| HGF (hereditary gingival fibromatosis) is characterized by diffusive and progressive gingival fibrous hyperplasia. It can affect the whole oral cavity or just regional area. It can appeared in the form of syndrome or nonsyndrome. The inheritance of HGF can be autosomal dominant, autosomal recessive, or just isolated disease. We mainly focus on the nonsyndromic HGF in this thesis, most of which are of autosomal dominant inheritance. The genetic heterogenity of HGF can be assessed from4genetic loci concluded from only a few nonsyndromic HGF families. c.3248-3249insC mutation located in the exon21of SOS1gene screened from GINGF1locus is the only fully elaborated pathogenic mechanism that caused nonsyndromic HGF.Our study group continuously collect nonsyndromic HGF families and sporadic cases.3loci have been successfully mapped using linkage analysis based on3HGF families. GINGF3loci was localized for yunnan HGF family using linkage studies by Ye xiaoqian in our study group。Objects:Mutation screening was performed in yunnan HGF family to find pathogenic mutation and underlying pathogenic mechanism of pathological fibrosis caused by the mutation.Methods:1. PCR was routinely used to sequence the exons located in the GINGF3locus;2. Co-segregation analysis between the mutation and the HGF phenotype was perfomed and mutation screening was also conducted in1300control DNA samples from unrelated healthy individuals to eliminate the possibility of being SNP;3. According to the position of mutation, pathogenic mechanism was further investgated:whether subcellular location or miRNA regulation was influenced by the mutation, et al; Results:1. Mutation screening:c.4396C>G nucleotide change was found in the3’UTR of FOSL2in this kindred.2. Co-segregation analysis:all the available DNA samples collected from this kindred were used for co-segregation analysis. There is complete co-segregation between the c.4396C>G mutation and the phenotype of HGF in yunnan family. c.4396C>G mutation is not present in2600control chromosomes extracted from unrelated healthy individuals.3. The effect of mutation to the subcellular location of FOSL2:according to the results of immunofluorescence, the subcellular location of FOSL2with wild-type3’UTR is located in the nucleus and the subcellular location of FOSL2with mutant3’UTR also located in the nucleus;4. The effect of mutation to the regulation of miRNA:according to the prediction results of miRanda, c.4396C>G mutation site is located just in the target site of miR-151a-3p and such prediction result was further confirmed by dual luciferase reporter assay and western blot;5. The relationship between FOSL2and fibrosis:FOSL2overexpression lentivirus was transfected into primary gingival fibroblast, then qPCR was used to assess mRNA level of collagen; qPCR results demonstrated that, overexpressed FOSL2in primary gingival fibroblast drastically increase collagen content.Discussion:We found c.4396C>G mutation in the3’UTR region of FOSL2in GINGF3locus. And the mutaion in FOSL2abolished the binding of miR-151a-3p, thus preventing negative regulation of miR-151a-3p. As a result, the protein level of FOSL2was upregulated, and hence, collagen content was elevated, which leading to pathogenic fibrosis. TSCC (tongue squamous cell carcinoma) is one of the most prevalent tumors arised from head and neck region. Although the treatment has achieved great progress, the5-year survival rate of patients with TSCC has not greatly improved, which was mainly due to the lymph node metastasis. And the extent of lymph node metastasis serve as a marker to predict the prognosis. Thus, understanding the molecular mechanism of TSCC invasion and migration is of the utmost importance to prevent tumor metastasis.ADAM10fucntions as a proteolytic enzyme to cleave the ectodomain of miscellaneous transmembrane proteins——growth factors, adhesion molecules, cell surface receptors, and some other substrates. Due to the great number and diverse functions of the substrate, the abnormal expression of AD AM10bring about extensive collateral effects. ADAM10was confirmed to be upregulated in cancers located in stomach, colon, uterus and tongue. Thus, ADAM10not only serves as a biomarker for tumors, but also functions as a target for anti-cancer therapy. The expression profile of miRNAs in certain tumors is unique and specific, and miRNA thus not only serves as biomarker for certain tumors but also functions as a negative regulator to repress the invasion and migration of tumors.Objects:1. Bioinformatics were used to predict the miRNA that can directly target ADAM103’UTR. And the miRNA with an8mer site in the ADAM103’UTR was chosen for further analysis;2. Experiments were conducted to confirm the prediction result, that is, ADAM10is indeed a direct target of the chosen miRNA;3. To confirm the repressive effect of the miRNA on TSCC cell invasion, migration and proliferation.4. According to the results of previous step, DAVID database was used to predict which targets of the miRNA might involved in the processes of repression.5. Experiments were carried out to verify prediction results. Methods:1. Bioinformatics analysis:Targetscan program was used to predict potential targets of the miRNA; DAVID database was used to classify those targets;2. Dual luciferase reporter assay:3’UTRs of the predicted target genes were cloned into the dual luciferase vector; Then the sequence recognized by the seed sequence of the miRNA was mutated into the same sequence as the seed sequence. miRNA mimes and dual luciferase plasmids were co-transfected into Tca8113cell.48h later, luciferase activities were measured to assess the repression effect.3. Western blotting:miRNA mimics or miRNA negative control was transfected into CAL27cell, and48h later, western blot was used to assess the endogenous protein level of the target genes.4. Transwell invasion/migration assay:miRNA mimics or miRNA negative control was transfected into CAL27cell, transwell assay was used to assess repressive effect of miRNA on CAL27cell invasion and migration.5. Proliferation assay:miRNA mimics or miRNA negative control was transfected into CAL27cell, CCK-8was used to assess the repressive effect of miRNA on CAL27cell proliferation.Results:1. According to the prediction results of Targetscan, there is only one8mer site in the3’UTR of ADAM10recoginzed by miR-140-5p;2. According to the results of dual luciferase assay, relative luciferase activity with wild-type ADAM103’UTR was significantly repressed by miR-140-5p, while relative luciferase activity with mutant ADAM103’UTR was not responsible to the regulation of miR-140-5p;3. According to the results of western blot, endogenous protein level of ADAM10was significantly repressed by miR-140-5p;4. According to the results of transwell invasion/migration assay, ecotopic miR-140-5p inhibits CAL27cell invasion and has apparent effect on CAL27cell migration;5. According to the results of proliferation assay, miR-140-5p has no effect on CAL27cell proliferation;6. All potential targets of miR-140-5p, predicted by Targetscan, were submitted to DAVID database for classification; LAMC1, HDAC7, PAX6, IGF1R, PSEN1and ERBB4were classified into (positive regulation of) cell migration ralated genes and were chosen for further analysis;7. According to the results of western blot, endogenous protein level of LAMC1, HDAC7and PAX6were significantly repressed by miR-140-5p, while IGF1R and PSEN1were not responsible to the regulation of miR-140-5p; Surprisingly, endogenous protein level of ERBB4, predicted to be direct target of miR-140-5p, was upregulated by overexpressed miR-140-5p;8. According to the results of luciferase assay, relative luciferase activity with wild-type LAMC1, HDAC7, PAX6and ERBB43’UTR was significantly repressed by miR-140-5p, while relative luciferase activity with mutant LAMC1, HDAC7, PAX6and ERBB43’UTR was not responsible to the regulation of miR-140-5p;Discussion:1. miR-140-5p inhibits TSCC cell migration and invasion, but not proliferation;2. ADAM10, HDAC7, PAX6and LAMC1are direct targets of miR-140-5p.3. In TSCC cell, miR-140-5p has no effect on IGF1R and PSEN1;4. Even though the binding between miR-140-5p and ERBB43’UTR, the endogenous protein level of ERBB4was upregulated by ectopic miR-140-5p;5. Repressed ADAM10can enhance the effect of miR-140-5p to other target genes——ERBB4and PAX6;... |
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