microRNA-199a-5p regulates epithelial-to-mesenchymal transition in diabetic cataract by targeting SP1 gene

Abstract

Background: As a common ocular complication of diabetes mellitus, diabetic cataract is becoming a leading cause of visual impairment. The progression of diabetic cataract progression involves epithelial-to-mesenchymal transition (EMT), the precise role of which remains to be investigated. As microRNAs (miRNAs) are suggested to be involved in the pathogenesis of many diseases, identification of aberrantly expressed miRNAs in diabetic lens epithelial cells (LECs) and their targets may provide insights into our understanding of diabetic cataract and potential therapeutic targets.

Methods: Diabetic cataract capsules and LECs exposed to high glucose (25 mmol/L, 1-5 days) were used to mimic the model. Quantitative RT-PCR was performed to evaluate the differential expression of miRNA. Dual luciferase reporter assay was used to identify the binding target of miR-199a-5p. The expression of EMT-associated proteins was determined by immunofluorescence and Western blot analysis.

Results: Our results showed the differential expression of miR-9, -16, -22, -199a and -204. MiR-199a was downregulated in diabetic cataract capsule and hyperglycemia-conditioned human LECs. Specific protein 1 could be directly targeted and regulated by miR-199a in LECs and inhibit EMT in diabetic LECs.

Conclusion: Our findings implied miR-199a could be a therapeutic target by regulating SP1 directly to affect EMT in diabetic cataract and provided novel insights into the pathogenesis of diabetic cataract.

Keywords: Diabetic cataract; Epithelial-to-mesenchymal transition; MiR-199a-5p; Specific protein 1.

Fig. 1

EMT and miRNA expression changes in human diabetic cataract and hyperglycemic LECs. a, b Immunofluorescence analysis was performed to detect the protein expression of EMT markers in capsules. The staining intensity of alpha-SMA, FSP-1 and vimentin expression increased in capsule of diabetic cataract than that of ARC, and E-cadherin expression decreased (n = 6). c, d The protein expression of EMT markers detected by western blot. An increased expression of alpha-SMA, FSP-1 and vimentin, while decreased expression of E-cadherin in HG conditions could be found (n = 9). e The expression of miRNAs was detected by RT-qPCR. MiR-9, miR-16, miR-138, miR-195, miR-204 were upregulated significantly, while miR-15a, miR-29 and miR-199a were downregulated in the diabetic cataract capsules (n = 9). f In the HG-treated LECs, miR-9 was upregulated firstly and then downregulated, while the expression of miR-16 and miR-29 were increased with HG exposure time. The expression of miR-22 and miR-199a were downregulated after 3 days of HG treatment (n = 3) (*P < 0.05; ***P < 0.005). EMT epithelial-to-mesenchymal transition, LECs lens epithelial cells, DCC diabetic cataract capsule, ARC age related cataract, HG high glucose

Fig. 2

SP1 was the direct target of miR-199a. a Bioinformatics-based target analysis showed that SP1 is a potential target of miR-199a. b Luciferase reporter assay showed that the luciferase activity of SP1 3′UTR-wt significantly decreased with miR-199a mimic transfection, comparing to that of the NC mimic or SP1 3′UTR-mutant group (n = 3). c–e In the HG-treated cells, the mRNA and protein expression of SP1 increased significantly compared to NG control cells (n = 9). f The level of miR-199a was increased with miR-199a mimic transfection and decreased in the inhibitor group, compared with the normal cells, or negative control mimic/inhibitor. Meanwhile, lipofectamine (Lipo Ctr) did not affect miR-199a expression (n = 3). g–i SP1 mRNA and protein levels were downregulated significantly with the miR-199a mimic transfection, while they were upregulated when miR-199a was inhibited (n = 9). SP1 specific protein 1, HG high glucose, Lipo Ctr only lipofectamine treated as the lipofectamine control. *P < 0.05

Fig. 3

MiR-199a repressed EMT through SP1 in hyperglycemic LECs. a, b SP1 mRNA and protein expression were significantly decreased by SP1 siRNA transfection (n = 6). c–f After miR-199a mimic transfection, the protein expression of alpha-SMA, vimentin and FSP-1 was downregulated compared with NC mimic in HG condition, while the E-cadherin was upregulated. When SP1 siRNA was transfected, alpha-SMA, vimentin and FSP-1expression decreased while E-cadherin expression increased (n = 6). SP1 specific protein, HG high glucose, LECs lens epithelial cells. *P < 0.05

Fig. 4

MiR-199a participated in the suppression of EMT by targeting SP1 gene in diabetic cataract