【摘要】：成人膠質(zhì)母細胞瘤（GBM）是顱內(nèi)最常見的惡性腫瘤之一，其患病率及死亡率較高，盡管現(xiàn)在已有標準的手術(shù)及輔助放化療治療方法，但患者的中位生存期仍小于14個月。目前認為GBM的高復(fù)發(fā)率是導(dǎo)致其預(yù)后較差的主要原因，GBM極少出現(xiàn)遠隔轉(zhuǎn)移且多為原位復(fù)發(fā)，故該種高復(fù)發(fā)率可能暗示了腫瘤細胞對瘤旁組織固有的侵襲性及其自身極強的增殖，侵襲能力�，F(xiàn)有治療GBM的方式主要為手術(shù)切除腫瘤的實質(zhì)部分及瘤周的膠質(zhì)增生帶，以期有效緩解顱高壓癥狀及降低載瘤負荷再輔助以術(shù)后標準放化療，但該種治療方式并不能保證清除已侵襲至周圍正常腦組織中的腫瘤細胞，腫瘤仍有復(fù)發(fā)可能。臨床上不同級別膠質(zhì)瘤的術(shù)后復(fù)發(fā)時間不同，甚至同一級別的膠質(zhì)瘤經(jīng)同種方案治療后復(fù)發(fā)時間仍可能不同，這可能與腫瘤細胞不同的侵襲能力有關(guān)。因此抑制膠質(zhì)瘤細胞的增殖、遷移及侵襲能力將有可能成為一種輔助現(xiàn)有治療措施的新方式。 MicroRNAs是一種內(nèi)源性非編碼單鏈核苷酸，其長度約為19-25個核苷酸。microRNAs廣泛調(diào)控細胞增殖、遷移、侵襲、凋亡、自噬及衰老等多種細胞學行為�，F(xiàn)已證實microRNA的種子序列（seed sequence）能通過完全或不完全堿基互補配對的方式與目標mRNA3’UTR的特定序列相結(jié)合從而影響mRNA的穩(wěn)定性或抑制其翻譯過程，進而在轉(zhuǎn)錄后水平調(diào)節(jié)基因的表達。目前越來越多的證據(jù)顯示microRNA表達水平的變化與人類腫瘤性疾病的關(guān)系極為緊密，約50%microRNA定位于染色體上的脆性位點，這些位點在腫瘤細胞中通常缺失或者擴增，而且microRNA的異常表達與臨床患者的預(yù)后有一定的關(guān)系。miR-101是眾多microRNAs中的一員，近年來多有文獻報道其在腫瘤細胞中的表達量下降并能在體內(nèi)或體外抑制腫瘤的生長。綜合分析miRbase、TargetScanHuman等數(shù)據(jù)庫及文獻可知miR-101的靶基因包括EZH2、COX-2、SOX-9、MCL-2、APP等上千種，通過負性調(diào)控這些基因的表達miR-101可能參與多種和腫瘤發(fā)生、發(fā)展等相關(guān)的信號通路。 2011年在乳腺癌組織中發(fā)現(xiàn)了miR-101的三個新靶標，，STMN-1是其中之一。STMN-1(stathmin-1)又名OP（oncoprotein-18），是一種微管解聚磷蛋白，最早見于Sobel等人在1983年的報道。本課題組前期使用基因芯片技術(shù)分析了72例膠質(zhì)瘤組織標本并結(jié)合生物信息分析技術(shù)篩選出29個耐藥相關(guān)基因，STMN-1是其中之一。在細胞有絲分裂的前期，STMN-1蛋白第25、38號絲氨酸位點發(fā)生磷酸化，輔助紡錘體的正確形成及姐妹染色體的分離，這與腫瘤細胞的增殖能力有關(guān)；STMN-1還參與細胞凋亡及自噬等過程，我們前期的實驗結(jié)果表明干擾STMN-1表達后U251細胞對TMZ的敏感性增強且該現(xiàn)象可能與細胞凋亡、自噬有關(guān)；此外，在細胞遷移的過程中STMN-1第16位絲氨酸位點發(fā)生磷酸化并通過Rac1-Pak1途徑調(diào)控微管功能進而影響細胞運動。故STMN-1表達水平或磷酸化水平的改變在檢測腫瘤的發(fā)生、惡性演進及評估預(yù)后等方面有潛在意義。 本次實驗通過分析四中膠質(zhì)母細胞瘤細胞系中miR-101的表達差異以篩選實驗細胞系；隨后構(gòu)建miR-101、STMN-1過表達及干擾慢病毒載體，檢測轉(zhuǎn)染后細胞的遷移及侵襲能力；進一步建立包含STMN-1的3’UTR野生型及突變型的雙熒光慢毒載體，與miR-101過表達病毒共轉(zhuǎn)染GBM細胞后檢測熒光強度驗證在膠質(zhì)瘤細胞中miR-101與STMN-1的3’UTR直接結(jié)合；最后得出在膠質(zhì)瘤細胞中存在miR-101—STMN-1—細胞增殖、遷移、侵襲這一調(diào)控通路。實驗分為兩部分：第一部分，（1）收集膠質(zhì)瘤組織標本檢測其中miR-101的表達量并分析表達量與腫瘤級別的關(guān)系，（2）構(gòu)建相關(guān)慢病毒載體利用Q-PCR、WB、雙熒光靶標驗證等技術(shù)驗證在膠質(zhì)瘤細胞中存在miR-101與STMN-1直接負性調(diào)節(jié)關(guān)系；第二部分，膠質(zhì)瘤細胞株中分別轉(zhuǎn)染miR-101過表達及STMN-1干擾等慢病毒，隨后檢測細胞增殖、遷移及侵襲能力。 第一部分膠質(zhì)瘤組織標本中miR-101表達量的檢測，驗證膠質(zhì)瘤細胞株存在miR-101對STMN-1的直接負性調(diào)控關(guān)系 研究目的：檢測膠質(zhì)瘤組織標本中miR-101表達量與腫瘤級別的關(guān)系；分析四中膠質(zhì)瘤細胞系中miR-101的表達量；驗證膠質(zhì)瘤細胞中存在miR-101對STMN-1的直接負性調(diào)控關(guān)系。 研究內(nèi)容及結(jié)果：方法：應(yīng)用熒光定量PCR技術(shù)分析37例膠質(zhì)瘤組織標本中及四種膠質(zhì)瘤細胞系中miR-101的表達量；使用real-time PCR和western-blot技術(shù)檢測miR-101過表達或抑制后U251細胞內(nèi)STMN-1的mRNA水平及蛋白水平的變化；構(gòu)建pL/TO/IRES/Luc-STMN1-3’UTR和pL/TO/IRES/Luc-STMN1-3’UTR-mut慢病毒載體，利用雙熒光素酶報告系統(tǒng)技術(shù)驗證在膠質(zhì)瘤細胞中miR-101的種子序列能與STMN-1的3’UTR直接結(jié)合。結(jié)果：備選的四種GBM細胞系中miR-101的表達量依次為：U251＞A172＞U87＞T98；37例膠質(zhì)瘤組織經(jīng)實時熒光定量PCR分析后顯示miR-101的表達量在正常腦組織、低級別膠質(zhì)瘤（WHOⅠ-Ⅱ級）和高級別膠質(zhì)瘤（WHOⅢ-Ⅳ級）之間有明顯的統(tǒng)計學差異（P＜0.01），且隨著腫瘤級別的增高，細胞內(nèi)miR-101表達量逐漸下降，但WHOⅠ級與Ⅱ級之間或WHOⅢ級與Ⅳ級之間則無明顯統(tǒng)計學差異（P＞0.05）。U251細胞轉(zhuǎn)染miR-101過表達及miR-101干擾病毒后細胞內(nèi)STMN-1的mRNA水平及蛋白表達水平均明顯下降，顯示出miR-101對STMN-1明顯的負性調(diào)節(jié)作用；構(gòu)建好的雙熒光慢病毒，miR-101過表達及陰性對照慢病毒按照實驗分組轉(zhuǎn)染U251細胞后測定螢火蟲熒光酶素RLU和海腎熒光素酶素RLU，數(shù)據(jù)顯示miR-101能夠與STMN1-3’UTR直接作用。 結(jié)論：miR-101的表達量與腫瘤級別的增高呈負相關(guān)，在低級別膠質(zhì)瘤和高級別膠質(zhì)瘤之間有明顯的差異；miR-101的種子序列能與STMN1-3’UTR直接結(jié)合并降低STMN-1mRNA及蛋白的表達水平。 第二部分驗證miR-101能通過STMN-1調(diào)控U251細胞的增殖、遷移及侵襲能力 研究目的：證明miR-101能調(diào)控U251細胞的增殖、遷移能力并通過STMN-1來調(diào)控侵襲能力。 研究內(nèi)容及結(jié)果：方法：分別用構(gòu)建好的miR-101過表達、miR-101干擾、STMN-1-siRNA及相應(yīng)的陰性對照（NC）病毒轉(zhuǎn)染U251細胞株；用CCK-8法檢測細胞增殖能力并繪制增殖曲線；用細胞劃痕實驗評估細胞遷移能力；用Transwell法檢測細胞的侵襲能力。結(jié)果：加入CCK-8試劑后用酶標儀檢測的結(jié)果顯示U251+miR-101過表達組的細胞增殖能力較U251+miR-101干擾組，U251+miR-101過表達NC組，U251+miR-101干擾NC組及U251組明顯下降且有顯著地統(tǒng)計學差異（P＜0.05），但后三組之間沒有統(tǒng)計學差異。U251細胞分別轉(zhuǎn)染miR-101干擾，miR-101過表達NC及miR-101干擾NC慢病毒72h后做細胞劃痕實驗，24h后結(jié)果顯示U251+miR-101過表達組的細胞劃痕面積的變化值要明顯小于其余三組并存在統(tǒng)計學差異（P＜0.05）；U251細胞轉(zhuǎn)染miR-101過表達，NC，siRNA-STMN-1慢病毒后利用Transwell技術(shù)檢測細胞侵襲能力，18h后結(jié)果顯示U251+miR-101過表達組及U251+siRNA-STMN-1組細胞的侵襲能力下降，兩者分別與NC組及正常U251組比較均有統(tǒng)計學差異（P＜0.05），二者之間亦存在明顯統(tǒng)計學差異（P＜0.05）。 結(jié)論：miR-101能在表觀遺傳學水平抑制STMN-1的表達，從而抑制U251細胞的增殖，遷移及侵襲能力。
[Abstract]:Adult glioblastoma (GBM) is one of the most common intracranial malignancies. Its morbidity and mortality are high. Although there are standard surgical and adjuvant chemoradiotherapy methods, the median survival time of the patients is still less than 14 months. Because septal metastasis is mostly in situ recurrence, this high recurrence rate may indicate the intrinsic invasiveness of tumor cells to adjacent tissues and their own strong proliferation and invasiveness. Re-adjuvant radiotherapy and chemotherapy are standard, but this treatment does not guarantee the removal of tumor cells that have invaded the surrounding normal brain tissue, and tumor recurrence is still possible. Inhibition of proliferation, migration and invasion of glioma cells may be a new approach to adjuvant therapy.
MicroRNAs are endogenous non-coding single-stranded nucleotides with a length of about 19-25 nucleotides. MicroRNAs regulate cell proliferation, migration, invasion, apoptosis, autophagy and senescence. It has been demonstrated that microRNA seed sequences can complement target mRNA 3 through complete or incomplete base complementary pairing. More and more evidences show that changes in microRNA expression levels are closely related to human cancerous diseases. About 50% of microRNAs are located at fragile sites on chromosomes. MicroRNA-101 is a member of many microRNAs. In recent years, it has been reported that the expression of microRNAs in tumor cells decreases and can inhibit tumor growth in vivo or in vitro. According to uman and other databases and literatures, the target genes of microRNAs-101 include EZH2, COX-2, SOX-9, MCL-2, APP and so on. Through negative regulation of these genes, microRNAs-101 may participate in a variety of signaling pathways related to tumorigenesis and development.
STMN-1, also known as oncoprotein-18, is a microtubule depolymerized phosphoprotein. It was first reported by Sobel et al. in 1983. Seventy-two glioma specimens were analyzed by gene chip technique and bioinformatics. STMN-1 is one of the 29 drug-resistance-related genes screened by information analysis. At the early stage of cell mitosis, STMN-1 protein phosphorylated at the 25th and 38th serine sites, assisted spindle formation and sister chromosome isolation, which is related to the proliferation of tumor cells; STMN-1 is also involved in apoptosis and autophagy. In addition, the phosphorylation of the 16th serine site of STMN-1 during cell migration and the regulation of microtubule function via Rac1-Pak1 pathway affect cell movement. Changes in levels of phosphorylation or reaching levels are potentially useful in detecting tumorigenesis, malignant progression, and evaluating prognosis.
In this experiment, we screened the experimental cell lines by analyzing the expression difference of microRNAs-101 in the four glioblastoma cell lines; then constructed microRNAs-101, STMN-1 overexpression and interfering lentivirus vector to detect the migration and invasiveness of the transfected cells; further established the wild-type and mutant dual-fluorescent lentivirus vector containing STMN-1. After co-transfection with the virus, the fluorescence intensity was detected to verify the direct binding of the RNA-101 to the 3'UTR of STMN-1 in glioma cells. Finally, it was concluded that there was a regulation pathway of proliferation, migration and invasion in glioma cells. The experiment was divided into two parts: the first part, (1) collecting glioma tissue. The expression of microRNAs-101 in glioma cells was detected and the relationship between microRNAs-101 expression and tumor grade was analyzed. (2) Construction of lentiviral vectors was used to verify the direct negative regulatory relationship between microRNAs-101 and STMN-1 in glioma cells by Q-PCR, WB, double fluorescence target assay and other techniques. N-1 interference such as lentivirus, then detect cell proliferation, migration and invasion ability.
Part I Detection of the expression of microRNAs-101 in glioma tissue specimens to verify the direct negative regulation of microRNAs-101 on STMN-1 in glioma cell lines
Objective: To investigate the relationship between the expression of microRNAs-101 and tumor grade in glioma tissues, analyze the expression of microRNAs-101 in glioma cell lines, and verify the direct negative regulation of microRNAs-101 on STMN-1 in glioma cells.
RESULTS: The expression of microRNAs-101 in 37 glioma tissues and four glioma cell lines was analyzed by fluorescence quantitative PCR, and the mRNA and protein levels of STMN-1 in U251 cells were detected by real-time PCR and Western-blot. C-STMN1-3'UTR and pL/TO/IRES/Luc-STMN1-3'UTR-mut lentiviral vectors were used to verify that the seed sequence of microRNAs-101 in glioma cells could bind directly to the 3'UTR of STMN-1. Results: The expression of microRNAs-101 in the four GBM cell lines was U251 > A172 > U87 > T98 in turn. Real-time fluorescence quantitative PCR analysis showed that the expression of microRNA-101 was significantly different among normal brain tissues, low-grade gliomas (WHO grade I-II) and high-grade gliomas (WHO grade III-IV) (P There was no significant difference between grade III and grade IV (P > 0.05). The overexpression of miR-101 in U251 cells and the expression of STMN-1 protein were significantly decreased after interfering with the virus, which indicated that the expression of STMN-1 was negatively regulated by the virus. The constructed double fluorescent lentivirus, the overexpression of microwave-101 and the negative control were observed. Luciferin RLU and nephron luciferin RLU in U251 cells transfected by lentiviruses were measured. The data showed that microRNA101 could directly interact with STMN1-3'UTR.
Conclusion: The expression of microRNAs-101 is negatively correlated with the increase of tumor grade, and there is a significant difference between low-grade glioma and high-grade glioma. The seed sequence of microRNAs-101 can directly bind to STMN1-3'UTR and reduce the expression of STMN-1 mRNA and protein.
The second part verifies that miR-101 can regulate the proliferation, migration and invasion ability of U251 cells through STMN-1.
OBJECTIVE: To demonstrate that microRNAs-101 can regulate the proliferation, migration and invasion of U251 cells through STMN-1.
RESULTS: U251 cells were transfected with constructed over-expression of microRNAs-101, interference of microRNAs-101, STMN-1-siRNA and corresponding negative control (NC) virus, the proliferation ability of U251 cells was detected by CCK-8 assay, the cell migration ability was evaluated by cell scratch test, and the invasive ability of U251 cells was detected by Transwell assay. Results: After adding CCK-8 reagent, the proliferation ability of U251 + Mi-101 overexpression group was significantly lower than that of U251 + Mi-101 interference group, U251 + Mi-101 overexpression NC group, U251 + Mi-101 interference NC group and U251 interference NC group (P < 0.05), but there was no significant difference between the latter three groups. Mi-101 interference, Mi-101 overexpression NC and Mi-101 interference NC lentivirus 72 hours after the scratch test, 24 hours after the results showed that U251 + Mi-101 overexpression group of cell scratch area was significantly smaller than the other three groups and there were statistical differences (P < 0.05); U251 cells overexpression of Mi-101, NC, siRNA-STMN-1 lentivirus after transfection. Transwell technique was used to detect the invasive ability of U251+microRNA-101 cells. After 18 hours, the invasive ability of U251+microRNA-101 overexpression group and U251+siRNA-STMN-1 group decreased, and there were significant differences between them (P<0.05) and NC group and normal U251 group, respectively.
Conclusion: Mi-101 can inhibit the expression of STMN-1 at the epigenetic level, thereby inhibiting the proliferation, migration and invasion of U251 cells.
【學位授予單位】：第二軍醫(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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1 孫海兵;魏永長;涂宏蕾;杜寧;趙陽;胡麗娟;任宏;;COX-2、PKC-α和miR-101在胃癌中的表達及相關(guān)性[J];南方醫(yī)科大學學報;2013年04期

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