Characteristic Expression And Anti-ultraviolet Radiation Injury Role Of Nucleolar Protein 12 In The Rat Retina

Nucleolar protein 12(NOL12),also known as Nop25,is a highly conserved nucleolar localization protein in vertebrates.NOL12 is widely distributed in brain,lung,heart,kidney and spleen of mice.Viriato,a homologous protein of NOL12 in Drosophila melanogaster,is strongly expressed in the drosophila melanogaster eye.Our previous work found that NOL12 exists in the optic chiasm of rats,suggesting that NOL12 should be expressed in the retina.However,there is no report on the expression of NOL12 in mammalian retina.NOL12 plays a protective role in many kinds of cells: the decrease of NOL12 can induce cell apoptosis of HCT116 cells,and inhence the activities of ataxia telangiectasia and Rad3 related kinase(ATR)actting as a leader of DNA damage response(DDR),and cells become more sensitive to DNA damage caused by oxidative stress;in drosophila salivary gland and eye cells,knocking down the viriato(NOL12 homologous protein)can lead to cell apoptosis.Ultraviolet radiation(UV)often causes DNA damage and then activates ATR kinase pathway,leading to retinal neuron apoptosis.The protective effect of NOL12 and the mechanism of retinal damage induced by UV radiation suggest that NOL12 may play a protective role in retinal damage induced by UV radiation,and its protective role may be related to ATR kinase pathway.However,it is unclear whether NOL12 plays a protective role in retinal injury,and what is the relationship between its protective function and ATR.In this study,on the base of the expression of NOL12 in rat retina,we test the level of NOL12 in retina after UV radiation,and the anti-UV radiation damage role of NOL12 was analyzed.Expression of NOL12 in adult rat retina and human retinoblastoma cellsImmunohistochemical staining of NOL12 in the adult rat retina shows that NOL12 are expressed in adult rat retina;and NOL12 immunoreactivity is mainly distributed in retinal nerve cells,with strong positive reaction in ganglion cell layer and rod cone layer,weak positive reaction in inner and outer plexiform layer,and weak positive or negative reaction in other layers.NOL12 are mainly distributed in the cytoplasm as granular form,but no positive reaction products are found in the nucleus.NOL12 immunofluorescence staining of human retinoblastoma cells(WERI-Rb-1)showed that NOL12 is also expressed in WERI-Rb-1 cells.The NOL12 positive signals in WERIRb-1 cells are mainly located in nucleolus and less in cytoplasm.Western blot analysis showed that the molecular weight of NOL12 in WERI-Rb-1 cells are basically the same as that in rat retina.The weight of NOL12 of WERI-Rb-1 are slightly smaller than the retina.The different distributions of NOL12 in adult retinal cells and WERI-Rb-1 cells suggest that NOL12 may play a role in cell differentiation.UV radiation injures retinal cells through downregulation of NOL12 levelNOL12 can protect drosophila eyes cells and HCT116 cells,and the damage of cells caused by UV radiation is often accompanied by the decrease of protective protein level.To determine whether NOL12 in retina has anti-UV radiation effects,the effects of UV radiation on the expression of NOL12 in retina and WERI-Rb-1 cells are examined.Western blot analysis showed that the level of Cleaved Caspase-3 in retina and WERI-Rb-1 cells increased significantly after UV irradiation,while the level of NOL12 decreased significantly.Real-time PCR analysis showed that NOL12 RNA level in WERI-Rb-1 cells did not decrease but increased significantly after UV irradiation,suggesting that the decrease of NOL12 level in WERI-Rb-1 cells induced by UV irradiation is not caused by the decrease of NOL12 expression,but by a large number of degradation.These results suggest that UV radiation may induce the level of NOL12 and lead apoptosis of retina.To prove this hypothesis,we up-regulate the NOL12 level after UV radiation and down-regulate the NOL12 level in WERI-Rb-1 cells,and then analyze the apoptosis.Western blotting,nuclear staining and annexin V/PI flow cytometry analysis showed that the level of Cleaved Caspase-3 and the proportion of the nuclear fragmentation cells are increased after NOL12 were silenced by small interfering RNA in WERI-Rb-1 cells.Overexpression of NOL12 in WERIRb-1 cells significantly inhibit the level of Cleaved caspase-3,the proportion of the nuclear fragmentation cells and the proportion of the dead and late apoptotic cells induced by UV radiation.It is concluded that NOL12 can inhibit apoptosis,and UV radiation can induce apoptosis and lead to retinal damage by lowering the level of NOL12 and weakening its protective effect.NOL12 inhibits UV radiation-induced retinal injury through suppressing ATR kinase pathwayTo determine whether the protective effect of NOL12 on retina is achieved by affecting the ATR kinase pathway,we examined the level of ATR by changing the expression level of NOL12 and by UV radiation on WERI-Rb-1 cells,we examine the apoptosis of WERI-Rb-1 cells after inhibiting the activity of ATR kinase followed by silencing NOL12 and UV radiation,we also overexpress ATR kinase in oder to examine the effects of overexpression of NOL12 after UV radiation in WERI-Rb-1 cells.Western blot analysis showed that over-expression of NOL12 had no significant effect on ATR kinase level in WERI-Rb-1 cells,while silent NOL12 and UV irradiation significantly increased ATR kinase level in WERI-Rb-1 cells;ATR kinase specific inhibitor VE822 treatment inhibit the increase of Cleaved Caspase-3 level in WERIRb-1 cells induced by silent NOL12 and UV radiation;over-expression of NOL12 alone could inhibit UV radiation,but the significant inhibition could be weakened by cooverexpression of ATR kinase.These results suggest that the apoptosis induced by the reduction of NOL12 protein after UV radiation is depending on the activation of ATR kinase pathway,and NOL12 protects retinal cells by inhibiting ATR kinase pathway against UV radiation.Conclusion: NOL12 is expressing in the cytoplasm of cells of rat retinal ganglion cell layer,rod cone layer,inner reticular layer and outer reticular layer.UV radiation decrease the level of NOL12 protein and induce retina apoptosis.NOL12 protects the retina against UV radiation and its protective function is related to the inhibition of ATR kinase pathway.