Circ_0001953 contribute to retinal vascular endothelial cell injury induced by high glucose through regulating miR-186

Purpose: To investigate the effects of circ_0001953/miR-186 on human retinal vascular endothelial cell (HRVEC) injury evoked by high glucose. Methods: A cell model (HG group) was established using high glucose-treated HRVECs, while untreated HRVECs were used as the control group (Con group). The levels of endothelin-1 (ET-1), ICAM-1 and IL-6 were evaluated by ELISA and the content of malondialdehyde (MDA) and superoxide dismutase (SOD) in HRVECs were determined. Apoptosis rate was tested adopting flow cytometry. The interrelationship between circ_0001953 and miR-186 was assessed using dual luciferase reporter assay. Measurement of Bax and Bcl-2 was implemented via western blot. Results: In HG group, circ_0001953 increased while miR-186 was downregulated, ET-1, IL-6, ICAM-1, and apoptosis rate increased and accompanied with up-regulated Bax content and declined Bcl-2 protein level. Furthermore, the content of MDA increased and SOD decreased. MiR-186 was a target of circ_0001953. Conclusion: Inhibition of circ_0001953 can repress inflammation, oxidative stress and apoptosis in HRVECs by up-regulating the expression of miR-186.

It is known that the expression of circ_0001953 is increased in active tuberculosis, which makes it a possible biomarker for the diagnosis of active tuberculosis [8].Target gene prediction shows that circ_0001953 has a binding site with miR-186.MiR-186 is down-regulated in osteoarthritis mouse chondrocytes, and contributes to the inhibition of apoptosis via disturbing PI3K-AKT pathway [9].Nevertheless, the level of circ_0001953 and miR-186 in DR as well as their underlying mechanisms are still unclear.In this study, high glucose-treated HRVECs were used to establish a cell model to unveil the function of circ_0001953/miR-186 in cell injury caused by high glucose.

Materials and reagents
Human retinal vascular endothelial cells and DMEM medium were obtained from Gibco (Grand Island, NY, USA); reverse transcription and fluorescent quantitative PCR reagents were purchased from Thermo Fisher (Carlsbad); Trizol® was bought from Invitrogen (Carlsbad, CA, USA); Lipofectamine 2000 and apoptosis detection kit were purchased from Soleibao Technology (Beijing, China); ET-1, IL-6, ICAM-1, SOD and MDA detection kits were bought from Jiancheng Institute of Bioengineering (Nanjing, China); si-NC, si-circ_0001953, miR-NC, miR-186 mimics, anti-miR-NC, and anti-miR-186 were synthesized by Jima Pharmaceutical Technology (Shanghai, Chain); rabbit anti-human primary antibodies and HRP-labeled secondary antibody were provided by Abcam (Cambridge, MA, USA)

Experimental groups
HRVECs were cultured in medium containing 5.5 mmol/L or 30 mmol/L D-glucose for 24 h [10], as control group (Con group) and high glucose treated group (HG group), respectively.When HRVECs were 70% confluent in 24-well plates, transfection was carried out.In brief, an appropriate amount of Lipofectamine 2000 was diluted with 50 μL medium without serum, followed by an appropriate amount of oligonucleotide sequence or vectors diluted with 50 μL medium without serum, and then the two were mixed.The mixture (100 μL) and 400 μL serum-free medium were cultured with HRVECs and 6 h later, the serum-containing medium was utilized.Transfected HRVECs were stimulated with 5.5 mmol/L or 30 mmol/L D-glucose based on the experimental group.

qRT-PCR
Total RNA in HRVECs was gained from cells applying Trizol® in accordance with the guidance of manufacturer, the concentration of purified RNA was detected by an ultraviolet spectrophotometer (HiTaChi, Tokyo, Japan).The solution for reverse transcription PCR was prepared as in the manufacturer's guidelines.At last, qRT-PCR was performed as per the guidelines of the manufacturer [11].

ELISA
Cell supernatant was harvested and the content of ET-1, IL-6 and ICAM-1 of each group was measured using ELISA as per manufactuer's instructions.About 100 μL of each sample and the standard substance were inoculated into the ELISA plate for 90 min incubation.Then 100 μL biotinylated antibodies were added into each well after discarding the liquid, followed by incubation at 37 ℃ for 60 min, adding 100 μL horseradish peroxidase-conjugated working solution to each well for 30 min incubation at 37 ℃.Next, each well was incubated with 90 μL TMB substrate solution away from light and 15 min later, stop solution was utilized.Lastly, the absorbance was measured using a micropore reader.

The detection of SOD and MDA
Cultured cells were collected, and the measurement of the content of SOD and MDA was conducted using their respective commercial kits.

Flow cytometry
In brief, collected cells were digested with 0.25% trypsin and then suspended in PBS solution.About 1×10 6 cells were taken from each sample, and the cells were suspended in 500 μL binding buffer with 5 μL Annexin V-FITC and 5 μL PI (Beyotime, Shanghai, China), consecutively.Apoptosis was detected by flow cytometry.

Dual-luciferase reporter assay
Starbase was employed to analyze the candidate target miRNAs of circ_0001953.The sequences of circ_0001953 3' UTR contained miR-186 binding sites or mutant sequences in binding sites was cloned into pmirGLO plasmid (GenePharma, Shanghai, China).The constructed vectors (named as WT-circ_0001953 or MUT-circ_0001953, respectively) were transfected into HRVECs in combination with miR-186 mimics or miR-NC.Luciferase activity was evaluated 48 h later, and the activity of renal luciferase was regarded as an internal reference.

Statistical analysis
All data were presented as mean ± standard deviation (SD).SPSS statistics 21.0 version was employed for performing statistical Student's t-test or one-way analysis of variance was employed to check the difference between groups.P < 0.05 indicated statistically significant.

High glucose promoted the expression of circ_0001953 while inhibited miR-186 level
A cell injury model of DR was established with high glucose.Then relative to the Con group, HRVECs in HG group displayed lower circ_0001953 level and higher miR-186 level (Figure 1).

Circ_0001953 contributed to high glucose induced inflammation and oxidative damage in HRVECs
To probe the contribution of circ_0001953 in cell jury induced by high level of glucose, si-circ_0001953 was transfected into cells.The si-circ_0001953 markedly weakened the expression of circ_0001953 under the exposure of high glucose (Figure 2A).High glucose elevated the levels of ET-1, IL-6 and ICAM-1, which were reversed by circ_0001953 inhibition (Figure 2B).For oxidative damage, results showed increased MDA content and decreased SOD activity in HG group, and these effects were alleviated by si-circ_0001953 (Figure 2C-D).Above results suggested that facilitated high glucose stimulated inflammatory and oxidative damage.

Circ_0001953 had functions in high glucose evoked cell apoptosis in HRVECs
As shown in Figure 3A, apoptosis rate, enhanced by high glucose, was blocked after the infection of si-circ_0001953.Furthermore, HG group showed elevated Bax but reduced Bcl-2 in cells, which was reversed by si-circ_0001953 (Figure 3B).Thus, these results suggest that circ_0001953 could promote apoptosis in high glucose-treated HRVECs.

MiR-186 is targeted by circ_0001953
CircRNAs can generally serve as sponges of miRNAs.Circular RNA interactome exhibited there are binding sites between miR-186 and circ_0001953 (Figure 4A).To further confirm this connection, dual luciferase reporter assay was employed.MiR-186 overexpression considerably blocked the luciferase activity of WT-circ_0001953 reporter while no obvious change was observed in MUT-circ_0001953 reporter together with overexpressed miR186, suggesting the interrelationship between miR-186 and circ_0001953 (Figure 4B).Consistent with above results, the level of miR-186 was elevated when si-circ_0001953 was introduced (Figure 4C).Therefore, we verified circ_0001953 suppressed the expression of miR-186.For exploring the effect of miR-186 in HG-treated HRVECs, miR-186 mimics was introduced into cells.As expected, Figure 5A showed successful transfection of miR-186 mimics with the elevated miR-186 level in HG+miR-186 group.ELISA assay demonstrated that miR-186 introduction inhibited the content of ET-1, IL-6 and ICAM-1 (Figure 5B).Moreover, oxidative stress was relieved with additional miR-186 (Figure 5C-D).It was also observed that the apoptosis rate of HGtreated HRVECs was lowered as manifested by high-expressed Bcl-2 and low-expressed Bax (Figure 5E-F).Altogether, miR-186 inhibited inflammatory and apoptosis while relieved oxidative stress in HG-treated HRVECs.

MiR-186 absent reversed the effect caused by si-circ_0001953 in HG-treated HRVECs
To directly unveil whether miR-186 was a functional target for circ_0001953, si-circ_0001953 and/or anti-miR-186 was cotransfected into HRVECs.As described in Figure 6A, the expression of miR-186 decreased in HG+si-circ_0001953+anti-miR-186 group.In addition, the level of inflammatory factors, MDA, Bax as well as apoptosis rate were elevated while SOD and Bcl-2 decreased (Figured 6B-F).In all, anti-miR-186 abated the functions caused by si-circ_0001953 in HG-treated HRVECs.

CONCLUSION
This study has demonstrated that circ_0001953 increased in high glucose-challenged HRVECs, while the expression of miR-186 was decreased.Circ_0001953 insufficiency inhibited cell inflammation, oxidative stress and apoptosis to alleviate cell damage by up-regulating miR-186.The circ_0001953/miR-186 axis may be responsible for DR progression, and benefited for developing new strategy for DR prevention.

Figure 1 :
Figure 1: The expression of circ_0001953 and miR-186 in high glucose-treated HRVECs was measured.Relative to Con group, * P <0.05

Figure 2 :
Figure 2: The effect of circ_0001953 inhibition in high glucose-treated HRVECs.(A) Examination of interference efficiency of si-circ_0001953 via qRT-PCR.(B-D) cells were introduced with si-NC or si-circ_0001953, and then cultured with high glucose.(B) ELISA analysis for ET-1, IL-6 and ICAM-1 contents.(C-D) Detection of MDA and SOD levels.* P <0.05, # P <0.05

Figure 5 :
Figure 5: miR-186 inhibited inflammatory and apoptosis but relieved oxidative stress.(A-F) miR-NC and miR-186 were infected into cells.(A) Measurement of miR-186 via qRT-PCR.(B) The level of inflammatory factors evaluated by ELISA.(C, D) Measurement of MDA and SOD contents.(E) Flow cytometry for apoptosis rate detection.(F) Western blot for Bax and Bcl-2 levels.* P <0.05

Figure 6 :
Figure 6: Effect caused by si-circ_0001953 was blocked by anti-miR-186.(A-F) si-circ_0001953 and anti-NC or anti-miR-186 transfected HRVECs were treated with high glucose.(A) Measurement of miR-186 utilizing qRT-PCR.(B) Levels of inflammatory factors was evaluated by ELISA.(C, D) Measurement of MDA and SOD contents.(E) Analysis of apoptosis rate via flow cytometry.(F) Western blot for Bax and Bcl-2 levels.* P <0.05