The Role Of MTOR/p70S6K Signaling Pathway In The Regulation Of Pterygium Fibroblasts On Their Proliferation And Transdifferentiation

Background:Pterygium is a common chronic disease with the character of tumor.The mechanism of pterygium is still unclearly.The principal treatment for pterygium is surgical removal,but the rate of recurrence is 2% ~ 39%.More insights into the pathogenesis of pterygium are required.Fibrovascular proliferation plays a privital role in pterygium pathogenesis.The the environment and ocular surface inflammation can induce the activation of HPFs,then the activated fibroblast lead to proliferation,transdifferentiation and ECM accumulation.This process can be attributed to pterygium progress and recurrent.Many factors and signaling pathway take part in this process.mTOR/p70S6 K signaling pathway is important for cell growth and proliferation.Dysregulation of the pathway is associated with many types of tumors and fibrotic disorders.It is reported that mTOR/p70S6 K signaling pathway plays important role in corneal scar,the scarring with glaucoma surgery and proliferative vitreoretinopathy.It is still unknown that the role of the signaling pathway in the pathogenesis of HPFs proliferation and transdifferentiation.Objectives:To investigate the expression of mTOR/p70S6 K signaling pathway in pterygium tissue and pterygium fibroblasts,to analyse the correlation between mTOR/p70S6 K with PCNA and α-SMA in pterygium fibroblasts.To investigate the proliferation and transdifferentiation of HPFs by mTOR overexpression and mTOR silence.To investigate the mTOR special inhibitor on the proliferation and transdifferentiation of HPFs,to find evidence for the using of mTOR special inhibitor in the treatment for pterygium.Methods:1.Primary pterygium samples from 48 patients and normal conjunctival samples from 16 patients were surgically removed and analysed.The expression levels of p-mTOR and p-p70S6 K in the excised specimens were assessed using immunohistochemistry,RT-PCR and western blotting.2.Primary culture fibroblasts from pterygium tissues and normal conjunctival tissues were observed with inverted microscope and confirmed their identity by immunofluorescence staining with vimentin and Keratin.The expression levels of mTOR/70S6 K,PCNA and α-SMA in culture fibroblasts were assessed using immunofluorescence staining and western blotting.The expression levels of mTOR/p70S6 K,PCNA and α-SMA were analysed to their correlation.3.p EGFP-N3-mTOR overexpression plasmids were constructed and detected the expression levels of p70S6 K,PCNA and α-SMA.mTOR sh RNA plasmids were constructed and transfected into HPFs.The expression levels of p-p70S6 K,PCNA and α-SMA were determined using western blotting and immunofluorescence.4.Cell proliferation was evaluated using a CCK8 assay.HPFs were treated with rapamycin at concentrations of 0,1,10,100,1000,and 10000 ng/ml for 24,48,and 72 h.The absorbance(OD)value was measured at 450 nm on an automated microplate reader and the inhibitory rate of cell proliferation was calculated.HPFs were treated with rapamycin at concentrations of 0,100,and 500 ng/ml for 48 h.The expression levels of mTOR/p70S6 K,PCNA and α-SMA were determined using western blotting and immunofluorescence.Results:1.The expression levels of p-mTOR and p-p70S6 K were significantly higher in the pterygium tissues than that in normal conjunctiva tissues(p<0.001).The stainings were distributed sparsely in the epithelium,and preferentially in the fibroblasts of the stroma.mTOR and p70S6 K m RNA expression levels were increased in the pterygium tissues than that in normal conjunctiva tissues(p<0.05).p-mTOR and p-p70S6 K protein expression levels were increased in the pterygium tissues than that in normal conjunctiva tissues(p<0.05).2.HPFs were cultured and identify by vimentin and keratin.The expression levels of p-mTOR,p-p70S6 K,PCNA and α-SMA were significantly higher than that in HCFs.A significant positive correlation was detected between the expression of mTOR/70S6 K,PCNA and α-SMA(p<0.001).3.p EGFP-N3-mTOR overexpression plasmids were constructed successfully.The expression levels of p-p70S6 K,PCNA and α-SMA were upregulated in HPFs after p EGFP-N3-mTOR transfection(p<0.05).mTOR sh RNA plasmids were constructed successfully.The expression levels of p-p70S6 K,PCNA and α-SMA were downregulated in HPFs after mTOR shRNA transfection(p<0.05).4.The treatment of rapamycin inhibited the proliferation of HPFs at the concentration of 10、100、500、1000、10000ng/ml.The treatment of rapamycin inhibited the expression levels of p-mTOR,p-p70S6 K,PCNA and α-SMA at the concentration of 100ng/ml and 500ng/ml.Conclusions:1.mTOR/p70S6 K signaling pathway may play important role in the pathogenesis of pterygium.The pathway can regulate the proliferation and transdifferentiation of HPFs,thus lead to the development of pterygium.2.The proliferation and transdifferentiation of HPF was inhibited by RAPA.It provides a theoretical basis for finding new targets for pterygium treatment.