Study on mRNA expression and DNA methylation of Pax6 in form-deprived myopia in C57BL/6 mice

Abstract

Objective To investigate the changes in refraction and ocular biometric parameters in monocular form-deprivation myopia, and to investigate whether myopia development is associated with changes in the retinal levels of Pax6 messenger RNA (mRNA) and the DNA methylation state in cytosine-phosphate-guanine (CpG) sites in the Pax6 promoter. Methods Experimental study. A diffuser lens was worn over one eye of one group of C57BL/6 mice for 4 weeks to establish a monocular form deprivation (MD) myopia model, and another group wore a diffuser lens for 4 weeks, then the goggles were removed for 1 week to build a recovery model. A total of 36 C57BL/6 mice (postnatal day 21) were randomly assigned to the MD group (n=12) , a normal control (NC) group (n=12) ; and MD recovery group (n=6) , a corresponding control group (n=6) . Ocular biometry was determined in mice by an EIR (eccentric infrared photorefractor) and OCT (optical coherence tomography) . The Pax6 mRNA level in the retina was determined by real-time PCR, and the Pax6 methylation state was observed by bisulfite DNA sequencing. Data were analyzed using paired t test or independent t test. Results After 4 weeks of form deprivation, a significant myopic shift had been induced in the treated eyes (MD-T) (-3.27±0.52 D) compared to the contralateral eyes (MD-C) (1.13±1.17 D) (t=-12.726, P<0.01) and normal control eyes (NC) (1.65±1.69 D) (t=-6.832, P<0.01) . Pax6 mRNA in the MD-T eyes (0.495±0.247) decreased significantly compared to the MD-C eyes (1.011±0.477) (t=-3.16, P<0.05) and the NC eyes (1.071±0.401) (t=-2.99, P<0.05) . One week after the removal of the diffuser lenses, Pax6 mRNA expression in recovery eyes had been fully restored to levels in the control eyes. The methylation percentage in CpG sites of the Pax6 promoter in the MD-T eyes was not significantly different compared to MD-C and NC eyes. Conclusion Monocular form deprivation myopia can be induced in C57BL/6 mice. Retinal Pax6 mRNA was reduced in the MD-T eyes compared to MD-C and NC eyes, but the changes in Pax6 mRNA may not be caused by DNA methylation of the CpG island in the promoter region of Pax6. Key words: Myopia; Retina; Pax6; DNA methylation; CpG island; Mice