| Aim:Hereditary gingival fibromatosis(HGF)is a highly genetically heterogeneous disease.It is impractical to design a targeted drug for every HGF family.Despite of different causative genes of HGF,the eventually outcome is consistent.Profibrotic pathways were activated to promote fibrotic responses.Thus,if there is a molecule that can target these key profibrotic genes,it would have the potential to be a broad spectrum anti-fibrotic molecule.Micro RNAs(miRNAs)are able to fine-tune large-scale target genes.Our present study aims to establish a novel and efficient miRNA screening pipeline based on canonical miRNA targets prediction algorithms to pinpoint the most potent antifibrotic miRNA,and experimentally unravel the functional and mechanical effect of miR-335-3p in HGF.We tried to provide novel targets for treatment of HGF.Materials and methods:1.We summarized 85 experimentally corroborated profibrotic genes and established a novel and efficient miRNA screening pipeline based on canonical miRNA targets prediction algorithms to pinpoint the most potent antifibrotic miRNA which targeted most of the 85 genes.m RNA expression profiling was used to screen the up-regulated genes in HGF gingival tissues compared to healthy control.Up-regulated genes were analyzed with the same miRNA screening methods.2.RT-q PCR and WB were employed to detect the expression of miR-335-3p in gingival fibroblasts derived from HGF patients and healthy control,and the relationship between miR-335-3p and the widely acknowledged profibrotic cytokine TGF-β1.3.RT-q PCR,WB,Flow Cytometry and cell immunofluorescence were utilized to testify the antifibrotic potential of miR-335-3p in NHGFs.4.Biotin-labeled miR-335-3p pull-down assay,dual-luciferase reporter assay,RT-q PCR and WB were applied to identify the down-stream gene targets of miR-335-3p.5.WB was employed to detect expression changes of fibrotic markers after knock-down of SMAD2/3,SOS1 and CTNNB1 by small interfering RNAs,respectively.Results:1.Bioinformatic results showed that miR-335-3p was ranked number 1 among all the human miRNAs and could potentially repress 46 of the 85 profibrotic genes(46/85,54%).m RNA expression profiling indicated that 79 genes were up-regulated in HGF gingival tissues compared to healthy control(fold change≥3,p<0.05).MiR-335-3p was ranked number 4 and potentially targeted 48 of the79 up-regulated genes(48/79,61%).2.MiR-335-3p was down-regulated in gingival fibroblasts derived from HGF patients compared to NHGFs(Normal human gingival fibroblasts).TGF-β1induced profibrotic activities of NHGFs,meanwhile expression of miR-355-3p was suppressed in a time-dependent manner.3.MiR-335-3p inhibited base-line and TGF-β1 induced fibrotic responses in NHGFs.4.MiR-335-3p directly targets SMAD2/3,SOS1 and CTNNB1 by canonical seed-paring sites or noncanonical 3’-compensatory base-paring sites to exert its negative regulation.5.Different portfolios of fibrotic markers were suppressed when SMAD2/3,SOS1 or CTNNB1 was silenced by si RNAs,respectively.Conclusion:We firstly proposed a novel and efficient miRNA screening pipeline based on canonical miRNA targets prediction algorithms to pinpoint the most potent antifibrotic miRNA-miR-335-3p.Ectopic miR-335-3p significantly suppressed fibrotic responses in human gingival fibroblasts by directly targets multiple profibrotic genes(SMAD2/3、SOS1 and CTNNB1)with canonical seed-paring sites or noncanonical 3’-compensatory base-paring sites to inhibit different portfolios of fibrotic markers.Aim:Gingival overgrowth(GO)can be presented as an inherited disease(Hereditary gingival fibrosis,HGF),or a side effect of therapeutic drugs,its etiology is unclear.Extensive studies have reported that activated mutations of potassium channel genes were strongly correlated with gingival overgrowth(GO)or gingival fibromatosis phenotype.Mutations of KCNQ1,KCNH1,KCNJ8,ABCC9 and KCNK4 in different syndromes all presented GO or gingival fibromatosis phenotype.It is reasonable to suppose that activation of potassium channels may be involved in hereditary GO or gingival fibromatosis.In the present study,we choose KCNQ1,a voltage-dependent potassium channel,to further explore the role of potassium channel in HGF.We aimed to unravel novel molecular drivers of GO and offer new targets for its treatment.Materials and methods:1.NHGFs(Normal human gingival fibroblasts)were stimulated by KCNQ1 selective agonist ML277 and antagonist chromanol 293 B.WB,cell immunofluorescence and whole-cell patch clamp were used to detect KCNQ1 expressions and outward K+ currents.Transmembrane potential and intracellular K+ changes were determined by flow cytometry as labeled by Di BAC4(3)and Enhanced Potassium Green-4 AM(EPG-4)respectively.2.Expression of KCNQ1 was detected by immunohistochemistry in HGF patients and normal control.Whole-cell patch clamp analysis was applied to detect outward K+ currents in NHGFs and HGF gingival fibroblasts.Intracellular K+changes was determined by flow cytometry as labeled by EPG-4 in NHGFs and HGF gingival fibroblasts.3.NHGFs were stimulated by ML277 for 3,6,9,12 and 15 days.Accumulation of Extracellular Matrix(ECM)proteins and production of total soluble collagenswere measured by Western blotting and Sircol Soluble Collagen Assay,respectively.Overexpression of KCNQ1 was conducted by adenovirus infection,fibrotic markers were detected by WB.4.The relationship of TGF-β1 and KCNQ1 was determined by Enzyme-Linked Immunosorbent Assay(ELISA),WB,Whole-cell patch clamp and EPG-4/Di BAC4(3)flow cytometry.5.Biotin-labeled cell membrane protein pull-down assay,active RAS pull-down assay,WB and cell immunofluorescence were utilized to verify RAS activation.6.Activation of MAPK signaling pathways were determined by WB and cell immunofluorescence.JNK-IN-8 and GDC-0094,selective inhibitor of JNK and ERK,were applied to inhibit MAPK signaling,and fibrotic markers was detected by WB.Results:1.NHGFs expressed functional KCNQ1 channels,which responded to its selective agonist ML277 and antagonist chromanol 293 B.2.KCNQ1 was upregulated in gingival tissues derived from HGF patients and HGF gingival fibroblasts presented increased outward K+ currents than normal human gingival fibroblasts(NHGFs).3.ML277 promoted synthesis of ECM proteins and production of total soluble collagens in NHGFs.Overexpression of KCNQ1 significantly facilitated COL1A1 and FN expressions.4.TGF-β1 and KCNQ1 channels formed a positive feed-back loop,chromanol293 B partially attenuated TGF-β1 induced ECM synthesis and accumulation in NHGFs.5.ML277 generated lateral clustering and activation of RAS on plasma membrane for activation in NHGFs.6.After treatment with ML277 for 2h,p-ERK and p-JNK were upregulated,and ERK was translocated to the nucleus.Then expression of Fra-2 and p-c-Jun wereupregulated after 4h treatment with ML277.JNK-IN-8 and GDC-0994,selective inhibitor of JNK and ERK1/2 pathway,suppressed ML277 induced Fra-2,p-c-Jun,COL1A1 and FN upregulation.Conclusion:We firstly proposed that activation of KCNQ1 channel promoted fibrogenic responses in NHGFs via RAS/MAPK/AP-1 signaling.TGF-β1 and KCNQ1 channels formed a positive feed-back loop.In particular,increased KCNQ1 currents promoted the activation of RAS on plasma membrane for activation,which facilitated MAPK/AP-1 signaling pathway input and responsive ECM output in NHGFs.Our study refreshed understandings of molecular drivers of HGF,and offered novel target for its treatment. |
