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Previous studies have revealed that long intergenic non-coding RNA for kinase activation (LINK-A), a long non-coding RNA (lncRNA) promotes disease progression in triple-negative breast cancer by activating hypoxia-inducible factor 1α (HIF1α). However, the activation of HIF1α has also been demonstrated to improve diabetic nephropathy. It is therefore reasonable to expect that LINK-A may also participate in diabetic nephropathy. In the current study, the expression of LINK-A lncRNA and HIF1α was determined in renal biopsies of patients with diabetic nephropathy. LINK-A lncRNA and HIF1α expression levels were detected by reverse transcription quantitative (RT-q) PCR and ELISA in diabetic patients without complications and used as controls. Correlations between LINK-A lncRNA and HIF1α expression were analyzed using Pearson's correlation coefficient. Effects of lncRNA and HIF1α overexpression on LINK-A lncRNA expression, HIF1α expression and cell apoptosis were assessed using RT-qPCR, western blotting and a cell apoptosis assay. The results revealed that LINK-A lncRNA and HIF1α were downregulated in patients with diabetic nephropathy, as well as in diabetic patients without complications. The lowest expression of LINK-A lncRNA and HIF1α were observed in healthy controls. A positive correlation was identified between LINK-A lncRNA and HIF1α in both patients groups, but not in the control group. LINK-A lncRNA and HIF1α overexpression inhibited the apoptosis of mouse podocyte cells under a high glucose treatment. LINK-A lncRNA overexpression also promoted HIF1α expression in mouse podocyte cells, while HIF1α overexpression did not significantly affect LINK-A lncRNA expression. In conclusion, LINK-A lncRNA may activate HIF1α signaling resulting in the improvement of diabetic nephropathy treatment.
Diabetes mellitus, or diabetes, is a common metabolic disorder affecting a considerable portion of the worldwide population (
It has been reported that the activation of hypoxia-inducible factors (HIF) prevents diabetic nephropathy (
A total of 102 diabetic patients with nephropathy and 466 diabetic patients without complications were treated from January 2014 to March 2018 at Kunming Medical University (Kunming, China). Form those patients, a total of 32 patients with diabetic nephropathy and 28 diabetic patients without obvious complications were enrolled in the current study. The inclusion criteria were as follows: i) Patients who had not been treated within 3 months prior to admission; ii) patients with normal major organ function except kidney function in patients with diabetic nephropathy and; iii) patients received renal biopsies to detect potential renal lesions or if renal lesions were excluded in diabetic patients without complications. The exclusion criteria were as follows: i) patients who suffered from other severe diseases or chronic diseases, (63 patients were excluded based on this parameter); ii) patients who could not fully understand experimental protocol, (6 patients were excluded based on this parameter). During the same time period (January 2014 to March 2018), 56 individuals without diabetes also received renal biopsies to confirm suspected renal lesions, the results of which revealed that renal lesions were not present in 16 of them. Among these 16 people, 14 were included in the control group and 2 cases were excluded due to individuals suffering from additional severe diseases, including one patient with Hepatitis B and another patient with heart disease. The current study received ethical approval from the Ethics Committee of Kunming Medical University (Kunming, China). Renal biopsies used within the experiment were obtained from the specimen library of Kunming Medical University (Kunming, China). All patients and their families provided their written informed consent. See
Mouse podocyte cells (PrimCells, LLC) were used to perform
Total RNA was extracted from renal biopsies and cell lines using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the Reverse Transcriptase AMV kit (Sigma-Aldrich; Merck KGaA). qPCR was subsequently performed using the SuperScript III Platinum SYBR Green One-Step qPCR kit (Thermo Fisher Scientific Inc.). The following primer pairs were used for the qPCR: LINK-A IncRNA forward, 5′-TTCCCCCATTTTTCCTTTTC-3′ and reverse, 5′-CTCTGGTTGGGTGACTGGTT-3′; β-actin forward, 5′-GACCTCTATGCCAACACAGT-3′ and reverse, 5-AGTACTTGCGCTCAGGAGG-3′. The following thermocycling conditions were as follows: Initial denaturation at 95°C for 56 sec; 40 cycles of 95°C for 12 sec and 57.6°C for 40 sec. Data was quantified using the 2−ΔΔCq method and normalized to the internal reference gene β-actin (
Renal biopsies were used to measure levels of HIF1α using an ELISA kit (cat. no. EHIF1A; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Levels of HIF1α were normalized to ng/g.
Cell apoptosis under 20 mM D-glucose treatments were detected via a cell apoptosis assay. Mouse podocyte cell suspensions (5×104 cells/ml) were prepared in mouse podocyte cell culture complete medium with serum (Celprogen, Inc.). Cell suspensions (10 ml) were added to each well of a six-well plate, followed by the addition of 20 mM D-glucose (Sangon Biotech Co., Ltd.). Cells were cultured for 24 h at 37°C. Cells were digested using 0.25% trypsin, collected and mixed with Mouse Podocyte Cell Culture Complete medium with serum. Following centrifugation at 1,200 × g for 3 min at 22°C, cells were stained using Annexin V-FITC (Dojindo Molecular Technologies, Inc.) and propidium iodide at 22°C for 18 min. Apoptotic cells were detected using a CytoFLEX LX flow cytometer (Beckman Coulter, Inc.) and analyzed using FCSalyzer-0.9.15 (
Protein was extracted using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Inc.) and protein concentration was measured using a BCA assay. After denaturing at 95°C for 10 min, 20 µg protein was subjected to 12% SDS-PAGE gel electrophoresis. Following gel transfer to PVDF membranes, 5% skimmed milk was used to block the membranes at room temperature for 1 h. Membranes were then incubated with rabbit anti-human HIF1α (1:1,650; cat. no. ab2185; Abcam) and rabbit anti-human GAPDH (1:1,400; cat. no. ab8255; Abcam) primary antibodies overnight at 4°C. Membranes were incubated the next day with IgG-horseradish peroxide conjugated secondary antibodies (goat anti-rabbit; 1:1,500; MBS435036; MyBioSource, Inc.) at room temperature for 1 h. Signals were subsequently developed by ECL (Sigma-Aldrich; Merck KGaA). Data normalization was performed using ImageJ software (v.1.60; National Institutes of Health).
GraphPad Prism 6 software (GraphPad Software, Inc.) was used for all statistical analyses. Data were expressed as the mean ± standard deviation and analyzed using one-way analysis of variance followed by a least significant difference post-hoc test. The correlation between LINK-A lncRNA and HIF1α expression was analyzed using Pearson's correlation coefficient. Diagnostic values of LINK-A lncRNA for diabetic nephropathy was evaluated by receiver operating characteristic (ROC) curve analysis. P<0.05 was considered to indicate a statistically significant difference.
LINK-A lncRNA (
The diagnostic value of LINK-A lncRNA for diabetic nephropathy was analyzed via ROC curve analysis. With controls as references, the area under the curve (AUC) value was 0.9007 (standard error; 0.04579; 95% confidence interval; 0.8109–0.9904;
As presented in
Cell apoptosis under 20 mM D-glucose treatment was detected via a cell apoptosis assay. Compared with control and negative control cells with LINK-A lncRNA overexpression (
To further investigate the interactions between LINK-A lncRNA and HIF1α, LINK-A lncRNA and HIF1α expression vectors were transfected into mouse podocyte cells. The expression of LINK-A lncRNA and HIF1α was detected by RT-qPCR and Western blotting. As presented in
Human materials were used in the current study to assess the role of LINK-A lncRNA in diabetic nephropathy. The results of the present study indicated that LINK-A, as a recently identified lncRNA, may participate in diabetic nephropathy by interacting with HIF1α. Data from the present study also demonstrated that LINK-A may be a potential therapeutic target for the treatment of diabetic nephropathy.
The development of diabetic nephropathy can affect the expression pattern of a large set of lncRNAs (
The present study indicated that the downregulation of LINK-A may be used to distinguish patients with diabetic nephropathy from healthy controls and patients with diabetes but without obvious complications. These data indicate that LINK-A may serve as a potential diagnostic biomarker for the diagnosis of diabetic nephropathy. LINK-A, as a recently identified lncRNA, has an unknown expression in triple negative breast cancer (
The interaction between LINK-A and HIF1α has been revealed to occur in triple negative breast cancer (
The present study only performed
LINK-A overexpression inhibited the apoptosis of mouse podocyte cells under high glucose treatment in the current study. Therefore, LINK-A overexpression may serve as a therapeutic target for diabetic nephropathy. The present study is limited by the small sample size due to the difficulties in collecting renal biopsies. In the future, greater sample sizes should be used to further support results.
In conclusion, the current study revealed that LINK-A and HIF1α were upregulated in patients with diabetic nephropathy. LINK-A may also upregulate HIF1α to improve diabetic nephropathy and therefore may be a useful therapeutic target for potential treatments and therapies.
Not applicable
No funding was received.
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
JY and LL designed the study and performed all of the experiments. SH, ZZ and WF analyzed data. LL interpreted data and prepared the manuscript. All authors read and approved the final manuscript.
The present study was approved by the Ethics Committee of Kunming Medical University (Kunming, China). All patients and healthy volunteers provided written informed consent prior to their inclusion within the study.
The present study was approved by the Ethics Committee of Kunming Medical University (Kunming, China). All patients and healthy volunteers provided written informed consent prior to their inclusion within the study.
All patients provided informed consent for publication.
The authors declare that they have no competing interests.
Expression of LINK-A lncRNA and HIF1α were elevated in patients with diabetic nephropathy. Comparison of (A) LINK-A lncRNA and (B) HIF1α expression in 3 groups of participants. *P<0.05. HIF1α, hypoxia-inducible factor 1α; lncRNA, long non-coding RNA; LINK-A, long intergenic non-coding RNA for kinase activation.
Upregulation of LINK-A lncRNA distinguished patients with diabetic nephropathy from diabetic patients without complications and healthy controls. Receiver operating characteristic curve analysis of the diagnostic value of LINK-A lncRNA for patients with diabetic nephropathy, (A) patients with diabetes but without complications and (B) with healthy controls as references. LINK-A IncRNA, LINK-A long non-coding RNA; LINK-A, long intergenic non-coding RNA for kinase activation.
Expression of LINK-A lncRNA and HIF1α were positively correlated in 2 patients groups but not in the control group. Pearson's correlation coefficient analysis revealed a significantly positive correlation in (A) patients with diabetic nephropathy, (B) patients with diabetes but without complications and (C) healthy controls. HIF1α, hypoxia-inducible factor 1α; lncRNA, long non-coding RNA; LINK-A, long intergenic non-coding RNA for kinase activation.
LINK-A lncRNA and HIF1α overexpression inhibited the apoptosis of mouse podocyte cells under a high glucose treatment. Data of the cell apoptosis assay revealed that (A) LINK-A lncRNA and (B) HIF1α overexpression inhibited apoptosis of mouse podocyte cells under a high glucose treatment. *P<0.05. lncRNA, long non-coding RNA; HIF1α, hypoxia-inducible factor 1α; C, Control; NC, negative control; LINK-A, long intergenic non-coding RNA for kinase activation.
LINK-A lncRNA activates HIF1α in mouse podocyte cells. The effect of LINK-A lncRNA overexpression on (A) HIF1α expression and HIF1α overexpression on (B) LINK-A lncRNA expression in mouse podocyte cells was assessed. *P<0.05. lncRNA, long non-coding RNA; HIF1α, hypoxia-inducible factor 1α; C, Control; NC, negative control; LINK-A, long intergenic non-coding RNA for kinase activation.
Basic data of the study groups.
Sex | ||||
---|---|---|---|---|
Groups | Male, n | Female, n | Age range (years) | Average age (years) |
Diabetic nephropathy | 17 | 15 | 23–67 | 45.9±5.2 |
Diabetes | 15 | 13 | 25–69 | 46.4±6.7 |
Control | 11 | 13 | 24–69 | 46.1±5.5 |