Clinical case sample collection

A total of 45 T2DM patients treated in our hospital from March 2017 to March 2018 were enrolled. There were 22 males with an average age of (51.2±6.4) years old and 23 females with an average age of (48.6±3.8) years old. According to the World Health Organization in 1999, inclusion criteria of patients with T2DM were: (1) Fasting blood glucose is equal or higher than 7.0 mmol/L. (2) 2 hours postprandial blood glucose is >11.1 mmol/L. (3) Age >32 years old. (4) BMI<40 kg/m². (5) No abnormal glucose regulation or genetic history of diabetes [14]. Exclusion criteria: (1) Patients complicated with hypertension, lung diseases, malignant tumors, autoimmune diseases, metabolic disorders, etc. (2) Patients suffering from chronic inflammation and infectious diseases. (3) Those who participate in other research projects. At the same time, the serum of 32 healthy people who came to our hospital for physical examination was collected as a control (fasting blood glucose level is lower than 6.1 mmol/L, 2 h postprandial blood glucose level is lower than 7.8 mmol/L). All the volunteers enrolled in the study had not received systemic treatment and have signed written informed consent. This study was approved by the Ethics Committee of our hospital (Approval No.20180812).

Cell culture

Mouse pancreatic β cell INS-1 was purchased from Mingzhou Biotechnology Co., Ltd. (Ningbo, China). Normal INS-1 cells were cultured using the mouse pancreatic β cell complete culture medium provided by Mingzhou Biotechnology Co., Ltd. (Ningbo, China). The medium contains 15% fetal bovine serum, 100 U/mL penicillin and 0.1 mg/mL streptomycin. INS-1 cell injury model was established and INS-1 cells were cultured with 25 mM glucose Dulbecco’s modified medium DMEM (Wuhan Punosai Life Science and Technology Co., Ltd., China). The treated cells were called HG-INS-1. The medium was supplemented with 15% fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 50 mmol/L β-mercaptoethanol. All cells were incubated in a 37°C, 5% CO₂ incubator, and cell transfection was carried out when the cell density reached 80%-90%.

Cell transfection

The siRNA, pcDNA and negative controls used in this study were all provided by GenScript Biotechnology Co., Ltd. (Nanjing, China). miRNA mimics and negative controls were provided by GenePharma Co., Ltd. (Shanghai, China). The siRNA recombinant plasmid was used to knock down the expression of PTGS2, and pcDNA and miRNA mimics were used for over-expression transfection of PTGS2, RBP4 and miR-146a-5p. According to the grouping requirements, the cell transfection was carried out according to the Lipofectamine 3000 transfection reagent manual. The subsequent experiments were carried out 48 hours after transfection. See Table 1.

qRT-PCR

Total RNA was isolated from INS-1 cells by Trizol reagent (Sigma-Aldrich, USA). Reverse transcription and RNA quantitative analysis were carried out according to the instructions of the SuperScriptIV one-step PCR system (Thermo Fisher, USA). U6 and GAPDH were used as internal references, and the results were further calculated by 2-delta CT.

The above detection steps were performed following the instructions of the kit manufacturer. The primers were provided by GenScript Biotechnology Co., Ltd. (Nanjing, China). The detailed sequences are shown in Table 2.

Subcellular isolation

The nucleus and cytoplasm of INS-1 cells were separated using a nuclear/cytoplasmic separation kit (Biyuntian Biotechnology Co., Ltd., Shanghai, China) according to the instructions of the kit. Total RNA was extracted from the nucleus and cytoplasm respectively for qRT-PCR reaction. qRT-PCR steps are shown in the “qRT-PCR” part.

Dual-Luciferase reporter assay

The 3’UTR fragments of LncRNA PTGS2 and RBP4 were amplified. Using endonuclease sites SpeI and Hind III, LncRNA PTGS2 and RBP4 fragments were cloned into pmirGLO reporter vector (Haijihaoge Biotechnology Co., Ltd., Shanghai, China), named PTGS2-WT and RBP4-WT. The mutation sites were designed on the 3’UTR fragments of LncRNA PTGS2 and RBP4. After restriction endonuclease digestion, the mutations were inserted into the pmirGLO vector by t4DNA ligase, which was named PTGS2-MUT and RBP4-MUT. The mutant and wild type sequences were co-transfected with miR-146a-5pNC and miR-146a-5pmimic into 293T cells, respectively, and the transfection steps were completed according to the instructions of the Lipofectamine 3000 kit (ThermoFisher, USA). After 48 hours, the transfected cells were collected, lysed and centrifuged for 5 minutes, and the supernatant was collected. Dual-luciferase gene reporter kit (Yisheng Biotechnology Co., Ltd., Shanghai, China) was used to detect the relative luciferase activity of cells in each group, with Renilla luciferase activity as a reference, and the detection steps were carried out under the requirements of the kit instructions.

Western blot

Protein was extracted from cells using RIPA cell lysate (SevenBiotech, Beijing, China), and protein concentration was detected by a BCA protein determination kit (Solebo, Beijing, China). After adding 12% sodium dodecyl sulfate-polyacrylamide gel for 1-hour electrophoresis, the isolated protein was transferred to PVDF membrane (Solebo, Beijing, China), and the membrane was sealed by 5% skim milk. The membrane was incubated overnight at 4°C with an anti-RBP4 (1chro1000) and an anti-GAPDH (1Drex1000). Horseradish peroxidase-conjugated secondary antibody goat anti-rabbit IgG (1:1000, Abcam, UK) was added to the membrane and incubated for 2 hours. TBST was used to wash the membrane. ECL chemiluminescence reagent (Suolaibao, Beijing, China) was used to visualize the protein and the gel imaging system was used for capture. The protein expression was analyzed by Image J software. The ratio of the gray value of the target protein band to GAPDH protein band was used for comparison.

CCK8

The transfected INS-1 cells were inoculated into a 96-well culture plate and a CCK8 cell proliferation detection kit (SevenBiotech, Beijing, China) was used to carry out the testing. The experimental procedures were carried out following the kit factory instructions. Finally, the OD value was measured at the wavelength of 450 nm with the use of a multifunctional microplate reader (Thermo Fisher, USA).

Enzyme linked immunosorbent assay (ELISA)

The transfected INS-1 cells were inoculated in a 96-well culture plate, and the insulin level secreted by INS-1 cells was detected by a mouse insulin ELISA kit (KA3812, Emijie Technology, Wuhan, China). The supernatant of the cell culture medium was centrifuged to remove the precipitate, and the standard well, test sample well and control well were set on the pre-coated plate. Then 50 μL of the standard solution was put into the standard Wells and 50 μL of appropriately diluted cell culture supernatant was added into the sample wells, respectively. In the above wells, 50 μL HRP-labeled anti-MO insulin antibody was added and incubated at 37°C for 60 minutes. Washing liquid was added to the wells and vortex for 2 min. Then 50 μL TMB substrate A and 50 μL TMB substrate B was add to each well in turn, swirl gently and shake for 30 seconds. The plate was incubated in darkness at 37°C for 15 min. At last, 50 μL stop solution was put into each well and mixed. Within 30 minutes after adding the stop solution, OD value at 450 nm was assessed with a microplate reader.

Knockdown of LncRNA PTGS2 promoted the proliferation and insulin secretion of HG-INS-1 cells

The LncRNA PTGS2 knockdown plasmid was established and transfected into HG-INS-1 cells. Compared with the si-NC group, si-PTGS2 significantly inhibited the expression of LncRNA PTGS2 (Figure 2A; P<0.05). To detect the effect of si-PTGS2 on the proliferation of HG-INS-1 cells, CCK8 experimental data showed that compared with the INS-1 group, the cell proliferation of the HG-INS-1 group was reduced, and compared with the si-NC group, si-PTGS2 significantly increased the proliferation of INS-1 cells induced by high glucose (Figure 2B, all P<005). Compared with the INS-1 group, the decrease of insulin secretion of INS-1 cells induced by high glucose was detected by an ELISA kit. Si-PTGS2 promoted HG-INS-1 cells to secrete insulin (Figure 2C; all P<0.05). The above experimental data suggest that high glucose can induce functional damage of INS-1 cells, while knockdown of LncRNA PTGS2 can promote INS-1 cell proliferation and insulin secretion, and improve the functional damage of INS-1 cells induced by high glucose.

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Figure 2

Knockdown of LncRNA PTGS2 improved the functional damage of INS-1 cells induced by high glucose. A: Transfection efficiency of LncRNA PTGS2 knockdown cells; B: Comparison of INS-1 cell proliferation in each group; C: Comparison of insulin secretion level of INS-1 cells in each group. Compared with the INS-1 group, #P<0.05; compared with the si-NC group, &P<0.05. LncRNA: long non-coding RNA.

miR-146a-5p could be used as a target for LncRNA PTGS2

LncLocator database showed the highest expression of LncRNA PTGS2 in the cytoplasm (Figure 3A). The nuclear and cytoplasmic separation of INS-1 cells was performed, and the high expression of LncRNA PTGS2 in the cytoplasm was confirmed by qRT-PCR assay (Figure 3B; P<0.05). It was suggested that LncRNA PTGS2 could play a role as a competitive endogenous RNA. With the help of an RNA database, it was found that there were specific binding sites between miR-146a-5p and LncRNA PTGS2, and the dual-luciferase report assay showed that there was a targeted relationship between LncRNA PTGS2 and miR-146a-5p (Figure 3C; P<0.05). Knockdown of LncRNA PTGS2 could promote the expression of miR-146a-5p (Figure 3D; P<0.05). Compared with healthy human serum, the expression of miR-146a-5p in serum of T2DM patients was down-regulated (Figure 3E; P<0.0001), and the expression of miR-146a-5p in HG-INS-1 cells was also down-regulated compared with normal INS-1 cells (Figure 3F; P<0.05). Pearson correlation analysis showed that there was a negative correlation between miR-146a-5p expression and LncRNA PTGS2 expression in serum of patients with T2DM (Figure 3G; P<0.05). The above experimental data showed that miR-146a-5p, as a target of LncRNA PTGS2, was negatively regulated by LncRNA PTGS2.

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Figure 3

Verification of targeting relationship between LncRNA PTGS2 and miR-146a-5p. A: Results predicted by LncLocator database; B: Results of nuclear-cytoplasmic separation experiment; C: Results of dual luciferase reportor analysis; D: The effect of LncRNA PTGS2 knockdown on miR-146a-5p expression; E: Low expression of miR-146a-5p in serum of T2DM patients; F: Low expression of miR-146a-5p in INS-1 cells induced by high glucose; G: Results of Pearson correlation analysis. Compared with U6, ΔP<0.05; compared with the PTGS2-WT+miR-146a-5pNC group, @P<0.05; compared with the si-NC group, &P<0.05; compared with the normal group, *P<0.05; compared with the INS-1 group, #P<0.05. T2DM: Type 2 Diabetes Mellitus.

miR-146a-5p could partially reverse the damaging effect of LncRNA PTGS2 on INS-1 cells

LncRNA PTGS2 overexpression plasmid and miR-146a-5p mimics were transfected into HG-INS-1 cells, and their effects on INS-1 cells were observed. Compared with the NC group, OE-PTGS2 significantly increased the expression of LncRNA PTGS2 (Figure 4A; P<0.05). Compared with miR-NC group, miR-146a-5p mimic upregulated the expression of miR-146a-5p (Figure 4B; P<0.05). CCK8 experimental data showed that compared with the NC group, the OE-PTGS2 group could significantly inhibit the proliferation of INS-1 cells, while compared with the OE-PTGS2+miR-NC group, the proliferation of cells in the OE-PTGS2+miR-146a-5p mimic group was promoted (Figure 4C; all P<0.05). Insulin secretion levels of cells in each group were detected. Compared with the NC group, the insulin secretion level of the OE-PTGS2 group decreased, while compared with the OE-PTGS2+miR-N group, the insulin secretion level of the OE-PTGS2+miR-146a-5p mimic group increased (Figure 4D; all P<0.05). The above experiments suggest that overexpression of LncRNA PTGS2 can significantly inhibit the proliferation and insulin secretion of HG-INS-1 cells, while overexpression of miR-146a-5p can partially reverse this effect.

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Figure 4

miR-146a-5p could partially reverse the effect of LncRNA PTGS2 on INS-1 cells. A: Detection of OE-PTGS2 transfection efficiency; B: Detection of miR-146a-5pmimic transfection efficiency; C: Comparison of proliferation of INS-1 cells in each group; D: Comparison of insulin secretion of INS-1 cells in each group. Compared with the NC group, ^P<0.05; compared with the miR-NC group, ▲P<0.05; compared with the INS-1 group, #P<0.05; compared with OE-PTGS2+miR-NC group, P<0.05.

RBP4 was proved to be the downstream target of miR-146a-5p

Through the Starbase online prediction website, we found the downstream targets of miR-146a-5p and performed GO analysis on potential downstream targets. Among them, the insulin secretion regulation pathway has attracted our attention, which is enriched in PFKFB2, GLUD1, RBP4, TARDBP genes (Figure 5A). The protein interaction analysis between these four genes and the risk genes related to the onset of T2DM revealed that there was a strong relationship between RBP4 and these proteins (Figure 5B). Some studies have found that RBP4 is upregulated in T2DM nephropathy [15]. Analysis of relative luciferase activity was done using base complementary sequences of RBP4 and miR-146a-5p. The experiment shows that compared with the RBP4-MUT+miR-146a-5p NC group, the relative luciferase activity of the RBP4-WT+miR-146a-5p mimic group was significantly inhibited (Figure 5C; P<0.05). MiR-146a-5p mimic could significantly inhibit the expression of RBP4 (Figure 5D; all P<0.05). The targeting relationship between RBP4 and miR-146a-5p was confirmed. The expression of RBP4 was upregulated in serum of T2DM patients and HG-INS-1 cells (Figure 5E and ​and5F;5F; all P<0.05). The expression of RBP4 in serum of T2DM patients was negatively correlated with that of miR-146a-5p but positively correlated with that of LncRNA PTGS2 (Figure 5G; all P<0.05). The above experimental data showed that RBP4 was the downstream target of miR-146a-5p.

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Figure 5

Verification of targeting relationship between 5RBP4 and miR-146a-5p. A: GO analysis; B: Protein interaction network map; C: Results of dual luciferase reporter analysis; D: Effect of miR-146a-5p on the expression of RBP4; E: High expression of RBP4 in serum of patients with T2DM; F: High expression of RBP4 in INS-1 cells induced by high glucose; G: Results of Pearson correlation analysis. Compared with the RBP4-WT+miR-146a-5pNC group, P<0.05; compared with the miR-NC group, ▲P<0.05; compared with the normal group, *P<0.05; compared with the INS-1 group, #P<0.05. T2DM: Type 2 Diabetes Mellitus.

This work was supported by the General Project of Hubei Provincial Natural Science Foundation for MRI in vivo imaging to assess the capacity of resting pancreatic islet β cells in rats (2020CFB589).