遗传性牙龈纤维瘤致病机理研究及miR-140-5p抑癌机制探讨
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摘要
遗传性牙龈纤维瘤是一种以牙龈组织弥漫性、渐进性纤维增生为特征的遗传疾病；可累及全口、亦可只病发于局部；可以以综合征的形式出现,亦可以非综合征型出现；可以以常染色体显性方式遗传或者常染色体隐形方式遗传,亦可呈现为散发病例而无遗传史；本文主要研究的是非综合征型遗传性牙龈纤维瘤,其遗传方式以常染色体显性遗传为主,并呈现出高度的遗传异质性：目前根据所报道的不多的几个非综合征型遗传性牙龈纤维瘤家系,所定位的致病区域就有4个：GINGF1-GINGF4:而唯一一个致病机制得到完整阐述的,也仅仅是GINGF1区域内的致病基因SOS1第21个外显子所发生的插入突变：c.3248-3249insC。
本课题组持续收集非综合征型遗传性牙龈纤维瘤家系及散发病例,并已成功对其中3个家系锁定其致病区域：其中GINGF3位点即为本课题组叶晓茜老师根据云南家系进行连锁分析所定位的致病区域。
实验目标：
对一个非综合征型遗传性牙龈纤维瘤大型家系——云南家系,进行突变筛查,并研究其导致病理性纤维化的致病机制；
材料和方法：
1.PCR：采用常规PCR测序的方法,筛查云南家系致病区域内(GINGF3)的基因外显子区域；
2.共分离及正常人对照验证：根据发现的突变,在家系内所有病人及所有正常人中,确定突变是否与遗传性牙龈纤维瘤表型共分离；然后在1300例正常人DNA对照中,确认突变是否会在正常对照中发现,排除其是SNP的可能性；
3.根据突变位置,进一步进行功能研究：是否改变亚细胞定位,是否影响miRNA的调控,等等等等。
结果：
1.突变筛查结果：在云南家系FOSL2基因的3'UTR区域,发现c.4396C>G碱基改变;
2.共分离及对照验证：在所有采集到血液并成功提取DNA的云南家系成员中,c.4396C>G碱基改变在所有病人中都有发现,在所有正常人中都没有发现；在1300例对照正常人中也没有发现；
3.突变是否影响FOSL2蛋白的细胞定位：免疫荧光结果表明,包含有正常型FOSL23'UTR的质粒所表达的蛋白,定位于细胞核,包含有突变型FOSL23'UTR的质粒所表达的蛋白,同样定位于细胞核；
4.突变是否影响miRNA的调控：根据niRanda软件预测结果,c.4396C>G突变位点正好处于miR-151a-3p的靶位点中；双荧光素酶检测以及western blot结果,进一步证实了该预测结果。
FOSL2与纤维化的关系：通过转染Fosl2过表达慢病毒到原代牙龈成纤维细胞,然后用qPCR检测胶原基因,结果发现,相对于阴性对照,牙龈成纤维细胞中过表达FosL2,会导致胶原含量明显升高。
结论：
FosL2基因3'UTR区域,c.4396C>G碱基突变,破坏了miR-151a-3p对于该处靶位点的粘附,导致其负性调控作用失效,进而导致FOSL2蛋白表达升高,胶原含量也随之升高,最终导致病理性纤维化。
舌鳞状细胞癌(TSCC, tongue squamous cell carcinoma)是头颈部区域最常见的肿瘤之一；尽管治疗手段在不断发展,但是病人的五年存活率却并没有得到明显改善,这主要是由于肿瘤的转移所造成的——淋巴结转移的程度被用来作为预测预后的标志；因此,深入了解舌癌侵袭和迁移的分子机制就显得尤为重要。
ADAM10主要发挥蛋白水解各类跨膜蛋白胞外域的功能,底物众多——包括各类生长因子、粘附分子、细胞表面受体以及其他各类分子；正是由于其底物众多,底物所发挥的功能各异,因此ADAM10的异常表达所导致的附加影响也更加广泛。ADAM10不仅在胃癌、结肠癌、子宫癌等各类癌症中,被证实是高表达的,而且在口腔/舌鳞状细胞癌中也呈现出高表达。因此ADAM10不仅可以作为肿瘤的标志性分子之一,也可以作为抗肿瘤的靶标。而miRNA不仅在特定的癌症中,呈现出特定的表达谱,从而作为肿瘤标志物发挥作用,而且作为负性调节因子,还可以通过对于肿瘤的侵袭和迁移发挥抑制作用,使其成为癌症治疗的重要分子。
实验目标：
1.采用生物信息学的方法,对ADAM103'UTR可能受到哪些miRNA的调控进行预测；并选择其中8mer靶位点上的miRNA,作为研究对象；
2.对于预测结果进行实验确认,确定ADAM10的确是该miRNA直接调控的靶基因；
3.确定此miRNA是否可以抑制舌癌细胞系的侵袭、迁移和增殖；
4.根据上一步的实验结果,利用DAVID数据库来预测,其中可能是哪些靶基因在发挥作用；
5.进一步实验验证这些靶基因；
材料和方法：
1.生物信息学分析：采用Targetscan预测miRNA可能的靶基因；采用DAVID将miRNA的靶基因进行功能分类；
2.双荧光素酶检测：将所预测靶基因的3’UTR区域克隆到双荧光素酶载体,再根据所预测的靶位点,将种子序列所对应的靶位点碱基突变成和种子序列一样的碱基；将miRNA mimics和双荧光素酶质粒转染到舌鳞状细胞癌细胞系Tca8113,转染48h以后检测荧光素酶活性；
3. Western blotting:将miRNA及阴性对照转染到舌鳞状细胞癌细胞系CAL27,48h后检测所预测的靶基因的内源性蛋白是否有降低；
4. Transwell侵袭／迁移实验：将miRNA及阴性对照转染到CAL27以后,用Transwell实验检测CAL27细胞的侵袭和迁移能力是否减弱；
5.细胞增殖实验：同样是瞬时转染miRNA及阴性对照到CAL27以后,使用CCK-8来检测miRNA对于CAL27细胞的增殖能力是否有影响；
结果：
1.根据Targetscan对于ADAM103'UTR的预测结果：只有一个8mer靶位点,可能受到miR-140-5p的调控；
2.根据双荧光素酶结果：含有正常型ADAM103'UTR的荧光素酶活性受到miR-140-5p的显著抑制；而突变型ADAM103'UTR的荧光素酶活性则不受miR-140-5p的影响；
3.根据Western blot结果：内源性ADAM10蛋白表达受到miR-140-5p的显著抑制；
4.根据Transwell侵袭和迁移实验结果：miR-140-5p可以抑制CAL27细胞的侵袭和迁移能力,并且对于迁移能力抑制效果明显；
5.根据细胞增殖实验结果：miR-140-5p并不影响CAL27细胞的增殖能力；
6.根据DAVID对于miR-140-5p所有可能靶基因的功能分类结果：LAMC1、 HDAC7、PAX6、IGF1R、PSEN1和ERBB4被归类为与细胞迁移相关,因此被挑选出来作进一步分析；
7.根据Western blot结果：内源性LAMC1、HDAC7和PAX6蛋白表达受到miR-140-5p的显著抑制,而IGF1R和PSEN1则不受影响；令人惊奇的是,被Targetscan预测为miR-140-5p靶基因的ERBB4,其内源性蛋白表达受到miR-140-5p的正性调控；
8.根据双荧光素酶检测结果：含有正常型LAMC1、HDAC7、PAX6和ERBB4-3'VTR的荧光素酶活性受到miR-140-5p的显著抑制；而突变型LAMC1、HDAC7、PAX6和ERBB4-3'UTR的荧光素酶活性则不受miR-140-5p的影响；结论：
1. miR-140-5p可以抑制TSCC细胞的侵袭和迁移能力,但并不影响其增殖能力：
2. ADAM10、LAMC1、HDAC7、PAX6是miR-140-5p直接作用的靶基因；
3. IGF1R和PSEN1在TSCC细胞中,并没有受到miR-140-5p的直接调控影响；
4.即使ERBB4的3’UTR受到miR-140-5p的直接粘附,但是最终蛋白表达量却是受到miR-140-5p的正性调控；
5.被抑制的ADAM10增强miR-140-5p对于其他靶基因(PAX6、ERBB4)的调控作用；
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;
本课题组持续收集非综合征型遗传性牙龈纤维瘤家系及散发病例,并已成功对其中3个家系锁定其致病区域：其中GINGF3位点即为本课题组叶晓茜老师根据云南家系进行连锁分析所定位的致病区域。
实验目标：
对一个非综合征型遗传性牙龈纤维瘤大型家系——云南家系,进行突变筛查,并研究其导致病理性纤维化的致病机制；
材料和方法：
1.PCR：采用常规PCR测序的方法,筛查云南家系致病区域内(GINGF3)的基因外显子区域；
2.共分离及正常人对照验证：根据发现的突变,在家系内所有病人及所有正常人中,确定突变是否与遗传性牙龈纤维瘤表型共分离；然后在1300例正常人DNA对照中,确认突变是否会在正常对照中发现,排除其是SNP的可能性；
3.根据突变位置,进一步进行功能研究：是否改变亚细胞定位,是否影响miRNA的调控,等等等等。
结果：
1.突变筛查结果：在云南家系FOSL2基因的3'UTR区域,发现c.4396C>G碱基改变;
2.共分离及对照验证：在所有采集到血液并成功提取DNA的云南家系成员中,c.4396C>G碱基改变在所有病人中都有发现,在所有正常人中都没有发现；在1300例对照正常人中也没有发现；
3.突变是否影响FOSL2蛋白的细胞定位：免疫荧光结果表明,包含有正常型FOSL23'UTR的质粒所表达的蛋白,定位于细胞核,包含有突变型FOSL23'UTR的质粒所表达的蛋白,同样定位于细胞核；
4.突变是否影响miRNA的调控：根据niRanda软件预测结果,c.4396C>G突变位点正好处于miR-151a-3p的靶位点中；双荧光素酶检测以及western blot结果,进一步证实了该预测结果。
FOSL2与纤维化的关系：通过转染Fosl2过表达慢病毒到原代牙龈成纤维细胞,然后用qPCR检测胶原基因,结果发现,相对于阴性对照,牙龈成纤维细胞中过表达FosL2,会导致胶原含量明显升高。
结论：
FosL2基因3'UTR区域,c.4396C>G碱基突变,破坏了miR-151a-3p对于该处靶位点的粘附,导致其负性调控作用失效,进而导致FOSL2蛋白表达升高,胶原含量也随之升高,最终导致病理性纤维化。
舌鳞状细胞癌(TSCC, tongue squamous cell carcinoma)是头颈部区域最常见的肿瘤之一；尽管治疗手段在不断发展,但是病人的五年存活率却并没有得到明显改善,这主要是由于肿瘤的转移所造成的——淋巴结转移的程度被用来作为预测预后的标志；因此,深入了解舌癌侵袭和迁移的分子机制就显得尤为重要。
ADAM10主要发挥蛋白水解各类跨膜蛋白胞外域的功能,底物众多——包括各类生长因子、粘附分子、细胞表面受体以及其他各类分子；正是由于其底物众多,底物所发挥的功能各异,因此ADAM10的异常表达所导致的附加影响也更加广泛。ADAM10不仅在胃癌、结肠癌、子宫癌等各类癌症中,被证实是高表达的,而且在口腔/舌鳞状细胞癌中也呈现出高表达。因此ADAM10不仅可以作为肿瘤的标志性分子之一,也可以作为抗肿瘤的靶标。而miRNA不仅在特定的癌症中,呈现出特定的表达谱,从而作为肿瘤标志物发挥作用,而且作为负性调节因子,还可以通过对于肿瘤的侵袭和迁移发挥抑制作用,使其成为癌症治疗的重要分子。
实验目标：
1.采用生物信息学的方法,对ADAM103'UTR可能受到哪些miRNA的调控进行预测；并选择其中8mer靶位点上的miRNA,作为研究对象；
2.对于预测结果进行实验确认,确定ADAM10的确是该miRNA直接调控的靶基因；
3.确定此miRNA是否可以抑制舌癌细胞系的侵袭、迁移和增殖；
4.根据上一步的实验结果,利用DAVID数据库来预测,其中可能是哪些靶基因在发挥作用；
5.进一步实验验证这些靶基因；
材料和方法：
1.生物信息学分析：采用Targetscan预测miRNA可能的靶基因；采用DAVID将miRNA的靶基因进行功能分类；
2.双荧光素酶检测：将所预测靶基因的3’UTR区域克隆到双荧光素酶载体,再根据所预测的靶位点,将种子序列所对应的靶位点碱基突变成和种子序列一样的碱基；将miRNA mimics和双荧光素酶质粒转染到舌鳞状细胞癌细胞系Tca8113,转染48h以后检测荧光素酶活性；
3. Western blotting:将miRNA及阴性对照转染到舌鳞状细胞癌细胞系CAL27,48h后检测所预测的靶基因的内源性蛋白是否有降低；
4. Transwell侵袭／迁移实验：将miRNA及阴性对照转染到CAL27以后,用Transwell实验检测CAL27细胞的侵袭和迁移能力是否减弱；
5.细胞增殖实验：同样是瞬时转染miRNA及阴性对照到CAL27以后,使用CCK-8来检测miRNA对于CAL27细胞的增殖能力是否有影响；
结果：
1.根据Targetscan对于ADAM103'UTR的预测结果：只有一个8mer靶位点,可能受到miR-140-5p的调控；
2.根据双荧光素酶结果：含有正常型ADAM103'UTR的荧光素酶活性受到miR-140-5p的显著抑制；而突变型ADAM103'UTR的荧光素酶活性则不受miR-140-5p的影响；
3.根据Western blot结果：内源性ADAM10蛋白表达受到miR-140-5p的显著抑制；
4.根据Transwell侵袭和迁移实验结果：miR-140-5p可以抑制CAL27细胞的侵袭和迁移能力,并且对于迁移能力抑制效果明显；
5.根据细胞增殖实验结果：miR-140-5p并不影响CAL27细胞的增殖能力；
6.根据DAVID对于miR-140-5p所有可能靶基因的功能分类结果：LAMC1、 HDAC7、PAX6、IGF1R、PSEN1和ERBB4被归类为与细胞迁移相关,因此被挑选出来作进一步分析；
7.根据Western blot结果：内源性LAMC1、HDAC7和PAX6蛋白表达受到miR-140-5p的显著抑制,而IGF1R和PSEN1则不受影响；令人惊奇的是,被Targetscan预测为miR-140-5p靶基因的ERBB4,其内源性蛋白表达受到miR-140-5p的正性调控；
8.根据双荧光素酶检测结果：含有正常型LAMC1、HDAC7、PAX6和ERBB4-3'VTR的荧光素酶活性受到miR-140-5p的显著抑制；而突变型LAMC1、HDAC7、PAX6和ERBB4-3'UTR的荧光素酶活性则不受miR-140-5p的影响；结论：
1. miR-140-5p可以抑制TSCC细胞的侵袭和迁移能力,但并不影响其增殖能力：
2. ADAM10、LAMC1、HDAC7、PAX6是miR-140-5p直接作用的靶基因；
3. IGF1R和PSEN1在TSCC细胞中,并没有受到miR-140-5p的直接调控影响；
4.即使ERBB4的3’UTR受到miR-140-5p的直接粘附,但是最终蛋白表达量却是受到miR-140-5p的正性调控；
5.被抑制的ADAM10增强miR-140-5p对于其他靶基因(PAX6、ERBB4)的调控作用；
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;
引文
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