Mechanism of cyclic RNA0001287 and miR-21 in the pathogenesis of diabetic retinopathy / 眼科新进展

ABSTRACT

Objective To investigate the action mechanism of cyclic RNA0001287(circ_0001287)and miR-21 in the pathogenesis of diabetic retinopathy(DR).Methods Primary human retinal pigment epithelium(phRPE)cells were iso-lated for circRNA microarray analysis.Arising retinal pigment epithelium(ARPE)-19 cells were cultured in vitro and divid-ed into the blank group,high-glucose group,negative group,si-circ group,circ_0001287 group,circ_0001287+negative group,and circ_0001287+miR-21 group.Small interfering RNA(siRNA)oligonucleotides against circ_0001287,mimics containing miR-21 sequences and miR-21 mimic plasmids were constructed.In the negative group,si-circ group,circ_0001287 group,circ_0001287+negative group and circ_0001287+miR-21 group,the empty plasmid,circ_0001287 siRNA,circ_0001287 mimics,circ_0001287 mimics+miRNA disordered sequence,and circ_0001287 mimics+miR-21 mimic plasmid were transfected into ARPE-19 cells using Lipofectamine 2000 Transfection Reagent.After transfection for 6 h,the Opti-MEM medium was replaced with a fresh normal medium.Cells in the blank group and the high-glucose group were not transfected.Cells in the blank group were cultured with culture solution containing 5.5 mmol·L-1 glucose,and cells in the high-glucose group were cultured with culture solution containing 15.5 mmol·L-1,25.5 mmol·L-1 and 35.5 mmol·L-1 glucose,respectively.Cells in other groups were treated with 35.5 mmol·L-1 glucose for 48 h.The expressions of circ_0001287 and miR-21 were detected by reverse transcription polymerase chain reaction(RT-PCR),cell proliferation activity was detected by Cell Counting Kit-8,and the targeting relationship between circ_0001287 and miR-21 was detected by Dual Luciferase Reporter Assay.RNA immunoprecipitation(RIP)assay and biotin-coupled probe pull-down assay were used to verify the targeting relationship between circ_0001287 and miR-21.Western blot was used to detect protein expression.Re-sults After screening by circRNA,the expression of hsa_circ_0001287 in phRPE cells was significantly reduced.RT-PCR detection showed that compared with the blank group,circ_0001287 expression in ARPE-19 cells in the high-glucose group decreased(P＜0.05)in a dose-dependent manner,and miR-21 expression in ARPE-19 cells gradually increased with the in-crease of glucose concentration(P＜0.05).After co-transfection of siRNA with circ_0001287 mimics,siRNA also reduced circ_0001287 expression,and the relative expression of circ_0001287 in the circ_0001287+negative group(0.70±0.03)was significantly lower than that in the negative group(0.98±0.04,P＜0.05).For cells transfected with the circ_0001287-WT plasmid,compared with the control simulation group(0.98±0.03),the relative luciferase activity of the miR-21 simulation group(0.59±0.02)decreased(P＜0.05).However,for cells transfected with circ_0001287-MUT plasmid,the relative ac-tivity of luciferase was almost the same in the control simulation group(0.96±0.05)and the miR-21 simulation group(1.00±0.04,P＞0.05).In the anti-Ago RIP experiment,miR-21 was significantly enriched in the circ_0001287 group com-pared with the control group,indicating that miR-21 could be significantly pulled down by the biotinylated circ_0001287 probe.Pull-down analysis demonstrated that compared with the control IgG,circ_0001287 specific probe pull-down sam-ples showed significant enrichment of circ_0001287 and miR-21.In this experiment,the cell proliferation rate of the circ_0001287+miR-21 group(78.25％±3.01％)was lower than that of the circ_0001287+negative group(90.88％±3.51％,P＜0.05).Compared with the blank group,the expression of PTEN protein in ARPE-19 cells in the high-glucose group treated with 35.5 mmol·L-1 glucose was significantly down-regulated(P＜0.05),the expression of PTEN protein in ARPE-19 cells in the circ_0001287 group was higher than that in the negative group(P＜0.05),and the expression of PTEN protein in ARPE-19 cells in the circ_0001287+miR-21 group was higher than that in the circ_0001287 group(P＜0.05).Conclusion The expression of circ_0001287 is down-regulated in phRPE cells and high-glucose induced ARPE-19 cells,and up-regulated circ_0001287 can inhibit the injuiy of diabetic RPE cells by adsorption of miR-21 and activation of PTEN expression.