本文選題：遺傳性牙齦纖維瘤 + FOSL2��； 參考：《武漢大學(xué)》2014年博士論文

【摘要】：遺傳性牙齦纖維瘤是一種以牙齦組織彌漫性、漸進(jìn)性纖維增生為特征的遺傳疾��；可累及全口、亦可只病發(fā)于局部；可以以綜合征的形式出現(xiàn),亦可以非綜合征型出現(xiàn)；可以以常染色體顯性方式遺傳或者常染色體隱形方式遺傳,亦可呈現(xiàn)為散發(fā)病例而無遺傳史；本文主要研究的是非綜合征型遺傳性牙齦纖維瘤,其遺傳方式以常染色體顯性遺傳為主,并呈現(xiàn)出高度的遺傳異質(zhì)性：目前根據(jù)所報(bào)道的不多的幾個(gè)非綜合征型遺傳性牙齦纖維瘤家系,所定位的致病區(qū)域就有4個(gè)：GINGF1-GINGF4:而唯一一個(gè)致病機(jī)制得到完整闡述的,也僅僅是GINGF1區(qū)域內(nèi)的致病基因SOS1第21個(gè)外顯子所發(fā)生的插入突變：c.3248-3249insC。 本課題組持續(xù)收集非綜合征型遺傳性牙齦纖維瘤家系及散發(fā)病例,并已成功對(duì)其中3個(gè)家系鎖定其致病區(qū)域：其中GINGF3位點(diǎn)即為本課題組葉曉茜老師根據(jù)云南家系進(jìn)行連鎖分析所定位的致病區(qū)域。 實(shí)驗(yàn)?zāi)繕?biāo)： 對(duì)一個(gè)非綜合征型遺傳性牙齦纖維瘤大型家系——云南家系,進(jìn)行突變篩查,并研究其導(dǎo)致病理性纖維化的致病機(jī)制； 材料和方法： 1.PCR：采用常規(guī)PCR測(cè)序的方法,篩查云南家系致病區(qū)域內(nèi)(GINGF3)的基因外顯子區(qū)域； 2.共分離及正常人對(duì)照驗(yàn)證：根據(jù)發(fā)現(xiàn)的突變,在家系內(nèi)所有病人及所有正常人中,確定突變是否與遺傳性牙齦纖維瘤表型共分離；然后在1300例正常人DNA對(duì)照中,確認(rèn)突變是否會(huì)在正常對(duì)照中發(fā)現(xiàn),排除其是SNP的可能性； 3.根據(jù)突變位置,進(jìn)一步進(jìn)行功能研究：是否改變亞細(xì)胞定位,是否影響miRNA的調(diào)控,等等等等。 結(jié)果： 1.突變篩查結(jié)果：在云南家系FOSL2基因的3'UTR區(qū)域,發(fā)現(xiàn)c.4396CG堿基改變; 2.共分離及對(duì)照驗(yàn)證：在所有采集到血液并成功提取DNA的云南家系成員中,c.4396CG堿基改變?cè)谒胁∪酥卸加邪l(fā)現(xiàn),在所有正常人中都沒有發(fā)現(xiàn)；在1300例對(duì)照正常人中也沒有發(fā)現(xiàn)； 3.突變是否影響FOSL2蛋白的細(xì)胞定位：免疫熒光結(jié)果表明,包含有正常型FOSL23'UTR的質(zhì)粒所表達(dá)的蛋白,定位于細(xì)胞核,包含有突變型FOSL23'UTR的質(zhì)粒所表達(dá)的蛋白,同樣定位于細(xì)胞核； 4.突變是否影響miRNA的調(diào)控：根據(jù)niRanda軟件預(yù)測(cè)結(jié)果,c.4396CG突變位點(diǎn)正好處于miR-151a-3p的靶位點(diǎn)中；雙熒光素酶檢測(cè)以及western blot結(jié)果,進(jìn)一步證實(shí)了該預(yù)測(cè)結(jié)果。 FOSL2與纖維化的關(guān)系：通過轉(zhuǎn)染Fosl2過表達(dá)慢病毒到原代牙齦成纖維細(xì)胞,然后用qPCR檢測(cè)膠原基因,結(jié)果發(fā)現(xiàn),相對(duì)于陰性對(duì)照,牙齦成纖維細(xì)胞中過表達(dá)FosL2,會(huì)導(dǎo)致膠原含量明顯升高。 結(jié)論： FosL2基因3'UTR區(qū)域,c.4396CG堿基突變,破壞了miR-151a-3p對(duì)于該處靶位點(diǎn)的粘附,導(dǎo)致其負(fù)性調(diào)控作用失效,進(jìn)而導(dǎo)致FOSL2蛋白表達(dá)升高,膠原含量也隨之升高,最終導(dǎo)致病理性纖維化。 舌鱗狀細(xì)胞癌(TSCC, tongue squamous cell carcinoma)是頭頸部區(qū)域最常見的腫瘤之一；盡管治療手段在不斷發(fā)展,但是病人的五年存活率卻并沒有得到明顯改善,這主要是由于腫瘤的轉(zhuǎn)移所造成的——淋巴結(jié)轉(zhuǎn)移的程度被用來作為預(yù)測(cè)預(yù)后的標(biāo)志；因此,深入了解舌癌侵襲和遷移的分子機(jī)制就顯得尤為重要。 ADAM10主要發(fā)揮蛋白水解各類跨膜蛋白胞外域的功能,底物眾多——包括各類生長(zhǎng)因子、粘附分子、細(xì)胞表面受體以及其他各類分子；正是由于其底物眾多,底物所發(fā)揮的功能各異,因此ADAM10的異常表達(dá)所導(dǎo)致的附加影響也更加廣泛。ADAM10不僅在胃癌、結(jié)腸癌、子宮癌等各類癌癥中,被證實(shí)是高表達(dá)的,而且在口腔/舌鱗狀細(xì)胞癌中也呈現(xiàn)出高表達(dá)。因此ADAM10不僅可以作為腫瘤的標(biāo)志性分子之一,也可以作為抗腫瘤的靶標(biāo)。而miRNA不僅在特定的癌癥中,呈現(xiàn)出特定的表達(dá)譜,從而作為腫瘤標(biāo)志物發(fā)揮作用,而且作為負(fù)性調(diào)節(jié)因子,還可以通過對(duì)于腫瘤的侵襲和遷移發(fā)揮抑制作用,使其成為癌癥治療的重要分子。 實(shí)驗(yàn)?zāi)繕?biāo)： 1.采用生物信息學(xué)的方法,對(duì)ADAM103'UTR可能受到哪些miRNA的調(diào)控進(jìn)行預(yù)測(cè)；并選擇其中8mer靶位點(diǎn)上的miRNA,作為研究對(duì)象； 2.對(duì)于預(yù)測(cè)結(jié)果進(jìn)行實(shí)驗(yàn)確認(rèn),確定ADAM10的確是該miRNA直接調(diào)控的靶基因； 3.確定此miRNA是否可以抑制舌癌細(xì)胞系的侵襲、遷移和增殖； 4.根據(jù)上一步的實(shí)驗(yàn)結(jié)果,利用DAVID數(shù)據(jù)庫(kù)來預(yù)測(cè),其中可能是哪些靶基因在發(fā)揮作用； 5.進(jìn)一步實(shí)驗(yàn)驗(yàn)證這些靶基因； 材料和方法： 1.生物信息學(xué)分析：采用Targetscan預(yù)測(cè)miRNA可能的靶基因；采用DAVID將miRNA的靶基因進(jìn)行功能分類； 2.雙熒光素酶檢測(cè)：將所預(yù)測(cè)靶基因的3’UTR區(qū)域克隆到雙熒光素酶載體,再根據(jù)所預(yù)測(cè)的靶位點(diǎn),將種子序列所對(duì)應(yīng)的靶位點(diǎn)堿基突變成和種子序列一樣的堿基；將miRNA mimics和雙熒光素酶質(zhì)粒轉(zhuǎn)染到舌鱗狀細(xì)胞癌細(xì)胞系Tca8113,轉(zhuǎn)染48h以后檢測(cè)熒光素酶活性； 3. Western blotting:將miRNA及陰性對(duì)照轉(zhuǎn)染到舌鱗狀細(xì)胞癌細(xì)胞系CAL27,48h后檢測(cè)所預(yù)測(cè)的靶基因的內(nèi)源性蛋白是否有降低； 4. Transwell侵襲／遷移實(shí)驗(yàn)：將miRNA及陰性對(duì)照轉(zhuǎn)染到CAL27以后,用Transwell實(shí)驗(yàn)檢測(cè)CAL27細(xì)胞的侵襲和遷移能力是否減弱； 5.細(xì)胞增殖實(shí)驗(yàn)：同樣是瞬時(shí)轉(zhuǎn)染miRNA及陰性對(duì)照到CAL27以后,使用CCK-8來檢測(cè)miRNA對(duì)于CAL27細(xì)胞的增殖能力是否有影響； 結(jié)果： 1.根據(jù)Targetscan對(duì)于ADAM103'UTR的預(yù)測(cè)結(jié)果：只有一個(gè)8mer靶位點(diǎn),可能受到miR-140-5p的調(diào)控； 2.根據(jù)雙熒光素酶結(jié)果：含有正常型ADAM103'UTR的熒光素酶活性受到miR-140-5p的顯著抑制；而突變型ADAM103'UTR的熒光素酶活性則不受miR-140-5p的影響； 3.根據(jù)Western blot結(jié)果：內(nèi)源性ADAM10蛋白表達(dá)受到miR-140-5p的顯著抑制； 4.根據(jù)Transwell侵襲和遷移實(shí)驗(yàn)結(jié)果：miR-140-5p可以抑制CAL27細(xì)胞的侵襲和遷移能力,并且對(duì)于遷移能力抑制效果明顯； 5.根據(jù)細(xì)胞增殖實(shí)驗(yàn)結(jié)果：miR-140-5p并不影響CAL27細(xì)胞的增殖能力； 6.根據(jù)DAVID對(duì)于miR-140-5p所有可能靶基因的功能分類結(jié)果：LAMC1、 HDAC7、PAX6、IGF1R、PSEN1和ERBB4被歸類為與細(xì)胞遷移相關(guān),因此被挑選出來作進(jìn)一步分析； 7.根據(jù)Western blot結(jié)果：內(nèi)源性LAMC1、HDAC7和PAX6蛋白表達(dá)受到miR-140-5p的顯著抑制,而IGF1R和PSEN1則不受影響；令人驚奇的是,被Targetscan預(yù)測(cè)為miR-140-5p靶基因的ERBB4,其內(nèi)源性蛋白表達(dá)受到miR-140-5p的正性調(diào)控； 8.根據(jù)雙熒光素酶檢測(cè)結(jié)果：含有正常型LAMC1、HDAC7、PAX6和ERBB4-3'VTR的熒光素酶活性受到miR-140-5p的顯著抑制；而突變型LAMC1、HDAC7、PAX6和ERBB4-3'UTR的熒光素酶活性則不受miR-140-5p的影響；結(jié)論： 1. miR-140-5p可以抑制TSCC細(xì)胞的侵襲和遷移能力,但并不影響其增殖能力： 2. ADAM10、LAMC1、HDAC7、PAX6是miR-140-5p直接作用的靶基因； 3. IGF1R和PSEN1在TSCC細(xì)胞中,并沒有受到miR-140-5p的直接調(diào)控影響； 4.即使ERBB4的3’UTR受到miR-140-5p的直接粘附,但是最終蛋白表達(dá)量卻是受到miR-140-5p的正性調(diào)控； 5.被抑制的ADAM10增強(qiáng)miR-140-5p對(duì)于其他靶基因(PAX6、ERBB4)的調(diào)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 ) ;

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.8

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