ncRNA在乳腺癌和前列腺癌发生发展过程中的表达和作用研究

发布时间：2018-06-12 17:40

本文选题：miR-203 + SNAI2　；参考：《南开大学》2014年博士论文

【摘要】：乳腺癌和前列腺癌分别是女性和男性最常见的肿瘤疾病之一,具有很高的发病率,其发生、演进、浸润和转移是一个多基因(包括癌基因和抑癌基因)共同参与的渐进累积过程。其中的分子机制还需要更多的研究。在人类基因组序列中,编码序列不超过2%,其余大部分为非编码序列,其中可以产生稳定转录本的序列称之为非编码RNA (non-coding RNA, ncRNA)基因。非编码RNA (non coding RNA, ncRNA)种类广泛。在细胞中含量最多的ncRNA是tRNA和rRNA。除此之外,还包括snRNA,snoRNA,piRNA,lncRNA和microRNA等。目前研究显示,这些RNA的主要功能是参与mRNA的稳定和翻译水平的调节、参与蛋白质的运输、参与RNA的加工和修饰、影响染色质的结构等。一些ncRNA在恶性肿瘤的发生发展中发挥着重要的调控作用甚至起着关键的作用。 microRNA(miRNA)是长度为19-24碱基的内源性非编码RNA(non-coding RNA,ncRNA)。它通过碱基互补的方式结合到靶基因mRNA的3'非翻译区进而抑制其表达。研究发现miRNA序列极其保守而且在基于RNA水平上的基因调控过程中起到了关键作用。miRNA参与调节一系列重要的生物学过程,包括细胞增殖、分化、衰老、凋亡和细胞干性维持等等。miRNA的异常表达与包括癌症在内的许多病理学过程息息相关。miRNA与其靶基因所构成的调控网络在肿瘤的发生发展过程中扮演了十分重要的角色。长链非编码RNA(long ncRNA, lncRNA)是长度大于200碱基的非编码RNA。研究表明,lncRNA作为关键因子参与了一系列的细胞调控过程,例如基因转录调控,介导染色质重构及组蛋白修饰,干扰mRNA剪切和翻译,X染色体失活,基因组印记等等。大量由临床观察和实验研究所得的结果表明,lncRNA的异常表达会导致很多疾病的发生,这种机制在癌生物学中尤为重要。尽管到目前为止,与癌症相关的大多数lncRNA的作用机制还没得到准确阐明,但对部分lncRNA的研究表明,其所介导的染色质重构和组蛋白修饰等表观遗传学过程会影响肿瘤的发生和迁移。 miR-203是一个可以抑制表皮细胞干性的miRNA,它可以通过靶向细胞干性因子ΔNp63来抑制表皮细胞的增殖,进而促进其分化。在多种类型的癌症细胞中均已检测到miR-203的异常表达。在膀胱癌和卵巢癌中,癌变的发生伴随着miR-203的上调。然而在胰腺癌中,miR-203的过表达却预示着病人的不良预后。在气管癌、淋巴癌、肝癌和口腔癌中,miR-203表达下调,而且其下调可能归因于启动子区域的甲基化。在分子层面上,转录因子zinc-finger E-box binding homeobox1(ZEB1)已被鉴定为促进肿瘤细胞上皮间质转化(epithelial-mesenchymal transition, EMT)的重要因子,而这种促进作用部分归因于EMT所引起的一些miRNA包括miR-203的下调。上述结果表明miR-203在癌症的发生发展中可能起到十分重要的作用,而且其功能因肿瘤类型的不同而有所区别。 PVT1是位于人类染色体8q24区域的一个lncRNA,该区域在包括乳腺癌在内的多种癌症中均发生扩增。在乳腺癌和卵巢癌中,沉默PVT1的表达可以抑制细胞的增殖并诱导细胞凋亡。但是PVT1本身的调控机制尚未阐明。在本文的研究中,我们首先旨在阐明miR-203在乳腺癌发生发展中的作用。我们首先检测了miR-203在乳腺癌组织样本中的表达情况,发现与相应的癌旁组织相比,在大部分肿瘤样本中miR-203表达上调。在乳腺癌细胞系中,与正常或者永生化的乳腺细胞相比,miR-203在luminal A类型的乳腺癌细胞(转移能力低)中表达上调,而在basal-like类型的乳腺癌细胞(转移能力高)中表达下调。接下来,我们检测了在乳腺癌细胞系中,miR-203启动子区域的甲基化状态。我们发现在高转移的basal-like乳腺癌细胞中,miR-203的启动子区域存在一定程度的甲基化,而在低转移的luminal A乳腺癌细胞中则不存在。同时,用去甲基化药物5-aza-dCyd处理basal-like类型的乳腺癌细胞可以使miR-203的表达得到一定回复。因此,miR-203在basal-like类型的乳腺癌细胞中表达下调,可能部分归因于启动子区域的甲基化。我们接下来研究了miR-203在乳腺癌细胞中的功能。我们发现在basal-like类型的乳腺癌细胞中过表达miR-203明显抑制细胞增殖,而其抑制作用的机制包括影响细胞周期和促进凋亡,并且具有细胞特异性。同时,过表达miR-203可以抑制上述细胞的迁移和浸润。在分子层面上,我们发现并证实与EMT相关的转录因子SNAI2是miR-203的一个作用靶点。而且在功能上,SNAI2可以挽救miR-203对basal-like细胞EMT过程的抑制作用。以上结果表明在恶性乳腺癌细胞中,miR-203的表达因为启动子的甲基化而被抑制,进而上调转录因子SNAI2的表达,最终促进肿瘤细胞的生长和转移。之后,我们进一步检测了miR-203极其靶点SNAI2所构成的调节通路对前列腺癌细胞增殖和迁移的作用。与别人的报道类似,我们发现miR-203在前列腺癌细胞中表达下调,同时过表达miR-203可以抑制前列腺癌细胞的增殖与迁移。而且,在前列腺癌细胞中,miR-203同样可以靶向SNAI2,过表达SNAI2同样可以使miR-203对前列腺癌细胞迁移的抑制作用得到恢复。与此同时,我们发现我们实验室所感兴趣的转录因子KLF5可以结合到miR-203的启动子区域从而正向调节miR-203的表达。另外,KLF5可以负向调节miR-203的靶点SNAI2的表达。过表达SNAI2可以拯救KLF5对前列腺癌细胞迁移过程的抑制作用。综上所述,本文中的发现说明了miR-203在乳腺癌和前列腺癌的发生发展中起到重要的作用。作为一个抑癌因子,miR-203可以通过靶向SNAI2抑制乳腺癌和前列腺癌细胞的增殖和迁移。同时,miR-203-SNAI2这一调节路径在KLF5所介导的乳腺癌EMT过程中扮演了重要角色。另一方面,我们发现位于PVTl所在染色体区域附近的一个乳腺癌敏感的核苷酸多态性位点(Single nucleotide polymorphism,SNP) rs13281615的基因型与PVT1在乳腺癌中的表达变化显著相关。对于SNP rs13281615,带有危险性的GG基因型的乳腺癌患者其肿瘤组织中PVT1的表达量显著高于带有其它基因型的患者,并且高于带有任何基因型患者的正常癌旁组织。与此同时,SNPrs13281615的在乳腺癌的发生过程发生频繁的方向性突变(G→A)。而且这种方向性突变使得原本存在的GG基因型与肿瘤等级和增殖marker Ki67之间的相关性丧失。我们的发现为PVTl受调控的机制提供了新的思路。
[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.
【学位授予单位】：南开大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.9;R737.25

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