Immunoregulatory effects of miR-31-5p overexpression on peripheral blood Th17 cell in a rabbit model of autoimmune dry eye

Objective

To investigate the immunoregulatory effects of lentivirus-mediated microRNA (miR)-31-5p overexpression on peripheral blood T helper cell 17 (Th17) in a rabbit model of autoimmune dry eye.

Methods

The miR-31-5p recombinant lentiviral vector was constructed.Lentivirus overexpressing miR-31-5p and its control virus were packaged.The concentration measurement and lentiviral titer determination were carried out.A rabbit model of autoimmune dry eye was established and the peripheral blood mononuclear cells (PBMC) of the rabbits were isolated.PBMC infected with miR-31-5p and negative control lentivirus particles were assigned as the miR-31-5p overexpression group and control group, respectively.The miR-31-5p expression level was detected using quantitative real-time PCR (qRT-PCR). Then PBMC in the two groups were co-cultured with γ-ray irradiated lacrimal gland epithelial cells.The expressions of Th17 cell related transcription factor retinoic acid-receptor-related orphan receptor C (RORC) and interleukin-17 (IL-17) mRNA, IL-1β, IL-6 and IL-23 were determined by qRT-PCR.The IL-17 protein expression level was detected by Western blot.The use and care of animals complied with Regulation for the Administration of Affair Concerning Experiment Animals by State Science and Technology Commission.The study protocol was approved by the Ethics Committee of Tianjin Medical University Eye Hospital (No.TJYY20201221036).

Results

The construction of the miR-31-5p recombinant lentiviral vector was verified by DNA sequencing.The lentiviral titer of lentivirus overexpressing miR-31-5p and control lentivirus particles was 3.82×10⁷ TU/ml and 3.50×10⁷ TU/ml, respectively.The miR-31-5p relative expression level of PBMC was significantly increased in miR-31-5p overexpression group in comparison with control group, showing a statistically significant difference (t=-9.696, P<0.001). When PBMC were co-cultured with lacrimal gland epithelial cells in vitro, the relative expression levels of RORC and IL-17 mRNA in miR-31-5p overexpression group were 0.33±0.03 and 0.28±0.09, which were significantly decreased in comparison with 1.00±0.00 and 1.00±0.00 in control group, with statistically significant differences between them (t=46.256, 13.810; both at P<0.05). The relative expression level of IL-17 protein in miR-31-5p overexpression group was significantly reduced than control group (t=4.977, P=0.008). The relative expression levels of IL-1β, IL-6 and IL-23 mRNA were significantly lower in miR-31-5p overexpression group than control group (t=220.076, 6.641, 13.271; all at P<0.05).

Conclusions

The overexpression of miR-31-5p can inhibit the Th17-immune response via down-regulating the expression of IL-6, IL-1β and IL-23.

Dry eye; Sjögren syndrome; Th17 cells; miR-31-5p; Disease models, animal; Peripheral blood mononuclear cell