MicroRNAs As Biomarkers And Regulators In BHD-PSP

Birt-Hogg-Dube syndrome(BHDS),caused by folliculin(FLCN)germline mutations,is an autosomal dominant disease that is characterized by skin fibrofolliculomas,pulmonary cysts,primary spontaneous pneumothorax(PSP)and predisposition to kidney cancers.BHDS patients can manifest only pnenumothrax or lung cysts without the full spectrum of BHDS,and therefore are clinically indistinguishable from sporadic PSP,and they are named as BHD-PSP.BHD-PSP should be differentially diagnosis from sporadic PSP because of their high risk for renal cancer.To date,genetic testing for FLCN germline mutation is the only diagnostic criteria for BHD-PSP.However,only a part of PSP patients,about 10%,is associated with BHD mutations.Obviously,there are difficulties in differential diagnosis of the syndrome and consequent increase in a proportion of PSP patients who are suspected to have a BHD gene mutation.A rapid,convenient and cost-efficient method for screening and diagnosis of BHD-PSP should be developed.MicroRNAs in plasma or serum may serve as improved and noninvasive biomarkers for numerous pathologic conditions,including cancers,autoimmune diseases and even genetic disorders.In this study,we aim at finding out aberrant miRNA expression patterns in lung cyst lesions of BHD-PSP and validating the association between FLCN and miRNA.We inferred that the dysregulated expression of miRNAs in the BHD-PSP tissues could be reflected in the circulation system and be valuable biomarker for the identification of BHD-PSP.What is more,the BHD-associated miRNAs may target some genes involved in FLCN-associated signaling pathways,which may bring new clues for BHD-related diseases.Genetic testing for FLCN mutations were carried out in PSP population to set up BHD-PSP mutation database and samples database.Whole-genome microRNAs(miRNAs)screen was carried out in lung cyst lesions from BHD-PSP and nBHD-PSP patients for candidate diagnostic markers.Differentially expressed miRNAs were identified and validated by reverse transcription quantitative PCR in both cysts lesions and BEAS-2B cells with BHD gene knockdown.The expression levels of candidate miRNAs in the plasma of BHD-PSP,nBHD-PSP patients and 10 controls were measured and statistically analyzed to confirm their diagnostic accuracy.BHD-related signaling pathways and target genes were predicted by bioinformatics software analysis for differentially expressed miRNAs.Functional studies for the predicted pathways and target genes were carried out in vivo and in vitro.We identified 77 BHD-PSP,about 11%,from 697 PSP patients.Aberrant miRNA expression patterns of BHD-PSP were observed via miRNAs microarray analysis.And three miRNAs,let-7d,miR-424 and miR-199a-3p,were identified as candidate markers in the BHD-PSP lesions.Validation experiments further confirmed their upregulation in mutants in vivo and inducible knockdown cells of BHD gene in vitro.Plasma levels of all the three miRNAs were significantly higher in BHD-PSP than that in non BHD-PSP patients(P<0.001).The combination of let-7d and miR-424 yielded a better AUC value of 0.956,as well as the highest specificity(87.7%)and sensitivity(86.1%).There were six signaling pathways predicted by DIANA miRPath v2.0 and TargetScan Human 6.2 in BHD-associated miRNAs,including TGF-β and WNT pathway.Experiments in BHD-PSP cyst lesions showed that when FLCN expression was deficient,phosphorylated Smad3 was activated probably due to the inhibition of Smad7,subsequently expression of BIM was increased and then apoptosis was induced.Evidences in cells were found that TGF-β pathway was activated in both epithelial cells and fibroblast cells.Apoptosis was observed in epithelial cells but not in fibroblast cells,instead,mesenchymal-epithelial transition like phenomenon was observed in fibroblast cells.In conclusion,we showed that circulating BHD-associated miRNAs,let-7d and miR-424,were suitable biomarkers for the screening and the identification of BHD-PSP in this study.We also found the abnormal expression of BHD-associated miRNAs might target Smad7 to activate TGFβ signaling pathway and induce apoptosis of epithelial cells,and to inhibit WNT signaling pathway in stromal cells to induce MET-like change,which may be a mechanism for cyst formation in BHD-PSP patients.