發(fā)布時間：2018-06-12 17:40

  本文選題：miR-203 + SNAI2�。� 參考：《南開大學(xué)》2014年博士論文

【摘要】：乳腺癌和前列腺癌分別是女性和男性最常見的腫瘤疾病之一,具有很高的發(fā)病率,其發(fā)生、演進、浸潤和轉(zhuǎn)移是一個多基因(包括癌基因和抑癌基因)共同參與的漸進累積過程。其中的分子機制還需要更多的研究。 在人類基因組序列中,編碼序列不超過2%,其余大部分為非編碼序列,其中可以產(chǎn)生穩(wěn)定轉(zhuǎn)錄本的序列稱之為非編碼RNA (non-coding RNA, ncRNA)基因。非編碼RNA (non coding RNA, ncRNA)種類廣泛。在細胞中含量最多的ncRNA是tRNA和rRNA。除此之外,還包括snRNA,snoRNA,piRNA,lncRNA和microRNA等。目前研究顯示,這些RNA的主要功能是參與mRNA的穩(wěn)定和翻譯水平的調(diào)節(jié)、參與蛋白質(zhì)的運輸、參與RNA的加工和修飾、影響染色質(zhì)的結(jié)構(gòu)等。一些ncRNA在惡性腫瘤的發(fā)生發(fā)展中發(fā)揮著重要的調(diào)控作用甚至起著關(guān)鍵的作用。 microRNA(miRNA)是長度為19-24堿基的內(nèi)源性非編碼RNA(non-coding RNA,ncRNA)。它通過堿基互補的方式結(jié)合到靶基因mRNA的3'非翻譯區(qū)進而抑制其表達。研究發(fā)現(xiàn)miRNA序列極其保守而且在基于RNA水平上的基因調(diào)控過程中起到了關(guān)鍵作用。miRNA參與調(diào)節(jié)一系列重要的生物學(xué)過程,包括細胞增殖、分化、衰老、凋亡和細胞干性維持等等。miRNA的異常表達與包括癌癥在內(nèi)的許多病理學(xué)過程息息相關(guān)。miRNA與其靶基因所構(gòu)成的調(diào)控網(wǎng)絡(luò)在腫瘤的發(fā)生發(fā)展過程中扮演了十分重要的角色。 長鏈非編碼RNA(long ncRNA, lncRNA)是長度大于200堿基的非編碼RNA。研究表明,lncRNA作為關(guān)鍵因子參與了一系列的細胞調(diào)控過程,例如基因轉(zhuǎn)錄調(diào)控,介導(dǎo)染色質(zhì)重構(gòu)及組蛋白修飾,干擾mRNA剪切和翻譯,X染色體失活,基因組印記等等。大量由臨床觀察和實驗研究所得的結(jié)果表明,lncRNA的異常表達會導(dǎo)致很多疾病的發(fā)生,這種機制在癌生物學(xué)中尤為重要。盡管到目前為止,與癌癥相關(guān)的大多數(shù)lncRNA的作用機制還沒得到準(zhǔn)確闡明,但對部分lncRNA的研究表明,其所介導(dǎo)的染色質(zhì)重構(gòu)和組蛋白修飾等表觀遺傳學(xué)過程會影響腫瘤的發(fā)生和遷移。 miR-203是一個可以抑制表皮細胞干性的miRNA,它可以通過靶向細胞干性因子ΔNp63來抑制表皮細胞的增殖,進而促進其分化。在多種類型的癌癥細胞中均已檢測到miR-203的異常表達。在膀胱癌和卵巢癌中,癌變的發(fā)生伴隨著miR-203的上調(diào)。然而在胰腺癌中,miR-203的過表達卻預(yù)示著病人的不良預(yù)后。在氣管癌、淋巴癌、肝癌和口腔癌中,miR-203表達下調(diào),而且其下調(diào)可能歸因于啟動子區(qū)域的甲基化。在分子層面上,轉(zhuǎn)錄因子zinc-finger E-box binding homeobox1(ZEB1)已被鑒定為促進腫瘤細胞上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition, EMT)的重要因子,而這種促進作用部分歸因于EMT所引起的一些miRNA包括miR-203的下調(diào)。上述結(jié)果表明miR-203在癌癥的發(fā)生發(fā)展中可能起到十分重要的作用,而且其功能因腫瘤類型的不同而有所區(qū)別。 PVT1是位于人類染色體8q24區(qū)域的一個lncRNA,該區(qū)域在包括乳腺癌在內(nèi)的多種癌癥中均發(fā)生擴增。在乳腺癌和卵巢癌中,沉默PVT1的表達可以抑制細胞的增殖并誘導(dǎo)細胞凋亡。但是PVT1本身的調(diào)控機制尚未闡明。 在本文的研究中,我們首先旨在闡明miR-203在乳腺癌發(fā)生發(fā)展中的作用。我們首先檢測了miR-203在乳腺癌組織樣本中的表達情況,發(fā)現(xiàn)與相應(yīng)的癌旁組織相比,在大部分腫瘤樣本中miR-203表達上調(diào)。在乳腺癌細胞系中,與正�；蛘哂郎娜橄偌毎啾�,miR-203在luminal A類型的乳腺癌細胞(轉(zhuǎn)移能力低)中表達上調(diào),而在basal-like類型的乳腺癌細胞(轉(zhuǎn)移能力高)中表達下調(diào)。接下來,我們檢測了在乳腺癌細胞系中,miR-203啟動子區(qū)域的甲基化狀態(tài)。我們發(fā)現(xiàn)在高轉(zhuǎn)移的basal-like乳腺癌細胞中,miR-203的啟動子區(qū)域存在一定程度的甲基化,而在低轉(zhuǎn)移的luminal A乳腺癌細胞中則不存在。同時,用去甲基化藥物5-aza-dCyd處理basal-like類型的乳腺癌細胞可以使miR-203的表達得到一定回復(fù)。因此,miR-203在basal-like類型的乳腺癌細胞中表達下調(diào),可能部分歸因于啟動子區(qū)域的甲基化。 我們接下來研究了miR-203在乳腺癌細胞中的功能。我們發(fā)現(xiàn)在basal-like類型的乳腺癌細胞中過表達miR-203明顯抑制細胞增殖,而其抑制作用的機制包括影響細胞周期和促進凋亡,并且具有細胞特異性。同時,過表達miR-203可以抑制上述細胞的遷移和浸潤。在分子層面上,我們發(fā)現(xiàn)并證實與EMT相關(guān)的轉(zhuǎn)錄因子SNAI2是miR-203的一個作用靶點。而且在功能上,SNAI2可以挽救miR-203對basal-like細胞EMT過程的抑制作用。 以上結(jié)果表明在惡性乳腺癌細胞中,miR-203的表達因為啟動子的甲基化而被抑制,進而上調(diào)轉(zhuǎn)錄因子SNAI2的表達,最終促進腫瘤細胞的生長和轉(zhuǎn)移。 之后,我們進一步檢測了miR-203極其靶點SNAI2所構(gòu)成的調(diào)節(jié)通路對前列腺癌細胞增殖和遷移的作用。與別人的報道類似,我們發(fā)現(xiàn)miR-203在前列腺癌細胞中表達下調(diào),同時過表達miR-203可以抑制前列腺癌細胞的增殖與遷移。而且,在前列腺癌細胞中,miR-203同樣可以靶向SNAI2,過表達SNAI2同樣可以使miR-203對前列腺癌細胞遷移的抑制作用得到恢復(fù)。 與此同時,我們發(fā)現(xiàn)我們實驗室所感興趣的轉(zhuǎn)錄因子KLF5可以結(jié)合到miR-203的啟動子區(qū)域從而正向調(diào)節(jié)miR-203的表達。另外,KLF5可以負向調(diào)節(jié)miR-203的靶點SNAI2的表達。過表達SNAI2可以拯救KLF5對前列腺癌細胞遷移過程的抑制作用。 綜上所述,本文中的發(fā)現(xiàn)說明了miR-203在乳腺癌和前列腺癌的發(fā)生發(fā)展中起到重要的作用。作為一個抑癌因子,miR-203可以通過靶向SNAI2抑制乳腺癌和前列腺癌細胞的增殖和遷移。同時,miR-203-SNAI2這一調(diào)節(jié)路徑在KLF5所介導(dǎo)的乳腺癌EMT過程中扮演了重要角色。 另一方面,我們發(fā)現(xiàn)位于PVTl所在染色體區(qū)域附近的一個乳腺癌敏感的核苷酸多態(tài)性位點(Single nucleotide polymorphism,SNP) rs13281615的基因型與PVT1在乳腺癌中的表達變化顯著相關(guān)。對于SNP rs13281615,帶有危險性的GG基因型的乳腺癌患者其腫瘤組織中PVT1的表達量顯著高于帶有其它基因型的患者,并且高于帶有任何基因型患者的正常癌旁組織。與此同時,SNPrs13281615的在乳腺癌的發(fā)生過程發(fā)生頻繁的方向性突變(G→A)。而且這種方向性突變使得原本存在的GG基因型與腫瘤等級和增殖marker Ki67之間的相關(guān)性喪失。我們的發(fā)現(xiàn)為PVTl受調(diào)控的機制提供了新的思路。
[Abstract]:Breast and prostate cancer are one of the most common cancer diseases in women and men. They have a high incidence, and their occurrence, evolution, infiltration and metastasis are the gradual accumulation of a multi gene (including oncogene and tumor suppressor gene). The molecular mechanism of which needs more research.
In the human genome sequence, the coding sequence is not more than 2%, and the rest of the majority are non coded sequences, in which the stable transcriptional sequence is called the non coded RNA (non-coding RNA, ncRNA) gene. The non coded RNA (non coding RNA, ncRNA) is widely used. The ncRNA of the most content in the cell is tRNA and rRNA., but also includes the exception. NRNA, snoRNA, piRNA, lncRNA and microRNA, etc. current research shows that the main functions of these RNA are to participate in the stability of mRNA and the regulation of the level of translation, participate in the transport of protein, participate in the processing and modification of RNA, affect the structure of chromatin, and so on. Some ncRNA play an important regulatory role in the development of malignant tumors and even play a key role. The function of the bond.
MicroRNA (miRNA) is an endogenous non coded RNA (non-coding RNA, ncRNA) with a length of 19-24 bases. It combines the base complementary way to the 3'non translation region of the target gene mRNA and then inhibits its expression. The study found that the miRNA sequence is extremely conservative and plays a key role in the modulation process based on the RNA level. A series of important biological processes, including cell proliferation, differentiation, aging, apoptosis and cell stem maintenance, and so on, the abnormal expression of.MiRNA is closely related to many pathological processes including cancer. The regulatory network of.MiRNA and its target genes plays a very important role in the development of tumor.
Long chain non coding RNA (long ncRNA, lncRNA) is a non coded RNA. study of length greater than 200 bases, indicating that lncRNA as a key factor participates in a series of cell regulation processes, such as gene transcription regulation, mediating chromatin remodeling and histone modification, interfering with mRNA shear and translation, X chromosome inactivation, genomic imprinting and so on. The results of bed observation and experimental study showed that abnormal expression of lncRNA could lead to many diseases. This mechanism is particularly important in cancer biology. Although the mechanism of most lncRNA related to cancer has not been accurately elucidated, the study on the division of lncRNA shows that its chromatin weight is mediated. Epigenetic processes such as constitutive and histone modifications affect the occurrence and migration of tumors.
MiR-203 is a miRNA that inhibits the dry nature of epidermal cells. It can inhibit the proliferation of epidermal cells by targeting the cell stem factor, Delta Np63, and then promote its differentiation. The abnormal expression of miR-203 has been detected in various types of cancer cells. In bladder and ovarian cancer, the occurrence of cancer is accompanied by the up-regulation of miR-203. In pancreatic cancer, the overexpression of miR-203 indicates a poor prognosis. In tracheal, lymphatic, liver and oral cancers, the expression of miR-203 is down, and its downregulation may be attributed to the methylation of the promoter region. At the molecular level, the transcription factor zinc-finger E-box binding homeobox1 (ZEB1) has been identified as the promotion of tumor cells. The important factor of epithelial-mesenchymal transition (EMT), which is partly attributable to the downregulation of some miRNA including miR-203, caused by EMT, suggests that miR-203 may play a very important role in the development of cancer, and its function is different from the type of tumor.
PVT1 is a lncRNA located in the 8q24 region of the human chromosome. The region is amplified in a variety of cancers, including breast cancer. In breast and ovarian cancers, the expression of silent PVT1 can inhibit cell proliferation and induce apoptosis. However, the regulatory mechanism of PVT1 itself has not yet been elucidated.
In this study, we first aim to elucidate the role of miR-203 in the development of breast cancer. We first detected the expression of miR-203 in breast cancer tissue samples, and found that the expression of miR-203 was up-regulated in most of the tumor samples compared with the corresponding para cancerous tissue. In the breast cancer cell lines, it is normal or immortalized. Compared with mammary cells, miR-203 was up-regulated in the luminal A type of breast cancer cells (low metastatic capacity), and down regulated in the basal-like type of breast cancer cells (high metastatic capacity). Next, we detected the methylation status of the miR-203 promoter region in the breast cancer cell line. We found that the basal-like milk was highly transferred in the breast cancer cell line. In adenocarcinoma cells, there is a certain degree of methylation in the promoter region of miR-203, but it does not exist in the low metastatic luminal A breast cancer cells. At the same time, the expression of miR-203 in the basal-like type of breast cancer cells treated with the demethylation drug 5-aza-dCyd can restore the expression of the breast cancer. Therefore, miR-203 is in the basal-like type of breast cancer. Down regulation of cell expression may be partly attributed to methylation of promoter region.
We then studied the function of miR-203 in breast cancer cells. We found that overexpression of miR-203 in basal-like type breast cancer cells significantly inhibits cell proliferation, and the mechanism of its inhibition involves cell cycle and apoptosis and cell specificity. At the same time, overexpression of miR-203 inhibits the above cells. At the molecular level, we found and confirmed that the transcription factor associated with EMT, SNAI2, is a target for miR-203, and in function, SNAI2 can save the inhibitory effect of miR-203 on the EMT process of basal-like cells.
The above results indicate that in malignant breast cancer cells, the expression of miR-203 is inhibited by the promoter methylation, and then up regulation of the expression of the transcription factor SNAI2, which ultimately promotes the growth and metastasis of tumor cells.
After that, we further examined the effect of the regulatory pathway of the miR-203 extremely target SNAI2 on the proliferation and migration of prostate cancer cells. Similar to other reports, we found that the expression of miR-203 in the prostate cancer cells was downregulated and that overexpression of miR-203 could inhibit the proliferation and migration of prostate cancer cells. Moreover, in prostate cancer, it was found that the prostate cancer cells could inhibit the proliferation and migration of prostate cancer cells. In cells, miR-203 can also target SNAI2, and over expression of SNAI2 can also restore the inhibition of miR-203 on prostate cancer cell migration.
At the same time, we found that the transcription factor KLF5, which is interested in our laboratory, can bind to the promoter region of miR-203 to regulate the expression of miR-203 positively. In addition, KLF5 can negatively regulate the expression of target SNAI2, which regulates miR-203. Over expression of SNAI2 can save the inhibitory effect of KLF5 on the migration of prostate cancer cells.
In summary, the findings in this paper suggest that miR-203 plays an important role in the development of breast and prostate cancers. As a tumor suppressor, miR-203 can inhibit the proliferation and migration of breast and prostate cancer cells by targeting SNAI2. At the same time, the miR-203-SNAI2 regulation pathway is mediated by KLF5 in the EMT process of breast cancer. The role played an important role.
On the other hand, we found that the genotype of a breast cancer sensitive nucleotide polymorphic loci (Single nucleotide polymorphism, SNP) rs13281615, located near the region of the PVTl chromosome, is significantly related to the expression of PVT1 in breast cancer. For SNP rs13281615, a breast cancer patient with a dangerous GG genotype is a tumor of the breast. The expression of PVT1 in the tissue is significantly higher than those with other genotypes and is higher than the normal para cancerous tissue with any genotypes. Meanwhile, SNPrs13281615 has frequent directional mutations (G to A) in the process of breast cancer (G to A). And this directional mutagenesis makes the original GG genotypes and tumor grade The correlation between proliferation and marker Ki67 is lost. Our findings provide a new idea for the regulatory mechanism of PVTl.
【學(xué)位授予單位】：南開大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.9;R737.25

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本文編號：2010534