A Preliminary Research On The Mechanisms Of Corneal Opacity In Mice Caused By Mycn Knockout

Mycn is a member of the Myc proto-oncogene family and is involved in many cellular processes such as cell proliferation,metabolism,differentiation,and tumorigenesis.Previous studies on Mycn mainly focused on tumorigenesis.Only a few studies on eyes have shown that Mycn can affect the development of retina and lens.However,there is no report on the correlation between Mycn and corneal opacity.In our previous study,a C57BL/6(B6)background heterozygous corneal opacity mouse with Mycn mutation was obtained with an N-ethyl-N-nitrosourea(ENU)mutagenesis strategy.Therefore,we will further verify the correlation in this study by using Mycn knockout mice,and have a study on the histological characteristics of corneas and the mechanism of corneal opacity in MycnKO/+mice.1 Verification of correlation between Mycn and corneal opacity in miceBased on the principle of Cre-loxP system,Mycn knockout(MycnKO/+)mice were obtained in a cross with Mycnflox/+and EIIa-Cre from the Jackson Laboratory in the United States.Some of MycnKO/+ mice appeared corneal opacity after birth(the penetrance was about 68.4%).In addition,it was found that some of these mice with corneal opacity displayed eyelids open at birth,so these mice were divided into two types:one with eyelids closed at birth(ECB)and corneal opacity;another with eyelids open at birth(EOB)and corneal opacity.2 Histological characteristics of corneas in MycnKO/+miceHE staining results of ECB corneal opacity mice,EOB corneal opacity mice and littermate control mice corneas in 2 weeks old(2W),6 weeks old(6W)and 8 weeks old(8W)showed that(1)Compared with the control,the corneal epithelium of ECB corneal opacity mice was thickened at 2W but became thinner at 8W;(2)The corneal epithelium of EOB corneal opacity mice began to thicken significantly and keratinized from 2W.In addition,neovascularization and inflammatory cells can be observed in the stromal layer of corneal opacity miceImmunohistochemical analyze of CD31 in corneal stroma showed that:CD31 was absent in the stromal layer of the control,indicating no neovascularization;CD31 was present in the stromal layer of ECB corneal opacity mice from 2W,further indicating neovascularization in the stromal layer;CD31 was abundantly present in the stromal layer of EOB corneal opacity mice from 2W,further indicating neovascularization in the stromal layer.We also performed staining for the corneal epithelial cell differentiation marker keratin 12(K12),the epithelial cell differentiation marker keratin 14(K14),and a specific epidermal differentiation marker,keratin 10(K10),by immunohistochemistry in 2W,6W and 8W mice.Immunohistochemistry results showed that:(1)K12 was present and K10 was not detected in the corneal epithelium of the control corneas.The expression of K14 was restricted to basal epithelial cells,the level was high at 2W but decreased gradually;(2)There was no significant change in K12 expression in corneal epithelium of ECB corneal opacity mice at 2W and 6W.But at 8W,K12 expression in the thinned regions of corneal epithelium was decreased.K14 was highly expressed at 2W,6W and 8W.K10 was not detected;(3)K12 was not detected in the thickened corneal regions of EOB corneal opacity mice,but was observed in the non-thickening regions at 2W,6W and 8W.K14 was expressed most prominently in the corneal epithelial layer(except for the negative expression in the superficial cells in thickened regions).K10 was detected in the thickened regions from 2W.3 Possible mechanism of abnormal corneal epithelial differentiation in MycnKO/+corneal opacity mice.Immunohistochemical results of integrin ?1,integrin ?4,E-cadherin and Cyclin D1 in corneal epithelium of 2W,6W and 8W mice showed that Mycn knockout may cause abnormal expressions of integrin ?1,?4 and Cyclin D1 in corneal epithelium during corneal development,and had no effect on the expression of E-cadherin in corneal epithelium(except for the negative expression in the superficial epithelial layer of EOB corneal opacity mice).Conclusion:Firstly,our study further verified the correlation between Mycn and corneal opacity,and found that Mycn knockout can also cause EOB.The results of HE staining and immunehisto-chemistry showed that abnormal corneal epithelial differentiation and neovascularization in the stromal layer were the key factors for corneal opacity.Preliminary research on the mechanism of abnormal corneal epithelial differentiation revealed that Mycn may affect the differentiation of corneal epithelium by regulating the expressions of integrin ?1,integrin ?4 and Cyclin D1 in corneal epithelium.