoptosis of HGMCs and HRGECs (Fig. 3e). Furthermore, the fibrosis related proteins FN, Col-4 and TGF-β1 were significantly decreased after transfection with si-SORBS2 (Fig. 3f). These data indicated that downregulation of SORBS2 suppressed proliferation and fibrosis and promoted apoptosis in DN in vitro.

To investigate the relationship between KCNQ1OT1 and SORBS2 in DN cells, the mRNA and protein levels of SORBS2 were detected in high glucose-induced HGMCs and HRGECs after KCNQ1OT1 was silenced or overexpressed. The qRT-PCR result suggested the mRNA level of SORBS2 was downregulated by knockdown of KCNQ1OT1 while increased by overexpression of KCNQ1OT1 in HGMCs and HRGECs (Fig. 4a, b). Similarly, the protein level of SORBS2 was reduced by si-KCNQ1OT1 but elevated by pcDNA-KCNQ1OT1 in HGMCs and HRGECs (Fig. 4c, d). Additionally, pcDNA-SORBS2 was used to overexpress SORBS2, and the western blot result showed that the protein level of SORBS2 was increased in HGMCs and HRGECs transfected with pcDNA-SORBS2 (Fig. 4e). Then, proliferation and apoptosis was determined in HGMCs and HRGECs transfected with si-KCNQ1OT1 or si-KCNQ1OT1 + pcDNA-SORBS2. And the results exhibited cell proliferation was impeded by KCNQ1OT1 knockdown and rescued by SORBS2 overexpression in HGMCs and HRGECs (Fig. 4f, g). Inversely, apoptosis was promoted in HGMCs and HRGECs transfected with si-KCNQ1OT1 and reversed when co-transfected with pcDNA-SORBS2 (Fig. 4h, i). In addition, the expression levels of fibrosis-related proteins FN, Col-4 and TGF-β1 were markedly down-regulated after transfection with si-KCNQ1OT1, while overexpression of SORBS2 reversed the changes of fibrosis-related proteins caused by KCNQ1OT1 knockdown (Fig. 4j). These results manifested that KCNQ1OT1 affected proliferation, apoptosis and fibrosis through regulating SORBS2 expression in DN in vitro.

To further explore the regulatory mechanism between KCNQ1OT1 and SORBS2, the target of KCNQ1OT1 was predicated by starBase 3.0. MiR-18b-5p was predicated as a potential target and the putative binding sequences for KCNQ1OT1 were exhibited (Fig. 5a). WT-KCNQ1OT1 and MUT-KCNQ1OT1 was constructed for dual-luciferase reporter assay. The luciferase activity was remarkably decreased by miR-18b-5p in 293 T cells transfected with WT-KCNQ1OT1 and had no change in MUT-KCNQ1OT1 group (Fig. 5b). Moreover, the expression of miR-18b-5p was measured in HGMCs and HRGECs transfected with pcDNA-KCNQ1OT1, and the result indicated that overexpression of KCNQ1OT1 downregulated the expression of miR-18b-5p in HGMCs and HRGECs (Fig. 5c). Also, the expression of miR-18b-5p was greatly repressed by miR-18b-5p inhibitor (Fig. 5d). In order to investigate the interaction between KCNQ1OT1 and miR-18b-5p in DN cells, high glucose-induced HGMCs and HRGECs were transfected with si-KCNQ1OT1 or si-KCNQ1OT1 + miR-18b-5p inhibitor. Cell proliferation was inhibited by KCNQ1OT1 knockdown and reverted by miR-18b-5p inhibitor in HGMCs and HRGECs (Fig. 5e, f). Besides, the enhanced apoptosis of HGMCs and HRGECs by si-KCNQ1OT1 was abolished by inhibiting miR-18b-5p (Fig. 5g, h). Furthermore, the fibrosis-related proteins (FN, Col-4 and TGF-β1) were down-regulated by KCNQ1OT1 knockdown and reversed by transfection with miR-18b-5p inhibitor (Fig. 5i). Interestingly, starBase 3.0 showed that miR-18b-5p can bind to 3′UTR of SORBS2 (Fig. 5j). Dual-luciferase reporter assay presented that the luciferase activity was weakened by miR-18b-5p in 293 T cells transfected with WT-SORBS2 3′UTR rather than MUT-SORBS2 3′UTR (Fig. 5k). In addition, the mRNA and protein levels of SORBS2 were determined in high glucose-induced HGMCs and HRGECs transfected with miR-18b-5p or miR-18b-5p and pcDNA-KCNQ1OT1. The data displayed that the expression of SORBS2 was attenuated by miR-18b-5p but rescued by KCNQ1OT1 overexpression (Fig. 5l, m). These results suggested that KCNQ1OT1 served as a competing endogenous RNA (ceRNA) to upregulate SORBS2 expression by sponging miR-18b-5p, thereby affecting proliferation and apoptosis in DN cells.

The expression levels NF-ĸB pathway-related proteins (Twist, NF-κB and STAT3) were detected to assess NF-ĸB pathway. High glucose-induced HGMCs and HRGECs were transfected with si-KCNQ1OT1 for the knockdown of KCNQ1OT1. The results showed the mRNA and protein levels of Twist, NF-κB and STAT3 were all decreased in HGMC cells transfected with si-KCNQ1OT1 (Fig. 6a, b), and there was a same trend in HRGEC cells (Fig. 6c, d), which suggesting that knockdown of KCNQ1OT1 impeded NF-ĸB pathway in DN cells.