遗传性牙龈纤维瘤致病机理研究及miR-140-5p抑癌机制探讨

发布时间：2018-05-16 18:37

本文选题：遗传性牙龈纤维瘤 + FOSL2　；参考：《武汉大学》2014年博士论文

【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.4396CG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.4396CG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.4396CG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.4396CG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；
[Abstract]:hereditary gingival fibromatosis is a genetic disease characterized by diffuse and progressive fibrous hyperplasia of gingival tissue ;
can be used for the whole mouth , but also can only be transmitted to the local area ;
may occur in the form of a syndrome or may occur in a non - syndrome type ;
can be inherited in a autosomal dominant manner or a autosomal recessive manner , or may be presented as sporadic cases without a genetic history ;
In this paper , we mainly study the non - syndrome type hereditary gingival fibroids , which are inherited mainly by autosomal dominant inheritance and show a high degree of genetic heterogeneity : the only one pathogenic mechanism is GINGF1 - GINGF4 , but only one of the pathogenic genes located in GINGF1 region has been fully explained , and is only the insertion mutation which occurs in the 21 exons of the pathogenic gene SOS1 in the GINGF1 region : c . 3248 - 3249insC .

The study group continued to collect non - syngenetic gingival fibromatosis families and sporadic cases , and has succeeded in locking three of them to their pathogenic regions : the GINGF3 locus is the pathogenic region located by Ye Xiaoxi of the study group according to the linkage analysis of Yunnan ' s family .

Experimental Objective :

To investigate the pathogenesis of pathological fibrosis by mutation screening for a large family of non - syngenetic gingival fibroids .

Materials and methods :

1 . PCR : The gene exon region of GINGF3 was screened by PCR sequencing .

2 . Control and verification of co - separation and normal controls : according to the detected mutation , it was determined whether the mutation was associated with the phenotype of hereditary gingival fibromatosis in all patients and all normal persons at home .
Then , in the control of 1300 normal controls , it was confirmed whether the mutation would be found in the normal control and the possibility of SNP was excluded .

3 . According to the position of mutation , further functional research is carried out : whether the sub - cell positioning is changed or not , whether the regulation of miRNA is affected , or the like .

Results :

1 . Results of mutation screening : c . 4396CG base change was found in the 3 ' untranslated region of the FOSL2 gene in Yunnan .

2 . Total separation and control verification : c . 4396CG base change was found in all Yunnan family members who collected blood and successfully extracted DNA , and found no found in all normal controls ;
None of the 1300 controls were found in normal controls .

3 . Whether the mutation affects the cell localization of FOSL2 protein : the immunofluorescence results show that the protein expressed by the plasmid containing the normal type of FOSL23 ' is located in the nucleus , and the protein expressed by the plasmid containing the mutant FOSL23 ' is also located in the nucleus ;

4 . Whether the mutation affects miRNA regulation : according to niRanda software prediction result , the c . 4396CG mutation site is in the target site of miR - 151a - 3P ;
The prediction results were further confirmed by the double luciferase assay and western blot results .

The relationship between FOSL2 and fibrosis : By transfection of Fosl2 overexpression lentivirus to primary gingival fibroblasts , and then using qPCR to detect the collagen gene , it was found that the overexpression of Bcl L2 in the gingival fibroblasts with respect to the negative control would result in a significant increase in collagen content .

Conclusion :

The mutation of the 3 ' untranslated region , the c . 4396CG base mutation of the gene was destroyed , which destroyed the adhesion of the miR - 151a - 3P to the target site of the site , which resulted in the failure of negative regulatory action , which led to an increase in the expression of the FOSL2 protein and an increase in the collagen content , which eventually led to pathological fibrosis .

Tongue squamous cell carcinoma ( SCC ) is one of the most common tumors in the head and neck region .
Despite continuous development , the patient ' s five - year survival rate has not been significantly improved , mainly due to the extent of lymph node metastasis caused by metastasis of the tumor to be used as a marker for predicting prognosis ;
Therefore , it is especially important to understand the molecular mechanism of invasion and migration of tongue cancer .

The protein hydrolyzes the functions of various transmembrane protein extracellular domains , including various kinds of growth factors , adhesion molecules , cell surface receptors and other molecules .
Because of the many substrates , the functions of the substrate are different , and therefore , the additional influence caused by abnormal expression is more extensive . Therefore , it can be used not only in cancers such as gastric cancer , colon cancer , uterine cancer and the like , but also high expression in oral / tongue squamous cell carcinoma .

Experimental Objective :

1 . The method of bioinformatics is used to predict which miRNA ' s regulation can be predicted .
and selecting miRNA in the 8mer target site as the research object ;

2 . Make an experiment on the prediction results to determine that the target gene which is directly regulated by the miRNA is determined .

3 . determining whether the miRNA can inhibit invasion , migration and proliferation of the tongue cancer cell line ;

4 . Based on the results of the previous step , the DAVID database is used to predict which target genes are functioning ;

5 . Further experiments verify these target genes ;

Materials and methods :

1 . Bioinformatic analysis : Using Targetscan to predict the possible target gene of miRNA ;
carrying out functional classification on target genes of miRNA by adopting DAVID ;

2 . detecting the double - luciferase : cloning the 3 ' - region region of the predicted target gene to the dual - luciferase vector , and then mutation the target site corresponding to the seed sequence into the same base as the seed sequence according to the predicted target site ;
The expression of luciferase activity was detected after 48h after transfection of miRNA and dual luciferase into the tongue squamous cell carcinoma cell line Tca8113 .

3 . Western blotting : After transfection of miRNA and negative control into the tongue squamous cell carcinoma cell line CAL27 , the endogenous protein of the predicted target gene was detected after 48h .

4 . Transwell invasion / migration experiment : After transfection of miRNA and negative control to CAL27 , the invasion and migration ability of CAL27 cells were detected by Transwell experiment .

5 . Cell proliferation assay : After transient transfection of miRNA and negative control to CAL27 , CCK - 8 was used to detect whether miRNA had an influence on the proliferation ability of CAL27 cells ;

Results :

1 . According to the prediction result of the Targetscan for the ADU10gene , there is only one 8mer target site , which may be regulated by miR - 140 - 5p ;

2 . According to the results of the dual luciferase : luciferase activity containing the normal type of an expression of a 103 ' utu was significantly inhibited by miR - 140 - 5p ;
However , the luciferase activity of the mutant ' s 103 ' utu is not influenced by miR - 140 - 5p ;

3 . According to the results of Western blot , the expression of the endogenous protein 10 protein was significantly inhibited by miR - 140 - 5p .

4 . According to the results of invasion and migration of Transwell : miR - 140 - 5p can inhibit the invasion and migration ability of CAL27 cells , and has obvious inhibitory effect on migration ability ;

5 . According to the experimental results of cell proliferation , miR - 140 - 5p does not affect the proliferation ability of CAL27 cells ;

6 . According to the functional classification results of all possible target genes of miR - 140 - 5p according to DAVID : LAMC1 , HDAC7 , PAX6 , IGF1R , PSEN1 and ERBB4 are classified as related to cell migration and are therefore selected for further analysis ;

7 . According to Western blot , the expression of endogenous LAMC1 , HDAC7 and PAX6 was significantly inhibited by miR - 140 - 5p , while IGF1R and PSEN1 were unaffected ;
Surprisingly , the Targetscan is predicted to be the ERBB4 of the miR - 140 - 5p target gene whose endogenous protein expression is regulated by the positive control of miR - 140 - 5p ;

8 . According to the results of the double luciferase assay : luciferase activity containing normal LAMC1 , HDAC7 , PAX6 and ERBB4 - 3 ' VTR was significantly inhibited by miR - 140 - 5p ;
while the luciferase activity of mutant LAMC1 , HDAC7 , PAX6 and ERBB4 - 3 & # x2032 ; is not influenced by miR - 140 - 5p ;
Conclusion :

1 . miR - 140 - 5p can inhibit the invasion and migration ability of TSCC cells , but does not influence its proliferation ability :

2 . A target gene for the direct action of miR - 140 - 5p , which is a direct action of miR - 140 - 5p ;

3 . IGF1R and PSEN1 were not directly regulated by miR - 140 - 5p in TSCC cells .

4 . The expression of the final protein is controlled by miR - 140 - 5p , even if the 3 ' UUIR4 of ERBB4 is directly adhered to miR - 140 - 5p .

5 . Inhibition of the regulatory action of miR - 140 - 5p on other target genes ( PAX6 , ERBB4 ) ;

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.8

【参考文献】