本文選題：膠質(zhì)瘤 + MicroRNA�。� 參考：《第四軍醫(yī)大學》2014年碩士論文

【摘要】：【背景】 膠質(zhì)瘤是最常見的原發(fā)性中樞神經(jīng)系統(tǒng)腫瘤。根據(jù)膠質(zhì)瘤組織病理學特點和臨床標準，世界衛(wèi)生組織（WHO）將其分為I-IV級。I級膠質(zhì)瘤在兒童多發(fā)，且一般認為I級膠質(zhì)瘤為良性腫瘤，可通過手術切除治愈，很少發(fā)生惡性進展；相比之下II、III級膠質(zhì)瘤具有一定侵襲性且惡性程度更高，預后較差；惡性程度最高且預后最差的是IV級膠質(zhì)母細胞瘤(Glioblastoma multiforme,GBM)，根據(jù)其是否由低級別膠質(zhì)瘤進展而來，可將其分為原發(fā)性膠質(zhì)母細胞瘤和繼發(fā)性膠質(zhì)母細胞瘤。 MicroRNAs(miRNA)為普遍存在于動植物體內(nèi)的內(nèi)源性小片段非編碼RNA分子，可通過與多個靶基因信使RNA(mRNA)結合從而調(diào)控基因的表達。miRNAs在進化中高度保守性，提示其可能在生命功能的諸多方面發(fā)揮重要作用。與腫瘤相關的miRNAs可根據(jù)其對腫瘤發(fā)生發(fā)展的調(diào)節(jié)作用，分為促癌miRNAs和抑癌miRNAs。目前的研究主要集中于挖掘與正常組織或細胞系相比，腫瘤中miRNAs的差異表達，進而研究其功能及尋找下游調(diào)控靶基因。已有大量研究表明miRNAs在包括膠質(zhì)瘤在內(nèi)的腫瘤中發(fā)生差異表達，這些miRNAs在腫瘤的發(fā)生和進展中發(fā)揮重要作用。 MiR-9在動植物中高度保守，且已被證實在多種腫瘤的發(fā)生與發(fā)展中發(fā)揮了重要作用。然而，其在膠質(zhì)瘤中的功能卻不明確。MiR-9在膠質(zhì)瘤中的表達有何特點？其異常表達對腫瘤的發(fā)生發(fā)展有何影響？MiR-9又是通過調(diào)節(jié)哪些重要靶基因來影響膠質(zhì)瘤的進展過程？在膠質(zhì)瘤中miR-9異常表達的機制是什么？上述問題的研究將有助于探索膠質(zhì)瘤的惡性行為機制，探究將miR-9作為治療靶點的可行性，為膠質(zhì)瘤的臨床治療提供了新策略。 【目的】 探究膠質(zhì)瘤臨床樣本及其膠質(zhì)瘤細胞系中miR-9的表達情況；研究miR-9在膠質(zhì)瘤惡性進展中的生物學作用；尋找及鑒定miR-9調(diào)節(jié)的功能相關靶基因；尋找調(diào)控miR-9的轉(zhuǎn)錄因子和表觀遺傳學調(diào)節(jié)因素，為膠質(zhì)瘤的預防、早期診斷以及治療靶點選擇提供新的理論依據(jù)。 【方法】 1.通過qRT-PCR檢測臨床組織樣本和膠質(zhì)瘤細胞系中miR-9的表達水平；2.體外A172細胞轉(zhuǎn)染miR-9mimic和U251細胞轉(zhuǎn)染miR-9inhibitor分別上調(diào)和下調(diào)miR-9的表達，通過MTT實驗、流式細胞術、劃痕實驗、Transwell實驗、血管生成實驗和粘附實驗分析miR-9對膠質(zhì)瘤細胞多種生物學行為的影響；3、運用生物信息學分析軟件預測miR-9潛在的功能靶基因，分子克隆技術將預測靶基因3’UTR結合區(qū)域連接至PGL3報告基因質(zhì)粒；4、利用熒光素酶報告基因?qū)嶒炶b定miR-9直接調(diào)控靶基因，并用qRT-PCR、Western blot和ELISA實驗在mRNA和蛋白水平上進行驗證；5、利用生物信息學網(wǎng)站預測對miR-9調(diào)節(jié)的轉(zhuǎn)錄因子及H3K27組蛋白甲基化，qRT-PCR檢測轉(zhuǎn)錄因子與H3K27組蛋白甲基化狀態(tài)；6.采用siRNA干擾技術和瞬時轉(zhuǎn)染過表達質(zhì)粒下調(diào)/上調(diào)轉(zhuǎn)錄因子和組蛋白去甲基化酶，分析miR-9的表達情況。 【結果】 1.qRT-PCR分析結果顯示，與正常腦組織相比，在膠質(zhì)瘤組織樣本中miR-9的表達水平增高；與正常膠質(zhì)細胞HEB相比，miR-9在膠質(zhì)瘤細胞系中均高表達，其中在膠質(zhì)瘤細胞A172中相對表達較低，在U251細胞系中表達較高；與正常腸上皮細胞相比，miR-9在結直腸癌中表達較高；而與正常腎上皮細胞相比，在腎癌中miR-9呈現(xiàn)低表達，表明miR-9的表達具有組織特異性；2.A172細胞轉(zhuǎn)染mimic后可有效上調(diào)miR-9的表達水平，MTT結果顯示miR-9促進膠質(zhì)瘤細胞增殖能力，細胞周期分析過表達miR-9組細胞進入S期細胞增多，G1期細胞數(shù)減少；上調(diào)miR-9表達后，A172膠質(zhì)瘤細胞遷移和侵襲細胞數(shù)明顯增高，過表達miR-9組條件培養(yǎng)基培養(yǎng)內(nèi)皮細胞HUVEC，增強了HUVEC細胞體外成管能力；而同時對U251細胞系轉(zhuǎn)染inhibitor后，細胞增殖能力下降、細胞周期S期細胞數(shù)量顯著減少，G1期細胞增多，Transwell結果可見下調(diào)miR-9組穿過小室細胞數(shù)量明顯減少，條件培養(yǎng)液培養(yǎng)HUVEC細胞，，成管能力顯著減弱；3.外源轉(zhuǎn)染miR-9mimic可顯著提高HUVEC細胞miR-9的表達水平，Transwell結果顯示過表達miR-9組較多細胞穿至小室膜下，過表達miR-9HUVEC細胞成管數(shù)量相比NC組顯著增多；且miR-9組內(nèi)皮細胞粘附力增強；4.生物信息學預測提示，THBS2、COL18A1、PTCH1和PHD3可能為潛在miR-9的下游靶基因，從而影響膠質(zhì)瘤增殖能力、遷移和侵襲、血管生成等生物學行為；5.qRT-PCR、Western blot、ELISA和熒光素酶報告基因?qū)嶒炦M一步證實THBS2、COL18A1、PTCH1和PHD3確實為miR-9直接調(diào)控的下游靶基因；6、膠質(zhì)瘤細胞A172中過表達C-MYC后，qRT-PCR分析發(fā)現(xiàn)miR-9表達增高；在U251細胞中應用siRNA技術下調(diào)C-MYC后，miR-9表達下調(diào)，且均主要影響miR-9-2的表達；7、在膠質(zhì)瘤細胞A172中瞬時上調(diào)OCT4的表達，qRT-PCR結果顯示miR-9表達上調(diào)，而且siRNA干擾U251細胞中OCT4的表達，miR-9表達下調(diào)，但是主要調(diào)節(jié)miR-9-1/3的表達；8.在膠質(zhì)瘤細胞A172中過表達組蛋白去甲基化酶KDM6A/B，qRT-PCR結果顯示miR-9的表達增高，而在U251中干擾KDM6A/B表達則miR-9表達呈現(xiàn)降低，均主要調(diào)節(jié)miR-9-2的表達。 【結論】 1. MiR-9在膠質(zhì)瘤中高表達，且具有組織特異性；2. MiR-9促進膠質(zhì)瘤細胞增殖能力，促進細胞周期進入S期，增強了膠質(zhì)瘤細胞遷移和侵襲能力，促進膠質(zhì)瘤血管生成；3. MiR-9增強內(nèi)皮細胞遷移和侵襲能力，增強了血管生成能力和細胞粘附力；4.THBS2、COL18A1、PTCH1和PHD3為miR-9直接調(diào)控的下游靶基因；5. MiR-9的表達受轉(zhuǎn)錄因子C-MYC和OCT4調(diào)控；6.組蛋白H3K27的甲基化狀態(tài)參與對miR-9的表達調(diào)控。
[Abstract]:Background background

Gliomas are the most common primary central nervous system tumors . According to the pathological characteristics and clinical criteria of glioma , the World Health Organization ( WHO ) divides it into grade I - IV . Grade I glioma is multi - hair in children .
in contrast , grade II and grade III glioma have certain invasive and malignant degree and poor prognosis ;
The highest degree of malignancy and the worst prognosis is Glioblastoma multiforme , which can be divided into primary and secondary Glioblastoma , depending on whether it is progressing from low grade glioma .

MicroRNAs ( miRNA ) are non - coding RNA molecules of endogenous small fragments which are ubiquitous in animals and plants , and can regulate the expression of genes by combining RNA ( mRNA ) with multiple target genes .

MiR - 9 is highly conserved in animal and plant , and has been proved to play an important role in the genesis and development of various tumors . However , the function of MiR - 9 in glioma is not clear . What is the mechanism of abnormal expression of MiR - 9 in glioma ? What is the mechanism of abnormal expression of miR - 9 in glioma ? The research of the above - mentioned problems will help to explore the malignant behavior mechanism of glioma , and explore the feasibility of using miR - 9 as therapeutic target , and provide a new strategy for clinical treatment of glioma .

Purpose of the project

To investigate the expression of miR - 9 in glioma clinical samples and their glioma cell lines ;
To study the biological effects of miR - 9 in malignant progression of glioma ;
finding and identifying the function - related target gene regulated by miR - 9 ;
To find the transcriptional factor and epigenetics regulation factor of miR - 9 , provide a new theoretical basis for the prevention , early diagnosis and treatment target selection of glioma .

Methodology

1 . The expression level of miR - 9 in clinical tissue samples and glioma cell lines is detected by qRT - PCR ;
2 . The expression of miR - 9 was up - regulated and down - regulated by miR - 9 inhibitor transfected with miR - 9 inhibitor and U251 cells in vitro . The effects of miR - 9 on various biological behaviors of glioma cells were analyzed by MTT assay , flow cytometry , scratch test , Transwell experiment , angiogenesis experiment and adhesion experiment .
3 , using bioinformatics analysis software to predict the potential functional target gene of miR - 9 , and the molecular cloning technology is used for connecting the predicted target gene ' s 3 ' untranslated region to the PGL3 reporter plasmid ;
4 . Using luciferase reporter gene experiment , the target gene was identified directly by miR - 9 , and the mRNA and protein levels were verified by qRT - PCR , Western blot and ELISA .
5 , using bioinformatics website to predict the transcription factor of miR - 9 and histone methylation of H3K27 , and detecting the methylation state of transcription factor and H3K27 histone by qRT - PCR ;
6 . The expression of miR - 9 was analyzed by using siRNA interference technique and transiently transfected expression plasmid to downregulate / regulate transcription factor and histone demethylase .

The result is not valid .

Compared with normal intestinal epithelial cells , miR - 9 has high expression in colorectal cancer ;
Compared with normal glial cell HEB , miR - 9 is highly expressed in glioma cell line , wherein the relative expression of miR - 9 in glioma cell A172 is lower , and the expression of miR - 9 is higher in U251 cell line ;
1 . The results of qRT - PCR showed that miR - 9 expression was increased in glioma tissue samples compared with normal brain tissue ;
Compared with normal renal epithelial cells , miR - 9 exhibits low expression in renal cancer , suggesting that miR - 9 expression has tissue specificity ;
2 . After transfection with A172 cells , the expression level of miR - 9 can be increased effectively , and the MTT results show that miR - 9 promotes the proliferation of glioma cells , and the cell cycle analysis expresses that the miR - 9 group cells enter the S phase cell to increase , and the number of G1 phase cells is reduced ;
After up - regulation of miR - 9 expression , the number of migration and invasion cells of A172 glioma cells was obviously increased , and the cultured endothelial cells were cultured in the conditioned medium of miR - 9 group , thus enhancing the in vitro tube forming ability of HUVEC cells ;
At the same time , after transfection of the U251 cell line , the cell proliferation ability decreased , the number of cell cycle S phase cells decreased significantly , the number of G1 phase cells increased , Transwell results showed that the reduction of miR - 9 group through the small cell number was obviously reduced , the conditioned medium cultured HUVEC cells , and the ability of forming the tube was significantly reduced ;
3 . The expression level of miR - 9 in HUVEC was significantly increased by exogenous transfection of miR - 9 cells , and the results of Transwell showed that the expression of miR - 9 group was significantly increased compared with NC group compared with NC group .
and the adhesion of the endothelial cells of the miR - 9 group is enhanced ;
4 . Bioinformatic prediction suggests that THBS2 , COL18A1 , PTCH1 and PHD3 may be downstream target genes of potential miR - 9 , thus affecting the biological behavior of glioma proliferation , migration and invasion and angiogenesis ;
5 . qRT - PCR , Western blot , ELISA and luciferase reporter gene experiments further confirmed that THBS2 , COL18A1 , PTCH1 and PHD3 were indeed the downstream target genes which were directly regulated by miR - 9 ;
6 . After overexpression of C - MYC in glioma cells A172 , the expression of miR - 9 was increased by qRT - PCR analysis .
After downregulating the C - MYC by siRNA in U251 cells , the expression of miR - 9 was downregulated , and the expression of miR - 9 - 2 was mainly influenced ;
7 . The expression of OCT4 was transiently increased in glioma cells A172 , and the expression of miR - 9 was up - regulated by qRT - PCR , and the expression of OCT4 and miR - 9 expression in U251 cells were downregulated by siRNA , but the expression of miR - 9 - 1 / 3 was mainly regulated ;
8 . In the glioma cells A172 , the expression of miR - 9 was increased , and the expression of miR - 9 was decreased in U251 , and the expression of miR - 9 - 2 was mainly regulated by the expression of miR - 9 in U251 .

Conclusion

1.MiR - 9 is highly expressed in glioma , and has tissue specificity ;
2 . MiR - 9 promotes glioma cell proliferation ability , promotes cell cycle to enter S phase , enhances glioma cell migration and invasion ability , promotes glioma angiogenesis ;
3 . MiR - 9 enhanced endothelial cell migration and invasion ability , enhanced angiogenesis and cell adhesion ;
4 . THBS2 , COL18A1 , PTCH1 and PHD3 are downstream target genes which are directly regulated by miR - 9 ;
5 . Expression of MiR - 9 is regulated by transcription factor C - MYC and OCT4 ;
6 . The methylation status of histone H3K27 is involved in the expression regulation of miR - 9 .

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.41

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