长链非编码RNA LINC01133在结直肠癌转移中的作用

发布时间：2018-05-05 21:42

本文选题：长链非编码RNA + 结直肠癌　；参考：《浙江大学》2016年博士论文

【摘要】：结直肠癌是世界上最常见的威胁人类健康的恶性肿瘤之一。在所有的恶性肿瘤中,结直肠癌每年的发病率和死亡率都高居前列。在最近几年,全世界每年都有超过60万患者死于结直肠癌。超过90%的肿瘤患者死亡是由肿瘤转移引起的。近年来的研究显示：恶性肿瘤细胞发生上皮间质转化(EMT)是恶性肿瘤发生转移的重要机制。EMT是指细胞在内部因素和外界环境的综合作用下由上皮细胞表型转变成间质细胞的过程。通过发生上皮间质转化,上皮来源的恶性肿瘤细胞获得间质细胞表型,获得了侵袭和转移的能力。那么恶性肿瘤发生EMT的分子机制又是什么呢?之前的大多数研究都是集中在对蛋白质分子的研究上。然而蛋白质分子所对应的mRNA在人类转录组中只占不到20%,超过80%的转录组是不能翻译成蛋白质的。这部分转录组对应的RNA就是非编码RNA(noncoding RNA, ncRNA)。这其中大部分是长度超过200个核苷酸的长链非编码RNA (long noncoding RNA, lncRNA), lncRNA在转录组中占据了绝大部分,它们必将会发挥着不可替代的功能,否则数目众多的lncRNA对细胞就会是一种负担,与进化过程中生命对使用物质和能量节约原则相悖。所以lncRNA在调控结直肠癌发生上皮间质转化过程中也很可能发挥着重要作用。我们选用TGF-p刺激结直肠癌细胞来构建诱导EMT发生的模型。然后将TGF-p处理的结直肠癌细胞作为实验组,将未处理的结直肠癌细胞作为对照组。为了鉴定出参与结直肠癌发生上皮间质转化过程中的lncRNA,我们对两组细胞做了转录组微矩阵芯片分析。从中鉴定出了差异表达的长lncRNA LINC01133。 LINC01133在TGF-β诱导EMT过程中表达显著下调。于是我们选择LINC01133作为研究对象来研究其在结直肠癌EMT和转移中的作用,并得出了以下结论：一、LINC01133的表达受到TGF-β的抑制。得到芯片结果之后,我们重复了TGF-β刺激构建结直肠癌细胞EMT模型试验。并通过q-RTPCR对芯片结果做了验证,证明LINC01133的表水平确实能被TGF-β下调。并且当阻断TGF-β受体或者通过沉默TGF-β通路下游的smad2后,LINC01133的表达水平又能恢复。结果表明LINC01133是受TGF-β抑制的一条长链非编码RNA。二、LINC01133抑制结直肠癌细胞EMT和转移。为了研究长链非编码RNA LINC01133在结直肠癌细胞发生EMT和转移中的作用。我们使用小干扰RNA在高表达LINC01133的结直肠癌细胞中的敲低LINC01133表达水平,在低表达LINC01133的结直肠癌细胞HCT116中过表达LINC01133。当LINC01133被敲低后,结直肠癌细胞的迁移侵袭能力增强,上皮标志物E-cadherin表达水平下调,间质标志物Fibronectin、Twist表达上调；LINC01133过表达后,结直肠癌细胞的迁移侵袭能力减弱,上皮标志物E-cadherin表达水平上调,间质标志物Fibronectin表达下调。同时小鼠尾静脉注射肺转移模型显示,LINC01133敲低后结直肠癌细胞的转移能力增强。同时结直肠癌临床样本研究表明LINC01133在肿瘤组织中表达明显下调,肿瘤组织中LINC01133表达水平高的患者远处转移率低,生存预后好,并且肿瘤组织中LINC01133表达水平与E-cadherin呈正相关,与Vimentin呈负相关。以上结果表明LINC01133可以抑制结直肠癌发生EMT和转移。三、LINC01133与SRSF6相结合并且可能通过阻断SRSF6促EMT的作用来抑制结直肠癌EMT和转移(1)鉴定LINC01133的结合蛋白SRSF6。我们通过改良ChIRP实验结合质谱鉴定出LINC01133的结合蛋白SRSF6.并通过Western Blot实验用SRSF6的抗体进行了验证。此外,又通过RIP反向验证SRSF6蛋白对LINC01133的结合作用。经过正反两方面的验证,我们确认长链非编码RNA LINC01133 和 SRSF6 蛋白有相互作用。(2)SRSF6促进结直肠癌细胞发生EMT和转移。在鉴定出LINC01133的结合蛋白SRSF6之后,我们又验证了SRSF6对结直肠癌的EMT和转移的调控作用。我们在高表达LINC01133的HT29细胞系、低表达LINC01133的HCT116细胞系和不表达LINC01133 的 SW620细胞系中利用慢病毒介导的shRNA将S RSF6干扰敲低。我们发现当SRSF6表达水平敲低以后,结直肠细胞的迁移侵袭能力减弱,上皮标志物E-cadherin表达水平上调,间质标志物Fibronectin、Twist表达下调；LINC01133过表达后,结直肠细胞的迁移侵袭能力减弱,上皮标志物E-cadherin表达水平表达上调,间质标志物Vimentin、 Fibronectin等表达下调。以上结果表明SRSF6可以不依赖于LINC01133,单独地促进结直肠癌细胞EMT和转移。(3)LINC01133对结直肠癌细胞EMT和转移的抑制用依赖于SRSF6我们通过实验验证LINC01133对结直肠癌细胞EMT过程的调控作用是否依赖于SRSF6.当SRSF6的表达被敲低后,LINC01133对EMT相关的蛋白标志物的调控以及对结直肠癌细胞迁移能力的调控作用都减弱了。结果提示我们长链非编码RNALINC01133对结直肠癌发生EMT和转移的抑制作用依赖于其与SRSF6结合和对SRSF6促EMT功能的阻断。综合以上结果可以得出结论：长链非编码RNA LINC01133通过与SRSF6相互作用来抑制结直肠癌EMT和转移。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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