Study On The Regulation Of PRAS40 Expression By MTOR And The Molecular Mechanism

Background:Tuberous sclerosis is an autosomal dominant neurocutaneous syndrome,which often occurs in children and can cause tumors in multiple organs or systems,including sebaceous adenoma in the skin,oral-nasal triangle,retinal glioma,renal angiomyolipoma,cardiac rhabdomyoma and pulmonary lymphangiomyomatosis.The mutation of TSC1 on chromosome 9 or TSC2 on chromosome 16 is often seen in patients with this disease,which results in hamartin or tuberin dysfunction respectively.These two proteins can form complex and regulate the activity of mTOR,so the mutation of TSC1 or TSC2 can lead to the activation of mTOR and affect cell proliferation and differentiation.Therefore,mTOR inhibitors rapamycin and everolimus have been used in the clinical treatment of tuberous sclerosis-related tumors and abnormally activated tumors of mTOR.However,they have some limitations.It is necessary to further study the specific mechanism of mTOR activation leading to disease,in order to provide a new strategy for the treatment of disease.PRAS40（encoded by AKT1S1 gene）is the substrate of AKT and the subunit of mTORC1.PRAS40 can be Phosphorylated by AKT and mTOR at sites T246 and S183respectively.it can also affect the activation of these two signaling pathways.Therefore,PRAS40 plays an important role in PI3K and mTOR signaling pathways.MiR-142-3p is a non-coding small RNA molecule,which can regulate gene expression by binding to the target m RNA.In recent years,studies have shown that miR-142-3p can inhibit the occurrence and development of tumors,and the expression level of miR-142-3p is down-regulated in most tumors.Studies have shown that in the serum of tuberous sclerosis patients,the expression of miR-142-3p decreased.Our Previous studies showed that PRAS40 was one of the targets of miR-142-3p,and miR-142-3p inhibited its expression by binding to 3’UTR of PRAS40 gene.Cytosine C5 methylation is an epigenetic modification of mammalian genome and plays an important role in transcriptional regulation.TET family is a class of5-methylcytosine dioxygenases,including TET1,TET2 and TET3,which can convert5-methylcytosine（5m C）into 5-hydroxymethylcytosine（5hm C）,and then catalyze its transformation to 5-formylcytosine（5f C）and 5-hydroxycycytosine（5ca C）,and then demethylate DNA by DNA damage repair mechanism.Studies have shown that mutations in TET family proteins in tumors can decrease the expression of micro RNAs by up-regulating the DNA methylation level encoding micro RNAs;in addition,some scholars have found that rapamycin can induce the expression of TET2.Objective:The purpose of this study is to explore the relationship between PRAS40and tuberous sclerosis-related tumors;to study the role of PRAS40 in the occurrence and development of tuberous sclerosis-related tumors;to explore how mTOR inhibits the expression of miR-142-3p;the ultimate goal is to provide a new scheme for the diagnosis and treatment of tuberous sclerosis and related tumors.Methods:The expression of PRAS40 protein in Tsc2^-/-MEF cells and Tsc2^+/+MEF cells was detected by Western blot,and the expression of PRAS40 m RNA in Tsc2^-/-MEF cells and Tsc2^+/+MEF cells was detected by RT-qPCR.Tsc2^-/-MEF cells were treated with Rapamycin,and the expression of PRAS40 protein and the level of PRAS40 m RNA were detected by Western blot and RT-qPCR,respectively.PRAS40 was knocked down in Tsc2^-/-MEF cells,and the proliferation difference between experimental group and control group was detected by CCK-8 experiment.On the basis of the above experiment,the proliferation difference between experimental group and control group was observed by Rapamycin treatment.The difference of miR-142-3p expression between Tsc2^-/-MEF and Tsc2^+/+MEF cells was detected by RT-qPCR.After transfection of Tsc2^-/-MEF with miR-142-3p and negative control micro RNA,CCK-8 and Western blot were used to compare the proliferation and PRAS40 expression among the groups.The RNAseq data of renal,breast and lung cancer in TCGA database were used to analyze the relationship between mTOR signaling pathway activity and Akt1s1 expression by GSEA.The differences of TET1 and TET2 expression between Tsc2^-/-MEF and Tsc2^+/+MEF cells were detected by RT-qPCR,and the changes of TET1 and TET2expression levels after Tsc2^-/-MEF Rapamycin treatment.The relationship between the activity of mTOR signaling pathway and TET1 as well as TET2 was analyzed by GSEA.Results:1.Compared with Tsc2^+/+MEF cells,the expression of PRAS40 protein and m RNA in Tsc2^-/-MEF cells was up-regulated.After rapamycin treatment of Tsc2^-/-MEF cells,the expression of PRAS40 protein and m RNA was down-regulated.2.Knockdown PRAS40 in Tsc2^-/-MEF cells inhibits cell proliferation,and the inhibition is not affected by mTOR activity.3.The expression of AKT1S1 was positively correlated with the activity of mTOR signaling pathway in TCGA lung cancer,renal cancer and breast cancer tissue samples.4.Overexpression of miR-142-3p in Tsc2^-/-MEF cells inhibits PRAS40 expression and cell proliferation5.Compared with Tsc2^+/+MEF cells,the expression of miR-142-3p was down-regulated in Tsc2^-/-MEF cells.6.The expression of TET1 and TET2 was negatively correlated with the activity of mTOR signaling pathway in TCGA lung cancer and renal cancer tissues.Conclusion:1.mTOR inhibits the expression of TET/miR-142-3p and promotes the expression of PRAS40.2.Knockdown PRAS40 inhibits mTOR activation-dependent and independent cell proliferation...