本文選題：microRNA-195 + 膠質(zhì)瘤��； 參考：《山西醫(yī)科大學(xué)》2014年博士論文

【摘要】：惡性膠質(zhì)瘤是最常見(jiàn)的中樞神經(jīng)系統(tǒng)惡性腫瘤，具有復(fù)發(fā)率高、致死、致殘率高的特點(diǎn)，嚴(yán)重危害著人類的健康。盡管目前國(guó)內(nèi)外治療膠質(zhì)瘤采用手術(shù)后輔以放療、替莫唑胺化療相結(jié)合的綜合治療方案，高級(jí)別膠質(zhì)瘤尤其是膠質(zhì)母細(xì)胞瘤不僅仍然無(wú)法治愈，而且中位生存期沒(méi)有得到顯著延長(zhǎng)。這與膠質(zhì)瘤細(xì)胞增殖過(guò)度、凋亡抵抗、侵襲浸潤(rùn)以及對(duì)化療藥物產(chǎn)生耐受的生物學(xué)行為特點(diǎn)密切相關(guān)。因此，尋找能夠有效抑制膠質(zhì)瘤細(xì)胞惡性生物學(xué)行為的分子靶點(diǎn)，對(duì)于膠質(zhì)瘤的治療具有重要的科學(xué)意義和臨床應(yīng)用價(jià)值。 MicroRNA (miRNA)是一種長(zhǎng)約22個(gè)核苷酸的內(nèi)源性小分子非編碼RNA，主要通過(guò)與目的基因mRNA3’UTR區(qū)完全或者不完全結(jié)合，實(shí)現(xiàn)在轉(zhuǎn)錄后水平對(duì)基因表達(dá)的調(diào)控。miRNA的作用方式為“一對(duì)多”，與目標(biāo)基因組成調(diào)控網(wǎng)絡(luò)，參與胚胎發(fā)育，細(xì)胞增殖凋亡、新生血管生成等多種生物學(xué)過(guò)程。由于miRNAs的靶基因傾向富集于同一個(gè)或某幾個(gè)信號(hào)通路的特點(diǎn)，也使得它對(duì)細(xì)胞功能和表型的調(diào)節(jié)更為迅速和有力。miRNA表達(dá)還具有時(shí)序性、保守性和組織特異性，同時(shí)它能夠穩(wěn)定存在于各種體液甚至福爾馬林固定的石蠟組織標(biāo)本（FFPE）中，因此，它越來(lái)越成為腫瘤診斷、治療及預(yù)后相關(guān)的理想的分子標(biāo)記物。近期大量研究表明，特異性miRNA的表達(dá)異常與膠質(zhì)瘤細(xì)胞的無(wú)限增殖、凋亡受阻、侵襲遷移以及化療耐藥等惡性生物學(xué)行為調(diào)控密切相關(guān)。 MicroRNA-195（miR-195）是miR-15a/15b/16/195/497家族中的重要一員，基因定位于染色體17p13.1區(qū)域，距離抑癌基因P53僅650kb，該區(qū)域常發(fā)生雜合性缺失。研究顯示，miR-195在肝癌、肺癌、結(jié)腸癌、胃癌、乳腺癌、原發(fā)性腹膜癌、膀胱腫瘤等多種惡性腫瘤中廣泛下調(diào)，通過(guò)抑制腫瘤細(xì)胞的多種惡性生物學(xué)行為，發(fā)揮著抑癌基因的作用。但關(guān)于miR-195在膠質(zhì)瘤中的生物學(xué)作用及相關(guān)機(jī)制尚未明確。課題組根據(jù)miRNA靶基因數(shù)據(jù)庫(kù)miRDB，TargetScan和PicTar predictions預(yù)測(cè)和目前國(guó)內(nèi)外已確認(rèn)的miR-195作用靶點(diǎn)，推測(cè)miR-195在調(diào)控腫瘤細(xì)胞周期G1/S期轉(zhuǎn)換和DNA損傷應(yīng)答反應(yīng)中發(fā)揮著重要作用，進(jìn)而影響腫瘤細(xì)胞的惡性生物學(xué)行為。因此，本研究以miR-195為研究對(duì)象，分別檢測(cè)miR-195在膠質(zhì)瘤FFPE組織、膠質(zhì)瘤細(xì)胞株及膠質(zhì)瘤替莫唑胺耐藥株中的表達(dá)情況，然后通過(guò)改變miR-195的表達(dá)水平，觀察其對(duì)膠質(zhì)瘤細(xì)胞增殖凋亡、侵襲遷移、克隆形成、體內(nèi)成瘤以及耐藥等生物學(xué)行為的影響，進(jìn)而結(jié)合miRNA靶基因預(yù)測(cè)數(shù)據(jù)庫(kù)的結(jié)果，從分子水平研究miR-195影響膠質(zhì)瘤細(xì)胞惡性生物學(xué)行為可能的分子機(jī)制，這些研究將有助于深入理解miR-195的生物學(xué)功能和膠質(zhì)瘤細(xì)胞惡性生物學(xué)行為的調(diào)控機(jī)制，為提高膠質(zhì)瘤診療效果提供新的預(yù)測(cè)分子和干預(yù)靶點(diǎn)，具有重要的理論意義和應(yīng)用前景。 本課題將從三個(gè)方面進(jìn)行研究： 1. miR-195在人腦膠質(zhì)瘤FFPE組織及細(xì)胞系中的表達(dá)及意義： 目的：研究miR-195在人腦膠質(zhì)瘤福爾馬林固定石蠟包埋組織及細(xì)胞系中的表達(dá)情況，分析其與膠質(zhì)瘤臨床病理分級(jí)之間的關(guān)系，為進(jìn)一步探索miR-195在膠質(zhì)瘤體內(nèi)外中的生物學(xué)作用奠定基礎(chǔ)。 方法：采用實(shí)時(shí)熒光定量逆轉(zhuǎn)錄-聚合酶鏈?zhǔn)椒磻?yīng)（qRT-PCR）法檢測(cè)miR-195在人腦膠質(zhì)瘤細(xì)胞系SHG-44、U87、U251，人星形膠質(zhì)細(xì)胞系，28例不同病理級(jí)別人腦膠質(zhì)瘤福爾馬林固定石蠟包埋的組織及2例正常腦組織中的表達(dá)情況。 結(jié)果：（1）qRT-PCR法檢測(cè)結(jié)果顯示，與正常人星形膠質(zhì)細(xì)胞（HA）相比，三種細(xì)胞株SHG-44、U87、U251中miR-195含量均明顯下調(diào)，其中SHG-44下調(diào)程度最高，而U251下調(diào)程度最小，差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；28例膠質(zhì)瘤FFPE組織中miR-195含量均低于正常腦組織，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；（2）相關(guān)性分析：應(yīng)用LSD法對(duì)四個(gè)不同病理級(jí)別組進(jìn)行兩兩比較，結(jié)果顯示：Ⅰ級(jí)、Ⅱ級(jí)之間miR-195表達(dá)差別無(wú)統(tǒng)計(jì)學(xué)意義（P0.05），Ⅰ級(jí)、Ⅱ級(jí)分別和Ⅲ、Ⅳ級(jí)之間差異有統(tǒng)計(jì)學(xué)意義（P＜0.05），Ⅲ、Ⅳ級(jí)miR-195表達(dá)高于Ⅰ級(jí)、Ⅱ級(jí)。Spearman秩相關(guān)分析得出miR-195表達(dá)與膠質(zhì)瘤患者的臨床病理分級(jí)呈負(fù)相關(guān)（r=-0.798，P＜0.001）。 2. miR-195影響人腦膠質(zhì)瘤細(xì)胞增殖凋亡和侵襲遷移能力的體內(nèi)外研究： 目的：觀察上調(diào)miR-195表達(dá)水平對(duì)人腦膠質(zhì)瘤細(xì)胞增殖凋亡和侵襲遷移能力的影響。 方法：實(shí)驗(yàn)分為空白對(duì)照組（Blank）組，無(wú)義序列組（NC組）和miR-195過(guò)表達(dá)組（miR-195組）；首先利用陽(yáng)離子脂質(zhì)體LipofectamineTM2000將cy5標(biāo)記的miR-195的模擬物(mimics)、無(wú)義序列分別轉(zhuǎn)染入膠質(zhì)瘤細(xì)胞株U251和SHG-44細(xì)胞中，激光共聚焦觀察轉(zhuǎn)染情況，流式細(xì)胞儀檢測(cè)轉(zhuǎn)染效率并篩選最佳轉(zhuǎn)染濃度；實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(qRT-PCR)法測(cè)定miR-195的表達(dá)情況；CCK-8法和平板克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞增殖能力；AnnexinV-FITC/PI雙染法和Hoechst33342/PI雙染法檢測(cè)細(xì)胞凋亡變化；流式細(xì)胞儀檢測(cè)細(xì)胞周期；傷痕愈合實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力；Transwell小室檢測(cè)細(xì)胞侵襲能力；免疫細(xì)胞化學(xué)檢測(cè)Bcl-2的表達(dá)；最后我們進(jìn)一步評(píng)估了過(guò)表達(dá)miR-195對(duì)裸鼠皮下膠質(zhì)瘤模型生長(zhǎng)的影響。 結(jié)果:（1）miR-195mimics轉(zhuǎn)入到膠質(zhì)瘤細(xì)胞株U251和SHG-44后，在激光共聚焦下觀察到較強(qiáng)的紅色熒光，流式細(xì)胞儀篩選出miR-195mimics最佳轉(zhuǎn)染濃度為50nM，轉(zhuǎn)染效率達(dá)90%以上；（2）qRT-PCR結(jié)果顯示與Blank組相比，U251、SHG-44miR-195組膠質(zhì)瘤細(xì)胞內(nèi)miR-195含量分別上調(diào)9.98、65.97倍（P0.05）；（3）CCK-8法和平板克隆形成實(shí)驗(yàn)結(jié)果顯示，與NC組和Blank組相比，兩種細(xì)胞株過(guò)表達(dá)miR-195后，細(xì)胞增殖能力均明顯減弱，克隆形成能力降低（P0.05）；（4）流式細(xì)胞儀和Hoechst33342/PI雙染法檢測(cè)細(xì)胞凋亡，結(jié)果顯示與Blank組及NC組相比，miR-195組細(xì)胞凋亡和壞死比例明顯增高（P0.05）；（5）傷痕愈合實(shí)驗(yàn)結(jié)果顯示，在U251中，過(guò)表達(dá)miR-195可以抑制細(xì)胞遷移（P0.05），而在SHG-44中，miR-195組的細(xì)胞遷移率與其余兩組間差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）；（6）Transwell侵襲實(shí)驗(yàn)結(jié)果顯示，在U251中，過(guò)表達(dá)miR-195組可以減少穿過(guò)Matrigel膜的細(xì)胞數(shù)（P0.05），而在SHG-44中，miR-195組穿過(guò)Matrigel膜的細(xì)胞數(shù)多于其余兩組（P0.05）；(7)流式細(xì)胞儀檢測(cè)細(xì)胞周期結(jié)果顯示，與Blank組及NC組相比，miR-195組在G0/G1期細(xì)胞明顯增加，而S期細(xì)胞顯著減少（P0.05）；（8）利用microRNA靶點(diǎn)預(yù)測(cè)網(wǎng)站及相關(guān)生物學(xué)信息軟件，發(fā)現(xiàn)Bcl-2為miR-195的一個(gè)靶基因，免疫細(xì)胞化學(xué)結(jié)果顯示過(guò)表達(dá)miR-195可下調(diào)Bcl-2的表達(dá)；（9）利用裸鼠皮下膠質(zhì)瘤模型觀察miR-195對(duì)膠質(zhì)瘤形成的影響，結(jié)果顯示miR-195組裸鼠瘤結(jié)節(jié)出現(xiàn)較晚，生長(zhǎng)速度較慢，腫瘤剝離后重量和體積也明顯低于NC組和Blank組（P0.05）；（10）裸鼠腫瘤標(biāo)本免疫組化分析結(jié)果顯示，與NC組和Blank組相比，miR-195組Ki67表達(dá)下調(diào)（P0.05）。 3. miR-195在人腦膠質(zhì)瘤對(duì)替莫唑胺形成耐藥中的作用及機(jī)制研究： 目的：研究miR-195在人腦膠質(zhì)瘤對(duì)替莫唑胺形成耐藥中的作用及機(jī)制。 方法：使用TMZ濃度遞增的方法誘導(dǎo)構(gòu)建膠質(zhì)瘤耐藥株，qRT-PCR法明確膠質(zhì)瘤耐藥株中miR-195的表達(dá)變化情況，然后改變耐藥株中miR-195的表達(dá)水平，CCK-8檢測(cè)膠質(zhì)瘤細(xì)胞對(duì)TMZ耐藥性、增殖能力的改變情況；流式細(xì)胞儀檢測(cè)細(xì)胞凋亡和細(xì)胞周期變化；Western blot檢測(cè)MGMT、P21、Bcl-2及Cyclin D1的表達(dá)；進(jìn)一步構(gòu)建裸鼠皮下膠質(zhì)瘤模型，觀察腫瘤生長(zhǎng)情況以及對(duì)替莫唑胺的敏感性變化。 結(jié)果：（1）歷時(shí)6個(gè)月成功建立了對(duì)TMZ耐藥的U251、SHG44耐藥株，分別命名為U251R、SHG-44R；（2）qRT-PCR結(jié)果顯示，膠質(zhì)瘤TMZ耐藥株U251R、SHG-44R中miR-195含量分別下調(diào)至親本細(xì)胞的40%、48%，SHG-44R下調(diào)更為顯著（P0.05），因此選擇SHG-44行后續(xù)實(shí)驗(yàn)；（3）CCK-8法結(jié)果顯示上調(diào)miR-195增加SHG-44R對(duì)TMZ的敏感性，而敲低miR-195則一定程度上增加SHG44R對(duì)TMZ的耐藥性（P0.05）；（4）流式細(xì)胞儀檢測(cè)細(xì)胞凋亡結(jié)果顯示過(guò)表達(dá)miR-195可明顯增加膠質(zhì)瘤耐藥株的總凋亡率（P0.05），敲低miR-195可略降低總凋亡率，但與對(duì)照組相比，差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）；（5）細(xì)胞周期結(jié)果顯示上調(diào)SHG-44R細(xì)胞中的miR-195表達(dá)水平，膠質(zhì)瘤細(xì)胞生長(zhǎng)減慢，傳代時(shí)間延長(zhǎng)，G0/G1期細(xì)胞增加，S期細(xì)胞下降（P0.05），而敲低miR-195對(duì)膠質(zhì)瘤耐藥細(xì)胞株的細(xì)胞周期影響與對(duì)照組差異不明顯（P0.05）；（6）Western blot結(jié)果顯示過(guò)表達(dá)miR-195可以下調(diào)P21、Cyclin D1、Bcl-2的表達(dá)，而敲低miR-195后蛋白表達(dá)與之相反（P0.05），MGMT表達(dá)水平不受miR-195表達(dá)變化影響；（7）裸鼠皮下膠質(zhì)瘤模型和TUNEL染色結(jié)果顯示miR-195聯(lián)合TMZ組抑制膠質(zhì)瘤生長(zhǎng)及促進(jìn)耐藥的膠質(zhì)瘤細(xì)胞凋亡最為顯著（P0.05）。 結(jié)論:（1）miR-195在所有已檢測(cè)的膠質(zhì)瘤FFPE標(biāo)本及細(xì)胞系中均表達(dá)下調(diào)，并且表達(dá)程度與膠質(zhì)瘤病理分級(jí)呈負(fù)相關(guān)；提示我們可以將其作為膠質(zhì)瘤I-IV級(jí)新的有效的診斷標(biāo)記物，同時(shí)作為常規(guī)組織病理學(xué)的分級(jí)標(biāo)準(zhǔn)的補(bǔ)充；（2）過(guò)表達(dá)miR-195能夠減弱膠質(zhì)瘤細(xì)胞體內(nèi)外的增殖能力，誘導(dǎo)其G0/G1期阻滯，，下調(diào)Bcl-2表達(dá)，促進(jìn)膠質(zhì)瘤細(xì)胞凋亡和壞死；但是miR-195對(duì)膠質(zhì)瘤細(xì)胞侵襲遷移能力的影響與細(xì)胞類型有關(guān)，尚需進(jìn)一步研究；總體上講，miR-195能夠負(fù)性調(diào)控膠質(zhì)瘤細(xì)胞的惡性生物學(xué)行為；（3）miR-195在膠質(zhì)瘤耐藥株中低表達(dá)，上調(diào)miR-195含量，可以誘導(dǎo)耐藥細(xì)胞凋亡和增殖受限，下調(diào)P21、Cyclin D1、Bcl-2的表達(dá)，一定程度上恢復(fù)體內(nèi)外膠質(zhì)瘤細(xì)胞對(duì)替莫唑胺的化療敏感性，而這一作用與MGMT表達(dá)變化無(wú)關(guān)。綜上述，miR-195有望成為膠質(zhì)瘤基因治療新的靶點(diǎn)。
[Abstract]:Malignant glioma is the most common malignant tumor of the central nervous system, which has the characteristics of high recurrence rate, death and high disability rate, which seriously endangers human health. Although the treatment of glioma is combined with radiotherapy at home and abroad, combined with the combination of temozolomide chemotherapy, high grade glioma especially glioblastoma is used. Not only is it still incurable, but the median survival time has not been significantly prolonged, which is closely related to the excessive proliferation of glioma cells, resistance to apoptosis, invasion and infiltration, and the biological behavior of chemotherapeutic drugs. Therefore, the molecular target, which can effectively inhibit the malignant biological behavior of glioma cells, is found for glioma. The treatment has important scientific significance and clinical application value.
MicroRNA (miRNA) is a small endogenous small molecular non coding RNA with 22 nucleotides long, mainly through the complete or incomplete combination with the target gene mRNA3 'UTR region, realizing the "one to many" mode of action of.MiRNA at the post transcriptional level of gene expression, regulating the network with the target gene, participating in the embryonic development and increasing the cell growth. There are many biological processes, such as apoptotic, neovascularization, and other biological processes. Because the target gene of miRNAs tends to be enriched in the same or some signal pathways, it also makes its regulation of cell function and phenotype more rapid and powerful.MiRNA expression is sometimes ordered, conserved and group specific, and it can be stable in various kinds of bodies. The fluid and even the formalin fixed paraffin tissue specimens (FFPE), therefore, become more and more ideal molecular markers for the diagnosis, treatment and prognosis of the tumor. A large number of recent studies have shown that the abnormal expression of specific miRNA and the infinite proliferation of glioma cells, the inhibition of apoptosis, the invasion and migration, and the chemotherapeutic resistance of chemotherapeutic agents. It is closely related to regulation.
MicroRNA-195 (miR-195) is an important member of the miR-15a/15b/16/195/497 family. The gene is located in the chromosome 17p13.1 region and is only 650kb from the tumor suppressor gene P53. The region often lacks heterozygosity. The study shows that miR-195 is widely used in a variety of malignant tumors, such as liver cancer, lung cancer, colon cancer, gastric cancer, breast cancer, primary peritoneal carcinoma, bladder tumor and so on. Down regulation plays the role of tumor suppressor genes by inhibiting many malignant biological behaviors of tumor cells. But the biological role and mechanism of miR-195 in glioma have not been clear. The subject group is pretested by the miRNA target gene database miRDB, TargetScan and PicTar predictions and the existing miR-195 target target at home and abroad. It is speculated that miR-195 plays an important role in regulating the G1/S phase transition of tumor cell cycle and the response to DNA damage response, and then affects the malignant biological behavior of tumor cells. Therefore, this study uses miR-195 as the research object to detect the table of miR-195 in glioma FFPE tissue, glioma cell line and glioma temozolomide resistant strain, respectively. By changing the expression level of miR-195, the effects of miR-195 on the proliferation and apoptosis of glioma cells, invasion and migration, cloning, in vivo tumorigenesis, and drug resistance were observed, and then the miRNA target gene was used to predict the results of the database, and the possible effects of miR-195 on the malignant biological behavior of glioma cells were studied at the molecular level. Molecular mechanisms, these studies will help to understand the biological function of miR-195 and the regulatory mechanism of malignant biological behavior of glioma cells, and provide new predictor and target for improving the diagnosis and treatment of glioma. It has important theoretical significance and application prospects.
This topic will be studied from three aspects:
Expression and significance of 1. miR-195 in human glioma FFPE tissues and cell lines:
Objective: To study the expression of miR-195 in the paraffin embedded tissues and cell lines of human glioma, formalin, and to analyze the relationship with the clinicopathological classification of glioma, and to lay a foundation for the further exploration of the biological role of miR-195 in the internal and external glioma.
Methods: real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-195 in human glioma cell lines SHG-44, U87, U251, human astrocyte system, 28 cases of different pathological grades of human glioma, formalin, fixed paraffin embedded tissues and 2 normal brain tissues.
Results: (1) the results of qRT-PCR assay showed that compared with normal astrocytes (HA), the content of miR-195 in SHG-44, U87 and U251 of the three cell lines decreased obviously, and the downregulation of SHG-44 was the highest, but the decrease of U251 was the least, and the difference was statistically significant (P0.05). The miR-195 content of FFPE tissues in 28 cases of glioma was lower than that of the normal brain group. The difference was statistically significant (P0.05); (2) correlation analysis: LSD method was used to compare 22 different pathological grade groups. The results showed that there was no significant difference in miR-195 expression between grade I and class II (P0.05), grade I, grade II and grade III, IV (P < 0.05), and high expression of grade IV miR-195. At rank I and rank II.Spearman rank correlation analysis, the expression of miR-195 was negatively correlated with the clinicopathological grade of glioma patients (r=-0.798, P < 0.001).
2. the effect of miR-195 on proliferation, apoptosis and invasion and migration of human glioma cells in vivo and in vitro:
Objective: To investigate the effect of up regulation of miR-195 expression on proliferation, apoptosis, invasion and migration of human glioma cells.
Methods: the experiment was divided into the blank control group (Blank) group, the non sense sequence group (group NC) and the miR-195 overexpression group (group miR-195). First, the cationic liposome LipofectamineTM2000 was used to transfect the Cy5 labeled miR-195 analogue (mimics) and the nonsense sequence into the glioma cell line U251 and SHG-44 cells respectively. Laser confocal observation was used to observe the transfection situation. The transfection efficiency was detected by flow cytometry and the optimal transfection concentration was screened. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression of miR-195; CCK-8 and flat clones were used to detect cell proliferation; AnnexinV-FITC/PI double staining and Hoechst33342/PI double staining were used to detect cell apoptosis; flow cytometry was used. The cell cycle was detected, the scar healing test was used to detect the cell migration ability, the Transwell chamber was used to detect the cell invasiveness, and the expression of Bcl-2 was detected by immunocytochemistry. Finally, we further evaluated the effect of overexpression of miR-195 on the growth of the subcutaneous glioma model in nude mice.
Results: (1) after miR-195mimics was transferred into the glioma cell line U251 and SHG-44, the strong red fluorescence was observed under the laser confocal laser. The optimal transfection concentration of miR-195mimics was 50nM and the transfection efficiency was more than 90%. (2) qRT-PCR results showed that the miR-195 contained in U251 and SHG-44miR-195 group glioma cells compared with the Blank group. The amount of 9.98,65.97 times increased (P0.05); (3) the results of CCK-8 and flat clones showed that compared with the NC group and the Blank group, the cell proliferation ability of the two cell lines decreased significantly and the clone formation ability decreased (P0.05). (4) the flow cytometry and Hoechst33342/PI double staining were used to detect the apoptosis, and the result showed that the cell apoptosis was with Bl. The proportion of cell apoptosis and necrosis in group miR-195 was significantly higher in group ank than in group NC (P0.05); (5) the result of scar healing experiment showed that overexpression of miR-195 in U251 could inhibit cell migration (P0.05), but in SHG-44, there was no significant difference between the cell migration rate of the miR-195 group and the other two groups (P0.05); (6) Transwell invasion experiment results. In U251, over expression of miR-195 group could reduce the number of cells passing through the Matrigel membrane (P0.05), while in SHG-44, the number of cells passing through the Matrigel membrane in the miR-195 group was more than the other two groups (P0.05). (7) the cell cycle results of the flow cytometry showed that the miR-195 group was significantly increased in the G0/G1 phase compared with the Blank and NC groups. Significantly decreased (P0.05); (8) using the microRNA target prediction site and related biological information software, Bcl-2 was found as a target gene of miR-195. Immunocytochemical results showed that miR-195 could down regulate the expression of Bcl-2; (9) the effects of miR-195 on the formation of glioma were observed by subcutaneous glioma model in nude mice, and the results showed miR-195 group. The nodule of nude mice appeared late and the growth rate was slow, and the weight and volume of the tumor were significantly lower than that of the NC group and Blank group (P0.05). (10) the immunohistochemical analysis of the tumor specimens in nude mice showed that the Ki67 expression in the miR-195 group was lower than that of the NC group and the Blank group (P0.05).
The role and mechanism of 3. miR-195 in resistance to temozolomide in human gliomas:
Objective: To study the role and mechanism of miR-195 in the resistance of Tempe to drug resistance in human gliomas.
Methods: using the method of increasing the concentration of TMZ to induce the drug resistant strain of glioma, the expression of miR-195 in the drug resistant strain of glioma was determined by qRT-PCR, then the expression level of miR-195 in the drug resistant strain was changed. CCK-8 was used to detect the change of the drug resistance and proliferation of the glioma cells, and the flow cytometry was used to detect the apoptosis and the cells. Periodic changes; Western blot were used to detect the expression of MGMT, P21, Bcl-2 and Cyclin D1, and the subcutaneous glioma model of nude mice was further constructed to observe the tumor growth and the sensitivity to temozolomide.
Results: (1) TMZ resistant U251, SHG44 resistant strains were successfully established for 6 months, respectively named U251R, SHG-44R, and (2) qRT-PCR results showed that the TMZ resistance of glioma was U251R, miR-195 content in SHG-44R decreased to 40%, 48% and SHG-44R (P0.05) in SHG-44R (P0.05). The results showed that up regulation of miR-195 increased the sensitivity of SHG-44R to TMZ, while knocking low miR-195 increased the resistance of SHG44R to TMZ (P0.05). (4) flow cytometry showed that over expression of miR-195 could significantly increase the total apoptosis rate (P0.05) of the drug-resistant strain of glioma, and low miR-195 could decrease the total apoptosis rate. The difference was not statistically significant (P0.05), and (5) the cell cycle results showed that the expression level of miR-195 in SHG-44R cells was up, the growth of glioma cells slowed, the time of passage was prolonged, the cells of G0/G1 phase increased, and the cells of S phase decreased (P0.05), while the influence of the knock down miR-195 on the cell cycle of the glioma resistant cell lines was not distinct from the control group. (P0.05); (6) the results of Western blot showed that overexpression of miR-195 could downregulate the expression of P21, Cyclin D1, Bcl-2, and the expression of protein after knocking down miR-195 was opposite (P0.05) and MGMT expression level was not affected by miR-195 expression; (7) the subcutaneous glioma model in nude mice and the staining results showed that the combination group inhibited the growth and growth of glioma. Apoptosis of glioma cells was most significant (P0.05).
Conclusion: (1) miR-195 is down regulated in all detected glioma FFPE specimens and cell lines, and the expression level is negatively correlated with the pathological grade of glioma. It is suggested that we can use it as a new and effective diagnostic marker for glioma I-IV grade and supplement the classification standard of conventional histopathology; (2) overexpression of miR-1 95 can weaken the proliferation ability of glioma cells inside and outside of glioma cells, induce G0/G1 phase block, down regulation of Bcl-2 expression, promote apoptosis and necrosis of glioma cells, but the effect of miR-195 on the invasion and migration of glioma cells is related to cell types, and further research is needed. In general, miR-195 can negatively regulate the evil of glioma cells. Sexual biological behavior; (3) low expression of miR-195 in the drug resistant glioma and up regulation of miR-195 content, can induce apoptosis and proliferation restriction of drug-resistant cells, down regulate the expression of P21, Cyclin D1, Bcl-2, to a certain extent, to restore the chemensitivity of timozolamine by glioma cells in vivo and in vitro, and this effect is not related to the changes in MGMT expression. MiR above, miR -195 is expected to become a new target for gene therapy of glioma.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41

【共引文獻(xiàn)】

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