In view of the high incidence of diabetic retinopathy and the functionality of long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) in different disease models, the present study aimed to investigate the role of MEG3 in diabetic retinopathy. In the study, patients with diabetic retinopathy, diabetic patients without retinopathy as well as healthy people were included. Fasting blood was extracted from each participant. Serum MEG3 levels were detected by everse transcription-quantitative polymerase chain reaction (RT-qPCR) and serum vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1) levels were detected by ELISA. Also, the effects of high glucose treatment on the expression of MEG3 and VEGF and the effects of MEG3 overexpression on expression of VEGF and TGF-β1 in high glucose-treated ARPE-19 cells were detected by RT-qPCR and western blot analysis to determine the mRNA and protein levels, respectively. It was indicated that serum levels of MEG3 were significantly lower, while the serum levels of VEGF and TGF-β1 were significantly higher in patients with diabetic retinopathy and diabetic patients without retinopathy compared with the healthy controls. Furthermore, slight differences were found between patients with diabetic retinopathy and diabetic patients without retinopathy; however, these differences were not significant. The findings indicated that high glucose upregulated the expression of VEGF mRNA and downregulated the expression of MEG3, MEG3 overexpression reduced the increased expression levels of VEGF and TGF-β1 induced by high glucose treatment. Therefore, it was concluded that lncRNA MEG3 overexpression may inhibit the development of diabetic retinopathy by inhibiting TGF-β1 and VEGF expression.
With the development of modern society and changes in people's life style and diet structure, incidence of diabetes has been increased significantly during the last several decades (
Long non-coding RNA (lncRNA) refers to a group of RNA transcripts composed of more than 200 nucleotides that will not be translated into protein products (
In this study, serum levels of MEG3 in patients with diabetic retinopathy, diabetic patients without retinopathy as well as healthy people were measured. Interactions between MEG3, and vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1), which are two key players in diabetic retinopathy were explored. There report is as follow:
A total of 33 patients with diabetic retinopathy were selected in Affiliated Hospital of Beihua University (Jilin, China) from January 2014 to January 2017. All those patients were diagnosed according to the diagnostic criteria established by Chinese Medical Association in 2014. Patients with other types of retinopathy were excluded. Those patients included 15 males and 17 females, and age ranged from 44 to 71 years, with an average age of 56±11.3 years. During the same time period, 28 diabetic patients without retinopathy were also selected. Those diabetic patients included 12 males and 16 females, and age ranged from 39 to 77 years, with an average age of 53±14.1 years. At the same time, 30 healthy people were selected as control group. Control group included 11 males and 19 females, and the age ranged from 40 to 72 years, with an average age of 54±13.7 years. No significant differences in age and gender were found among three groups. This study was approved by the Ethics Committee of Affiliated Hospital of Beihua University. All patients signed informed consent.
Fasting blood (20 ml) was extracted from each participant in the morning. Blood samples were kept at room temperature for 1 h, followed by centrifugation at 2,500 × g for 20 min to collect serum samples. Serum samples were stored at −80°C before use.
Serum levels of VEGF and TGF-β1 were detected using corresponding ELISA kit provided by (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the instructions.
Human retinal pigment epithelial cell line ARPE-19 was provided by American Type Culture Collection (Manassas, VA, USA). ARPE-19 cells were cultured under conditions recommended by ATCC. Cells were harvested at logarithmic growth phase.
MEG3 cDNA (V0728, GeneCopoeia) was inserted into pIRSE2-EGFP vector (Clontech Laboratories, Inc., Mountainview, CA, USA) to make MEG3 expression vector. Cells were cultured overnight to reach 80–90% confluent, and transfection was performed using Lipofectamine 2000 reagent (11668-019; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Total RNA was extracted from serum and
Total protein extraction from
SPSS19.0 (SPSS, Inc., Chicago, IL, USA) was used. Normal distribution data were expressed as mean ± standard deviation), and comparisons among multiple groups were performed by one-way analysis of variance followed by the LSD test. P<0.05 was considered to indicate a statistically significant difference.
As shown in
VEGF and TGF-β1 play pivotal roles in the development of diabetic retinopathy. Therefore, serum levels of VEGF and TGF-β1 were detected by ELISA and compared among groups. As shown in
In this study, two different concentrations (15 and 30 mM) of d-glucose were used to treat ARPE-19 cells, and 5 mM d-glucose was used as control. Expression of VEGF, TGF-β1 and MEG3 in ARPE-19 cells was detected by qRT-PCR. As shown in
In this study, expression level of VEGF was increased for more than 5-fold after treatment with 30 mM d-glucose for 48 h. Previous studies have shown that MEG3 expression level is negatively correlated with the expression of VEGF, indicating that MEG3 may negatively regulate the expression of VEGF. In this study, MEG3 overexpression ARPE-19 cell line was established (
It is well known that high glucose can induced the expression of TGF-β1 (
At present, diabetes affects more than 350 million people worldwide, and the incidence of this disease is still increasing (
LnRNAs now are considered to be major plays in the development of various human diseases. A recent study has shown that the development of diabetic retinopathy is accompanied with the changes in genome-wide expression profile of lncRNA (
Vascular endothelial growth factor (VEGF), which is a key player in neovascularization, is closely correlated with the development of diabetic retinopathy (
In conclusion, serum levels of MEG3 were significantly reduced, while serum of VEGF and TGF-β1 were significantly increased in patients with diabetic retinopathy and diabetic patients without retinopathy compared with healthy controls. High glucose treatment upregulated the expression of VEGF mRNA and downregulated the expression of MEG3. MEG3 overexpression reduced the increased expression levels VEGF and TGF-β1 induced by high glucose. Our study first reported that lncRNA MEG3 is involved in the pathogenesis of diabetic retinopathy, and it may serve as a promising target for the treatment of this disease. However, this study is still limited by small sample size. In addition, all participants in this study are Asian, and the effects caused by ethnicity cannot be excluded. Therefore, further studies are still needed to confirm the conclusions in this study. Besides that, due to the limited resources, deeper investigations were not performed. We will solve those problems in our future study.
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.
DZ, YL and JW designed the experiments. DZ, HQ and LY performed the experiments. XL, LZ, DB and YM analyzed the data. YL wrote the manuscript. All authors have read and approved the final submitted manuscript.
This study was approved by the Ethics Committee of Affiliated Hospital of Beihua University. All patients signed informed consent.
Not applicable.
The authors declare that they have no competing interests.
Serum levels of MEG3 in three groups. *P<0.05 as indicated. DR, patients with diabetic retinopathy; D, diabetic patients; HC, healthy control; MEG3, maternally expressed gene 3.
Serum levels of VEGF and TGF-β1 in three groups. Serum levels of (A) VEGF and (B) TGF-β1 in three groups. *P<0.05 as indicated. DR, patients with diabetic retinopathy; D, diabetic patients; HC, healthy control. TGF-β1, transforming growth factor-β1; VEGF, vascular endothelial growth factor.
Glucose regulates the expression of VEGF, TGF-β1 and MEG3 in the ARPE-19 cell line. Serum levels of (A) VEGF, (B) TGF-β1 and (C) MEG3 in ARPE-19 cells treated with different concentrations of d-glucose at different time points. *P<0.05 as indicated. VEGF, vascular endothelial growth factor; TGF-β1, transforming growth factor-β1; MEG3, maternally expressed gene 3.
MEG3 overexpression reduces the increased expression level of VEGF in ARPE-19 cells induced with glucose. (A) MEG3 expression in cells with different treatments. (B) MEG3 overexpression reduced the increased serum level of VEGF mRNA. (C) MEG3 overexpression reduced the increased serum level of VEGF protein. *P<0.05 as indicated. C, control; G, 30 mM d-glucose; NC, negative control; O, MEG3 overexpression. VEGF, vascular endothelial growth factor; MEG3, maternally expressed gene 3.
MEG3 overexpression reduced the increased expression level of TGF-β1 in ARPE-19 cell induced by glucose. (A) MEG3 overexpression reduced the increased serum level of TGF-β1 mRNA. (B) MEG3 overexpression reduced the increased serum level of TGF-β1 protein. *P<0.05 as indicated. C, control; G, 30 mM d-glucose; NC, negative control; TGF-β1, transforming growth factor-β1; MEG3, maternally expressed gene 3.