Expression characteristics of long noncoding RNA uc.322 and its effects on pancreatic islet function

2.1 Animals and cell culture

All animal experiments were carried out in strict accordance with the rules of management used by the Experimental Animal Committee of Nanjing Medical University (Approval NO: IACUC-1781195). Five-week-old C57BL/6J male mice were purchased from the Model Animal Research Center of Nanjing Medical University. The mice were killed by the spinal dislocation method, and then the heart, liver, pancreas, spleen, kidney, muscle, and fat of the mice were removed. Total RNA of these tissues was extracted by Trizol reagent (Thermo Fisher Scientific, Shanghai, China) and the expression of lncRNA uc.322 in these tissues was detected by quantitative polymerase chain reaction (qPCR). The primers were designed by Primer 5.0 software and synthesized by Shanghai Generay Biotech Co, Ltd (Table 1).

Min6 cells are a mouse insulinoma cell line. The line was obtained from the Institute of Endocrinology and Metabolism, Ruijin Hospital. The cells were cultured in Dulbecco modified Eagle medium high-glucose medium containing 15% fetal bovine serum, 0.5% streptomycin and penicillin, and 50 μM of β-mercaptoethanol at 37°C in 5% CO₂.

2.2 Uc.322 overexpression and knockdown experiments via lentivirus transfection

For overexpression experiments, Min6 cells were infected by LV5-lncRNA uc.322, and the infection efficiency was confirmed by qPCR. For knockdown, cells at 60% confluency were transfected with uc.322-specific short hairpin RNA (shRNA) pool (LV3-uc322-shRNA-71 and LV3-uc322-shRNA-166). Control cells were treated with relative RNA negative lentivirus. The lentivirus was purchased from Shanghai GenePharma Pharmaceutical Technology Co, Ltd.

2.3 qPCR and Western blot analysis

The Trizol reagent (Thermo Fisher Scientific) was used to extract total RNA from Min6 cells, and the total RNA of the cells was reverse-transcribed into complementary DNA by a Revertra ace-m (TOYOBO Shanghai Biotech Co, Ltd) high-efficiency reverse transcription Kit in the PCR amplification instrument. The reaction condition was 37°C for 15 minutes, 85°C for 5 seconds, and preservation at 4°C. Then, complementary DNA was analyzed by qPCR on the ABI 7500 real-time qPCR to detect the expression of lncRNA uc.322, insulin TFs (PDX1 and FOXO1), and SOX6, which are the possible targets of lncRNA uc.322 in Min6 cells. β-Actin was used as the internal control, and the 2^−∆∆Ct method was used to calculate the relative value of gene expression. PCR primers were designed with Primer5.0 software and were synthesized by Shanghai Generay Biotech Co, Ltd (Table 1).

For Western blot analysis, protein was extracted from whole-cell lysates using RIPA buffer and quantified by the bicinchonininc acid (BCA) Protein Assay Kit. A 50 µg sample of the extracted protein was separated via 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22 µm nitrocellulose membranes (Sigma), and incubated with specific antibodies. The autoradiograms were quantified using densitometry. The rabbit primary antibodies were used at a dilution of 1:1000. The secondary antibody used in these assays was horseradish peroxidase conjugated goat anti-rabbit lgG (dilution of 1:1000). The β-actin antibody was used as the control. Antibodies used in this study are PDX1 (SAB2104577-50UG, Sigma), SOX6 (SAB2102269-50UG, Sigma), FOXO1 (2880P, Cell Signaling), β-actin (4970S, Cell Signaling). All of the experiments were repeated at least 3 times.

2.4 Detecting glucose-, arginine-, and KCl-stimulated insulin secretion by enzyme-linked immunosorbent assay

Min6 cells were digested and passaged in 96-well plates until the cells were 80% confluent, and 6 accessory holes were established in each group. Then, the culture medium was replaced by 200 μL of 0.25% BSA, 2.8 mM of glucose (Glu) KRB buffer to pre-incubate for 30 minutes. After that, the 0.25% BSA, 2.8 mM of Glu KRB buffer was replaced by new 200 μl of 0.25% BSA, 2.8 mM of Glu KRB buffer to incubate at 37°C for 1 hour. Then, 100 μL of culture solution was used to detect insulin secretion under low glucose stimulation. Afterward, we added new 100 μL of 0.25% bovine serum albumin (BSA), 2.8 mM of Glu kerbs-ringerbicarbonate (KRB) buffer, and 30 mM l-arginine into the surplus 100 μL of 0.25% BSA, 2.8 mM of Glu KRB buffer to incubate at 37°C for 1 hour. Next, 100 μL of culture solution was used to detect insulin secretion under l-arginine stimulation. Then, the 0.25% BSA, 2.8 mM of Glu KRB, and 30 mM l-arginine buffer was replaced by 200 μL of 0.25% BSA, 16.7 mM of Glu KRB buffer to incubate at 37°C for 1 hour. Next, 100 μL of culture solution was used to detect insulin secretion under high glucose stimulation. Then, the 0.25% BSA, 16.7 mM of Glu KRB buffer was replaced by 200 μL of 0.25% BSA, 30 mM of KCL KRB buffer to incubate at 37°C for 1 hour. Finally, 100 μL of culture solution was used to detect insulin secretion under KCL stimulation. The 4 types of culture solutions were centrifuged (200g, 20 minutes, 4°C) to remove some of the isolated cell debris or impurities. Enzyme-linked immunosorbent assay was used to detect insulin concentrations of different standard products, and we used 3 enzyme-linked immunosorbent assay wells per sample.

2.5 Detection of mitochondrial ATP production

The ATP bioluminescence assay kit and multifunctional enzyme marker were used to detect mitochondrial ATP concentration (Shanghai Beyotime Biotechnology Co, Ltd). Min6 cells were cultured to the logarithmic phase, and then the cells were passaged in 6-well plates until 80% confluency. After that, the cell culture fluid in the 6-well plate was removed, and 200 μL of lysate was added into each pore. The sample was continuously pipetted to ensure cell lysis and then centrifuged to separate the cell mass and supernatant. The centrifugal frequency was 12000 rpm for 5 minutes at 4°C. After centrifugation, the supernatant was collected. Then, 20 μL of the supernatant and different concentrations of the standard were added to the wells prepared with an ATP detection working fluid and rapidly mixed. We used 3 ATP detection wells per sample when measuring the ATP, and the numerical values of luminous intensity were measured by the liquid scintillation analyzer. Standard curves were plotted according to the luminous intensity of standard products. The amount of ATP in cells was calculated according to the standard curve.

2.6 Statistical analysis

SPASS l9.0 statistical software was used for statistical analyses. The data were presented as mean ± standard deviation and the P values of the experiment were calculated using 1-way analysis of variance or unpaired 2-tailed Student t test with a significance of P < .05. The experiment was repeated more than 3 times.

3.1 lncRNA uc.322 is an extremely conservative lncRNA and it is highly expressed in pancreas tissues of mice

Conservation analysis of the lncRNA uc.322 locus found it to be highly conserved in rat, human, dog, orangutan, horse, and opossum (Figure 1A). Sequence alignment by DNAMAN (http://www.lynnon.com) of lncRNA uc.322 between human and mouse genomes showed 100% identity. qPCR was performed to detect the expression of lncRNA uc.322 in different tissues of adult C57BL/6J male mice, including pancreas, fat, muscle, spleen, kidney, heart, and liver. As shown in Figure 1B, lncRNA uc.322 appeared to be highly expressed in pancreas tissue, followed by adipose tissue.

3.2 lncRNA uc.322 overexpression increases the expression of insulin TFs and insulin secretion

To estimate whether lncRNA uc.322 directly affected the expression of insulin TFs (PDX-1 and FOXO1) and insulin secretion, Min6 cells were transduced with a lentivirus expressing lncRNA uc.322. The expression of lncRNA uc.322 in Min6 cells was significantly increased after transfection (Figure 2A). qPCR and Western blot analysis revealed that lncRNA uc.322 overexpression resulted in elevated expression of the insulin TFs, PDX1 and FOXOl (Figure 2B,C).

The effects of lncRNA uc.322 overexpression on insulin secretion were detected by glucose, l-arginine, and KCL. As shown in Figure 2D, Insulin secretion in the lncRNA uc.322 overexpression group was significantly higher than that of the control group under high glucose, l-arginine, and KCL stimulation (P < .05). However, there was no significant difference in insulin secretion under low-glucose conditions (P > .05). It has been reported that Min6 cells have impaired insulin secretion after a high number of passages, and the overall response in insulin release in the Min6 cells under high glucose and low glucose was lower than expected. Considering that l-arginine and KCL are potentiators of glucose-stimulated insulin secretion, we used l-arginine and KCL to evaluate the insulin release response, and found that l-arginine- and KCL-stimulated insulin secretion in Min6 cells was significantly greater than with glucose (Figure 2D).

3.3 lncRNA uc.322 knockdown decreases the expression of insulin TFs and insulin secretion

To estimate the impact of lncRNA uc.322 loss of function on the expression of insulin TFs (PDX-1 and FOXO1) and insulin secretion, we performed an RNAi-knockdown experiment using a pool of shRNAs to transfect Min6 cells. As expected, lncRNA uc.322 shRNA significantly decreased the expression of lncRNA uc.322 in Min6 cells (Figure 3A). qPCR and Western blot analysis revealed that lncRNA uc.322 knockdown resulted in decreased expression of the insulin TFs, PDX1, and FOXO1 (Figures 3B and 2C).

The effects of lncRNA uc.322 knockdown on insulin secretion were also detected by glucose, l-arginine, and KCL. As shown in Figure 3D, insulin secretion in the lncRNA uc.322 knockdown group was significantly lower than that of the control group under high-glucose, low-glucose, l-arginine, and KCL stimulation (P < .05). Consistent with the above research, l-arginine and KCL-stimulated insulin secretion in Min6 cells was significantly greater than with glucose (Figure 3D).

3.4 lncRNA uc.322 is located in the exon region of the SOX6 gene, and it influences mitochondrial ATP production in Min6 cells

The full-length lncRNA uc.322 is 224 bp, which is located in the exon region of the SOX6 gene (chr7: 122, 893, 196-122, 893, 419, GRCH37/hg19; Figure 4A). qPCR and Western blot analysis revealed that uc.322 overexpression resulted in elevated expression of SOX6. However, lncRNA uc.322 knockdown resulted in decreased expression of SOX6 (Figure 4B,C). To estimate whether lncRNA uc.322 directly affected the expression of mitochondrial ATP production, luciferase assay was used to detect the changes in mitochondrial ATP production in Min6 cells. The results revealed that lncRNA uc.322 overexpression resulted in elevated content of ATP in the mitochondria of Min6 cells (P < .05). However, lncRNA uc.322 knockdown resulted in decrease of content of ATP in the mitochondria of Min6 cells (P < .05; Figure 4D).