Associations of Hsa-mir-328-3p and PAX6 Protein Expression with Ocular Refractive Errors

Abstract

Introduction Eyesight is one of our most important senses: 80% of what we perceive comes through our sense of sight [1]. Refraction disorders, such as myopia, hyperopia or astigmatism can negatively affect our quality of life, therefore it is important to understand how these problems develop so proper prevention tactics can be utilised in time. It is established that environmental and genetic factors have notable influence in the development of refraction disorders [2]. Among the many researched genes associated with these disorders is PAX6, a very important gene in eye development [3]. The importance of the gene led to the hypothesis that mutations of this particular gene, could be linked to multiple eye anomalies, particularly myopia. A case-control study published in 2011 found that rs662702, along with other single nucleotide polymorphisms (SNPs) had marginal significance (p = 0.063), and further analyses showed that it was associated with extreme myopia [4]. JASPAR computer program showed that in the 3'UTR region of the PAX6 gene, located at thers662702 single nucleotide polymorphism site, when there is a risk allele change, miR-328 binds to it, suppressing PAX6 protein expression. All the studies so far have been done in cells, we tried to study and evaluate the dependence of PAX6 protein on miR-328 in human and evaluate PAX6 protein and miR-328 as potential biomarkers. Aim To assess the association of hsa-mir-328-3p and PAX6 protein expression with refractive errors. Methods 69 myopes, 32 hyperopes and 43 emmetropics subjects participated in the study whose ages varied between 18 and 40 years. Subjects were included in the study according to the inclusion criteria: consent to participate in the study; individuals without any interventions that could lead to changes in refraction; age: ≥18 and ≤40; not having cataracts. Serum PAX6 protein levels were determined by ELISA. The expression of miR-328 was determined by real-time polymerase chain reaction (RT-PCR). Pearson's correlation coefficient r and Spearman's rank correlation coefficient rs were used to calculate correlation. The receiver operating characteristic - ROC was used to determine PAX6 protein concentration and miRNA-328 as a potential biomarker for refractive disorders. Results In the myopia group (from -2,6 to -7,9 D) there was a weak negative correlation between ΔΔCt miR-328 and PAX6 – as PAX6 increases ΔΔCt miR-328 decreases, and in the hyperopia group (age 18-40), there was a weak positive correlation between ΔΔCt miR-328 and PAX6, but the results were statistically insignificant for both groups. The miR-328 as a possible biomarker for myopia had an AUC = 0.56, p = 0,33 while the PAX6 protein had an AUC = 0.60 p = 0,20. MiR328 as a potential biomarker for farsightedness has an AUC = 0.51, p= 0,84 and PAX6 an AUC= 0.51. p = 0,88. Conclusions1. ΔΔCt miR-328 and PAX6 protein expression are not statistically significantly different between the refractive error groups and the control group. 2. The correlation calculations showed no statistically significant association between ΔΔCtmiR-328 and PAX6 protein expression in the refractive error groups and the controlgroup. 3. Based on the AUC plots of the ROC curves, ΔΔCt miR-328 and PAX6 protein as a potential prognostic biomarker are not suitable for the prediction of myopia or hyperopia.