Roles And Mechanisms Of MTORC1 Signaling Pathway In Primary Pterygium

Background and aimsPterygium is a very common ocular proliferative disease with the chararcteristics of wing-shaped tissue encroachment of conjunctival tssue over the cornea.The wing-shaped tissue after invasion the cornea may increases gradually,even can cover the pupil area,.Its existence not only affect the appearance of the patients,but also can caused eye discomfort,corneal astigmatism,eye movement disorders.If the influence of light into the eyes of pterygium covering pupil,will seriously affect the visual acuity.At present,the pathogeny and pathogensis of this disease is is not fully understood,involves many theories,such as cell apoptosis contro disorder,oxidative damage,virus infection,the effect of cytokines and so on.Now we also treat the pterygium by surgery,though the surgert method is improved,such as using limbal tissue,autologous conjunctival,or amniotic membrane,there also have a high level of recurrence rate.Some anti-tuma medicians such as mitomycin,pingyangmycin also be used in pterygium opteration in order to reduce the recurrence rate,but the effect is limited.And the anti-tuma meidcians may caused some serious complications,such as wound healing time delay,sclera dissolution.In recent years,with the development of anti-VEGF drugs,ranibizumab is also used for the treatment of pterygium,but its effectiveness is still controversial,and also lack of large multi center clinical trials.The incompletely understanding of the pathogenesis of pterygium is the root cause of the lack of effective prevention and control measures,therefore,further study of pathogenesis of pterygium is vital to its prevention and control.Mammalian target of rapamycin(mTOR)is a highly conserved Ser/Thr protein kinase and forms two distinct functional complexes,termed mTOR complex 1(mTORC1)and mTORC2.mTORCl integrates diverse signals including nutrients,growth factors,energy and stresses to regulate cell growth,proliferation,survival,and metabolism.At present many major diseases such as cancer,cardiovascular disease,diabetic retinopathy have be find involved in mTOR signaling pathways of imbalance,so in recent years the study of the mTOR signaling pathway has become a focus of proliferative disease targeted therapy,and the drug development,but the study on the pathway and pterygium has not yet been reported.A large number of epidemiology and basic research showed that pterygium is a disease closely related to the ultraviolet irradiation.Skin cancer is a kind of disease also closely related to the ultraviolet irradiation.A new study suggests that the skin cancer is the imbalance between cell proliferation and apoptosis caused due to ultraciolet stimulated the activation of mTORC1 signaling pathway.From Taiwan epidemiological investigation showed that compared with the normal people,the pterygium patients are associated with an increased risk of skin cancer,suggesting the pterygium and skin cancer may existence of similar pathogenesis.So we think that mTORC1 signaling pathway may play an important role in the pathogenesis of pterygium.CyclinDl and vascular endothelial growth factor(VEGF)are two important downstream of mTORC1 signaling pathway,their expression is regulated by mTORC1 signaling pathway and paly an important role in the formation of tumors.CyclinD1 is a kind of important cell regulatory proteins,has been confirmed in such as breast cancer,lung cancer,bile duct carcinoma,uterine fibroids,and other abnormal hyperplastic disease plays an important role.It promotes cell cycle progression through the G1-phase by forming active holoenzymes with CDK(cyclin-dependent kinase)4 and CDK6 and leads to phosphorylation of pRb(retinoblastoma protein).Phosphorylation causes pRb to release the E2F transcription factor,which can then activate genes essential for progression through the G1-S transition.Under normal circumstances,CyclinDl protein only expressed in G1 phase transient,if its excessive expression,can promote cell from G1 phase to S phase transformation,accelerate the process of cell cycle,due to constant proliferation of the cell.VEGF is a very powerful stimulate angiogenesis factor,and is closely related with the formation of tissue angiogenesis,the formation of new blood vessels provide oxygen and nutrients to the organisation.The combination of VEGF and endothelialcell receptor,can immediately cause the influx of calcium ions,the intracellular calcium concentration increased rapidly,and stimulated to produce the three inositol phosphate.VEGF and its receptor extracellular domain combination,can make the two receptordimers,induced tyrosine phosphorylation of receptor in the tyrosine residues autophosphorylation and intracellular proteins,intracellular signal conduction,promoting the proliferation of vascular endothelial cells,increase of vascular permeability,eventually leading to the formation of new blood vessels,promote the sustained growth of the abnormal hyperplasia tissue.This study aims investigate the expression of mTOR,P-S6,CyclinD1,VEGF,and then investigate the relationship between them,to explore the role of mTORC1 signaling pathway in the pathogenesis of pterygium.MethodsPtergyium specimens were obtained from 31 patients during surgery at the thire affliliated hospital,southern medical university in May 2013 to July 2014.Normal conjunctiva tissues taken from 13 cases of retinal detachment surgery patients,4 cases of patients with strabismus surgery.25 cases of pterygium and 11 cases of normal conjunctival were formalin-fixed and paraffin-embedded.6 cases of pterygium and 6 cases of normal conjunctival preserved in liquid nitrogen,Study on the following experiments and application of the above tissue specimens:1.Application of hematoxylin eosin staining observation of primary pterygium and normal conjunctiva organization histological differences.2.Using the immunohistochemical fluorescence staining detection the expression level of primary pterygium and normal conjunctiva.3.Using Western blot method and immunohistochemical staining method to detect the expression of P-S6,CyclinDl and VEGF in primary pterygium,and,and compared with normal conjunctiva,further explore mTORC1 signaling pathway in the mechanism in primary pterygixumResults1.Compared with normal conjunctiva,pterygium tissue epithelial hyperplasia was obvious,cell layers up to 10 or more,and with a large number of fibroblasts,new blood vessels and inflammatory cells.2.The expression of mTOR and P-S6 were higher in primary pterygium than normal conjunctiva.In primary,the mean IOD was 4207000±179900 for mTOR,and the mean IOD was 1872000±11817000 for P-S6.While in normal conjunctiva,the mean IOD was 185400±32330 for mTOR,and the mean IOD was 178500±27950 for P-S6.The difference was statistics significant(P<0.05).3.Western blot show in 6 cases of pterygium specimen the P-S6 protein/S6 protein(1.196±0.1015)was higher than normal conjunctiva tissues(0.2952±0.05613).The difference was statistics significant.The positive rate of P-S6 was 100%in pterygium(score:11.36±0.26)and was 18.2%in normal conjunctiva(score:2.09±0.31),showing significant differences in the expressions of P-S6 between pterygium and normal conjunctiva(P<0.05).4.Western blot show in 6 cases of pterygium specimen the CyclinD1 protein(1.229±0.07974)was higher than normal conjunctiva tissues(0.2327±0.03228).The difference was statistics significant(P<0.05).The positive rate of CyclinDl was 100%in pterygium(score:11.04±0.32)and was 9.1%in normal conjunctiva(score:1.73±0.27),The difference was statistics significant(P<0.05).5.Western blot show in 6 cases of pterygium specimen the VEGF protein(1.341 ±0.05731)was higher than normal conjunctiva tissues(0.2982±0.06974).The difference was statistics significant(P<0.05).The positive rate of VEGF was 100%in pterygium(score:10.92±1.71)and was 27.3%in normal conjunctiva(score:2.18±1.25),The difference was statistics significant(P<0.05).6.Used the pearson correlation analysis,the expression of P-S6 and CyclinD1 existed positive correlation(r=0.752,P<0.05),and there also existed positive correlation between the expression of P-S6 and VEGF in the pterygium(r=0.914,P<0.05).Conclusions1.The mTORC1 signaling pathway is activated in primary pterygium.2.The activation of mTORC1 signaling pathway may induce overexpression of CyclinDl and VEGF,which may be related to the occurrence and development of pterygium.