本文選題：長鏈非編碼RNA + 結(jié)直腸癌　； 參考：《浙江大學》2016年博士論文

【摘要】：結(jié)直腸癌是世界上最常見的威脅人類健康的惡性腫瘤之一。在所有的惡性腫瘤中,結(jié)直腸癌每年的發(fā)病率和死亡率都高居前列。在最近幾年,全世界每年都有超過60萬患者死于結(jié)直腸癌。超過90%的腫瘤患者死亡是由腫瘤轉(zhuǎn)移引起的。近年來的研究顯示：惡性腫瘤細胞發(fā)生上皮間質(zhì)轉(zhuǎn)化(EMT)是惡性腫瘤發(fā)生轉(zhuǎn)移的重要機制。EMT是指細胞在內(nèi)部因素和外界環(huán)境的綜合作用下由上皮細胞表型轉(zhuǎn)變成間質(zhì)細胞的過程。通過發(fā)生上皮間質(zhì)轉(zhuǎn)化,上皮來源的惡性腫瘤細胞獲得間質(zhì)細胞表型,獲得了侵襲和轉(zhuǎn)移的能力。那么惡性腫瘤發(fā)生EMT的分子機制又是什么呢?之前的大多數(shù)研究都是集中在對蛋白質(zhì)分子的研究上。然而蛋白質(zhì)分子所對應的mRNA在人類轉(zhuǎn)錄組中只占不到20%,超過80%的轉(zhuǎn)錄組是不能翻譯成蛋白質(zhì)的。這部分轉(zhuǎn)錄組對應的RNA就是非編碼RNA(noncoding RNA, ncRNA)。這其中大部分是長度超過200個核苷酸的長鏈非編碼RNA (long noncoding RNA, lncRNA), lncRNA在轉(zhuǎn)錄組中占據(jù)了絕大部分,它們必將會發(fā)揮著不可替代的功能,否則數(shù)目眾多的lncRNA對細胞就會是一種負擔,與進化過程中生命對使用物質(zhì)和能量節(jié)約原則相悖。所以lncRNA在調(diào)控結(jié)直腸癌發(fā)生上皮間質(zhì)轉(zhuǎn)化過程中也很可能發(fā)揮著重要作用。我們選用TGF-p刺激結(jié)直腸癌細胞來構(gòu)建誘導EMT發(fā)生的模型。然后將TGF-p處理的結(jié)直腸癌細胞作為實驗組,將未處理的結(jié)直腸癌細胞作為對照組。為了鑒定出參與結(jié)直腸癌發(fā)生上皮間質(zhì)轉(zhuǎn)化過程中的lncRNA,我們對兩組細胞做了轉(zhuǎn)錄組微矩陣芯片分析。從中鑒定出了差異表達的長lncRNA LINC01133。 LINC01133在TGF-β誘導EMT過程中表達顯著下調(diào)。于是我們選擇LINC01133作為研究對象來研究其在結(jié)直腸癌EMT和轉(zhuǎn)移中的作用,并得出了以下結(jié)論：一、LINC01133的表達受到TGF-β的抑制。得到芯片結(jié)果之后,我們重復了TGF-β刺激構(gòu)建結(jié)直腸癌細胞EMT模型試驗。并通過q-RTPCR對芯片結(jié)果做了驗證,證明LINC01133的表水平確實能被TGF-β下調(diào)。并且當阻斷TGF-β受體或者通過沉默TGF-β通路下游的smad2后,LINC01133的表達水平又能恢復。結(jié)果表明LINC01133是受TGF-β抑制的一條長鏈非編碼RNA。二、LINC01133抑制結(jié)直腸癌細胞EMT和轉(zhuǎn)移。為了研究長鏈非編碼RNA LINC01133在結(jié)直腸癌細胞發(fā)生EMT和轉(zhuǎn)移中的作用。我們使用小干擾RNA在高表達LINC01133的結(jié)直腸癌細胞中的敲低LINC01133表達水平,在低表達LINC01133的結(jié)直腸癌細胞HCT116中過表達LINC01133。當LINC01133被敲低后,結(jié)直腸癌細胞的遷移侵襲能力增強,上皮標志物E-cadherin表達水平下調(diào),間質(zhì)標志物Fibronectin、Twist表達上調(diào)；LINC01133過表達后,結(jié)直腸癌細胞的遷移侵襲能力減弱,上皮標志物E-cadherin表達水平上調(diào),間質(zhì)標志物Fibronectin表達下調(diào)。同時小鼠尾靜脈注射肺轉(zhuǎn)移模型顯示,LINC01133敲低后結(jié)直腸癌細胞的轉(zhuǎn)移能力增強。同時結(jié)直腸癌臨床樣本研究表明LINC01133在腫瘤組織中表達明顯下調(diào),腫瘤組織中LINC01133表達水平高的患者遠處轉(zhuǎn)移率低,生存預后好,并且腫瘤組織中LINC01133表達水平與E-cadherin呈正相關(guān),與Vimentin呈負相關(guān)。以上結(jié)果表明LINC01133可以抑制結(jié)直腸癌發(fā)生EMT和轉(zhuǎn)移。三、LINC01133與SRSF6相結(jié)合并且可能通過阻斷SRSF6促EMT的作用來抑制結(jié)直腸癌EMT和轉(zhuǎn)移(1)鑒定LINC01133的結(jié)合蛋白SRSF6。我們通過改良ChIRP實驗結(jié)合質(zhì)譜鑒定出LINC01133的結(jié)合蛋白SRSF6.并通過Western Blot實驗用SRSF6的抗體進行了驗證。此外,又通過RIP反向驗證SRSF6蛋白對LINC01133的結(jié)合作用。經(jīng)過正反兩方面的驗證,我們確認長鏈非編碼RNA LINC01133 和 SRSF6 蛋白有相互作用。(2)SRSF6促進結(jié)直腸癌細胞發(fā)生EMT和轉(zhuǎn)移。在鑒定出LINC01133的結(jié)合蛋白SRSF6之后,我們又驗證了SRSF6對結(jié)直腸癌的EMT和轉(zhuǎn)移的調(diào)控作用。我們在高表達LINC01133的HT29細胞系、低表達LINC01133的HCT116細胞系和不表達LINC01133 的 SW620細胞系中利用慢病毒介導的shRNA將S RSF6干擾敲低。我們發(fā)現(xiàn)當SRSF6表達水平敲低以后,結(jié)直腸細胞的遷移侵襲能力減弱,上皮標志物E-cadherin表達水平上調(diào),間質(zhì)標志物Fibronectin、Twist表達下調(diào)；LINC01133過表達后,結(jié)直腸細胞的遷移侵襲能力減弱,上皮標志物E-cadherin表達水平表達上調(diào),間質(zhì)標志物Vimentin、 Fibronectin等表達下調(diào)。以上結(jié)果表明SRSF6可以不依賴于LINC01133,單獨地促進結(jié)直腸癌細胞EMT和轉(zhuǎn)移。(3)LINC01133對結(jié)直腸癌細胞EMT和轉(zhuǎn)移的抑制用依賴于SRSF6我們通過實驗驗證LINC01133對結(jié)直腸癌細胞EMT過程的調(diào)控作用是否依賴于SRSF6.當SRSF6的表達被敲低后,LINC01133對EMT相關(guān)的蛋白標志物的調(diào)控以及對結(jié)直腸癌細胞遷移能力的調(diào)控作用都減弱了。結(jié)果提示我們長鏈非編碼RNALINC01133對結(jié)直腸癌發(fā)生EMT和轉(zhuǎn)移的抑制作用依賴于其與SRSF6結(jié)合和對SRSF6促EMT功能的阻斷。綜合以上結(jié)果可以得出結(jié)論：長鏈非編碼RNA LINC01133通過與SRSF6相互作用來抑制結(jié)直腸癌EMT和轉(zhuǎn)移。
[Abstract]:Colorectal cancer is one of the most common malignant tumors that threaten human health in the world. In all malignant tumors, the incidence and mortality of colorectal cancer are high in each year. In recent years, more than 600 thousand patients die from colorectal cancer every year in the world. More than 90% of the patients with swollen tumors are caused by tumor metastasis in recent years. Studies have shown that epithelial mesenchymal transformation (EMT) is an important mechanism for malignant tumor metastasis,.EMT is the process of cell transformation from epithelial cell phenotype to interstitial cell under the combined action of internal and external environment. The cell phenotype has the ability to invasion and metastasis. What is the molecular mechanism of EMT in malignant tumors? Most of the previous studies focus on the study of protein molecules. However, protein molecules correspond to only less than 20% of the mRNA in human transcripts, and more than 80% of the transcriptional groups cannot be translated into protein. Qualitative. The RNA of this part of the transcriptome is the non coded RNA (noncoding RNA, ncRNA). Most of them are long chain non coded RNA (long noncoding RNA, lncRNA) over 200 nucleotides, lncRNA in the transcriptional group, and they will certainly have an unreplaceable function, otherwise a large number of lncRNA pairs The cell will be a burden, which is contrary to the principle of the use of material and energy conservation in the process of evolution. So lncRNA is also likely to play an important role in regulating the epithelial mesenchymal transition in colorectal cancer. We use TGF-p to stimulate colorectal cancer cells to construct a model to induce the occurrence of EMT. Then, the treatment of TGF-p is made straight. In order to identify the lncRNA in the process of epithelial mesenchymal transition in colorectal cancer, we made a microarray microarray analysis of the two groups of cells in order to identify the process of epithelial mesenchymal transition in colorectal cancer. We identified the differential expression of long lncRNA LINC01133. LINC01133 in the TGF- beta induced EMT process. We chose LINC01133 as a study object to study its role in the EMT and metastasis of colorectal cancer, and we concluded that the expression of LINC01133 was inhibited by TGF- beta. After the results of the chip, we repeated the TGF- beta stimulation to construct the EMT model test of colorectal cancer cells and through q-RTPCR The results of the chip proved that the surface level of LINC01133 can be downregulated by TGF- beta. And the expression level of LINC01133 can be recovered after blocking the TGF- beta receptor or through the Smad2 downstream of the silent TGF- beta pathway. The result indicates that LINC01133 is a long chain non coding RNA. two controlled by TGF- beta, and LINC01133 inhibits colorectal cancer cell EMT. To study the role of long chain non coding RNA LINC01133 in EMT and metastasis of colorectal cancer cells. We use small interfering RNA in the low LINC01133 expression level in colorectal cancer cells with high expression of LINC01133, and overexpress LINC01133. in the colorectal cancer cell HCT116 with low expression of LINC01133 when LINC01133 is knocked down, The metastasis and invasion ability of colorectal cancer cells increased, the expression level of epithelial markers E-cadherin was down, the expression of interstitial markers Fibronectin and Twist were up regulated. After LINC01133 overexpression, the migration and invasion ability of colorectal cancer cells weakened, the expression level of E-cadherin in epithelial markers was adjusted, and the expression of interstitial marker Fibronectin was downregulated. At the same time, the expression of interstitial marker Fibronectin was down. The rat tail vein injection model of lung metastasis showed that the metastasis of colorectal cancer cells increased after LINC01133 knockout. At the same time, the colorectal cancer clinical sample study showed that the expression of LINC01133 was obviously downregulated in the tumor tissue. The distant metastasis rate of the patients with high LINC01133 expression level in the tumor tissues was low, the survival prognosis was good, and the tumor tissue was LINC01133 Expression level is positively correlated with E-cadherin and negatively correlated with Vimentin. The above results suggest that LINC01133 can inhibit the occurrence of EMT and metastasis in colorectal cancer. Three, LINC01133 is combined with SRSF6 and may inhibit SRSF6 by blocking the role of SRSF6 to inhibit the EMT and metastasis of colorectal cancer (1) to identify LINC01133 binding protein SRSF6.. The RP experiment combined with mass spectrometry to identify the binding protein SRSF6. of LINC01133 and the antibody of SRSF6 through the Western Blot experiment. In addition, the binding effect of SRSF6 protein on LINC01133 was verified by RIP. Through positive and negative two aspects, we confirmed that the long chain non coded RNA LINC01133 and the SRSF6 protein have interaction. (2) F6 promotes the occurrence of EMT and metastasis in colorectal cancer cells. After identifying the LINC01133 binding protein SRSF6, we also verified the regulatory role of SRSF6 on EMT and metastasis in colorectal cancer. We use the slow disease in the HT29 cell line that expresses the LINC01133, the HCT116 cell lines with low expression of LINC01133 and the SW620 cell line that is not up to the LINC01133. The toxic mediated shRNA interfered with the S RSF6 interference. We found that after the SRSF6 expression level was knocked down, the migration and invasion ability of the colorectal cells was weakened, the expression of E-cadherin in the epithelial markers was up-regulated, the interstitial marker Fibronectin, and the expression of Twist was down regulated. After LINC01133 overexpression, the migration and invasion ability of the colorectal cells weakened and the epithelial marker E was in E. The expression of -cadherin expression was up-regulated and the expression of interstitial markers Vimentin, Fibronectin and so on. The above results showed that SRSF6 could independently promote EMT and metastasis of colorectal cancer cells without LINC01133. (3) the inhibition of LINC01133 on the EMT and metastasis of colorectal cancer cells depends on SRSF6 we have experimentally verified LINC01133 for colorectal cancer. Whether the regulatory role of EMT process in cancer cells depends on the reduction of SRSF6. when the expression of SRSF6 is knocked down, the regulation of EMT related protein markers and the regulation of the migration ability of colorectal cancer cells are weakened. The results suggest that our long chain non coded RNALINC01133 is dependent on the inhibition of EMT and metastasis of colorectal cancer. In combination with SRSF6 and blocking the function of SRSF6 to promote EMT, the above results can be concluded that long chain non coded RNA LINC01133 inhibits colorectal EMT and metastasis by interacting with SRSF6.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.34

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