一种全新的长链非编码RNA IRAIN在乳腺癌中的印记表达及作用机制研究

发布时间：2018-05-26 10:14

本文选题：乳腺癌 + 肿瘤异质性　；参考：《吉林大学》2015年博士论文

【摘要】：研究背景：乳腺癌是一种严重威胁女性健康的恶性肿瘤，占女性全部恶性肿瘤的18%，已经成为35-65岁妇女第一位死亡原因。尽管个体化的靶向治疗和其他治疗手段的快速发展显著地延长了乳腺癌的生存期，但是我们仍然对乳腺癌治疗中靶向治疗耐药及三阴性乳腺癌无有效治疗药物等问题束手无策。这主要是由于乳腺癌本身具有高度异质性，发病因子、疾病演进、治疗反应和器官转移倾向等互异。要想彻底地战胜乳腺癌，需要更深入地研究乳腺癌发生、发展相关的信号通路，寻找新的个体化治疗靶点。鉴于PI3K/AKT/mTOR通路的上游成员例如ER、PR和HER2在乳腺癌的重要地位，同为上游成员的胰岛素样生长因子1受体（insulin-like growth factor1receptor，IGF-1R）引起研究者们的极大兴趣。IGF-1R是一种异四倍体的细胞质膜糖蛋白，为一种跨膜的酪氨酸蛋白受体。在结合胰岛素样生长因子配体后，通过自身磷酸化，激活PI3K以及MAPK信号通路，调控细胞的增殖、分化和衰老。该通路在乳腺癌中的高频率失调,并证实其介导的信号在乳腺癌的生长、演变、侵袭以及肿瘤血管生成、转移中均扮演着重要的角色。作为肿瘤靶向治疗最有前景的靶点之一，针对IGF-1R的靶向治疗药物也逐渐问世。早期的试验结果非常令人鼓舞，遗憾的是随后进行的试验中有很多结果是失败的，这说明我们对信号通路的认识并不充分,使得我们有必要更深层次地探讨其调控机制，并进一步寻找能够预测抗IGF-1R治疗效果的生物标志物，，进而选择潜在获益的正确的目标人群。新近研究发现，曾经被认为是基因转录“副产品”或“暗物质”的长链非编码RNA（long non-codingRNA,LncRNA）能够通过多种作用方式调控肿瘤细胞的基因表达，从而广泛参与肿瘤的发生及转移。LncRNA是指一类长度超过200个核苷酸的RNA分子的总称，已经成为当今分子生物学最热门的前沿研究领域之一。虽然在人类基因组中已经发现了一些LncRNA，但关于其对基因组的调控及具体机制尚不清楚，国际上亦没有任何关于LncRNA调控IGF-1R表达的报道。本课题客座导师胡继繁教授前期采用他在《Science》上发表的研究染色质空间结构方法，寻找参与IGF-1R启动子区域的调控因子时在IGF-1R基因区发现一条全新的lnRNA，命名为IRAIN。依据IGF-1R通路在乳腺癌中的重要地位及IRAIN处于IGF-1R基因区这一事实，我们推测IRAIN在乳腺癌的发生、发展中扮演一定的角色，然而IRAIN在乳腺癌领域的研究仍处于完全空白状态。研究目的：阐明一种全新的长链非编码RNA IRAIN在正常乳腺组织及各分子亚型乳腺癌组织的的表达水平、表达特点及其与IGF-1R基因表达的相关性；进一步明确调控IGF-1R信号通路的关键分子及其作用机制；揭示IRAIN与乳腺癌临床病理因素和预后的相关性。为乳腺癌寻找有预测、预后价值的生物标记物及个体化的乳腺癌治疗靶点提供全新的思路和实验依据。研究方法： 1.半定量逆转录聚合酶链反应（RT-PCR）确定IRAIN mRNA是否表达于乳腺癌组织中。2.应用实时荧光定量逆转录聚合酶链反应(qRT-PCR)方法检测IRAIN mRNA和IGF-1R mRNA在正常乳腺组织及不同分子亚型乳腺癌组织的表达水平、表达特点。3.应用链方向特异性RT-PCR（Strand-Specific RT-PCR,SSRT-PCR）绘制IRAIN转录方向。4.用聚合酶链反应-限制性酶切片段长度多态性分析法(PCR-RELP)及DNA测序方法分析乳腺细胞系及冰冻人乳腺癌组织中IRAIN SNP位点（rs8034564）杂合状态及等位基因表达特点，明确该基因是否呈单等位印记表达。5. DNA测序方法追踪印记亲本来源，分析正常组织与乳腺癌组织以及乳腺癌原发部位与转移部位印记的变化情况。6.结合重亚硫酸盐的测序法（Bisulfite Genomic Sequence，BSP）分析IRAIN启动子DNA甲基化状态，初探单等位印记表达及发生印记转换的机制。7.统计学分析IRAIN mRNA和IGF-1R mRNA表达与乳腺癌临床病理特征及预后之间的关系。研究结果： 1．半定量分析表明IRAIN在乳腺癌中广泛表达，表达水平各异。进一步应用实时荧光定量PCR方法对各分子分型乳腺癌及正常乳腺组织中IRAIN mRNA表达进行定量分析，实验结果显示，与正常乳腺组织相比,各分子亚型乳腺癌中IRAIN表达均降低,其中以三阴性乳腺癌降低最为明显，HER2阳性型乳腺癌IRAIN表达也明显低于正常乳腺组织，两者差异均有统计学意义（P＜0.05）。Luminal型同样低于正常乳腺组织，但无统计学意义。而对后三者做两两比较，无统计学意义（P＞0.05）。IGF-1R在预后最好的Luminal型中表达最高，与正常乳腺组织接近，而在预后差的TNB型和HER2+型表达明显低于正常乳腺组织及Luminal型，差异有统计学意义（P＜0.05）。对TNB型和HER2+型两者做比较，无统计学意义（P＞0.05）。 2.选取IRAIN基因上两个不同位置，在多个乳腺癌患者组织上应用SSRT-PCR方法确定IRAIN转录方向，其结果均是一致的，IRAIN转录方向与IGF-1R mRNA的方向相反，阐明IRAIN为IGF-1R的反义长链非编码RNA。 3．IRAIN在人正常乳腺组织中呈单等位基因表达，在乳腺癌组织中仍呈单等位基因表达。有趣的是，单等位基因呈不均衡表达，等位基因‘A’优势表达，18例SNP位点为杂合子的患者中，16例表达等位基因“A”，占89%，只有2例表达等位基因“G”占11%，目前还不清楚等位基因“A”的优势表达是否与这种非编码RNA的功能相关。 4．为了确定IRAIN亲本来源即是父源等位基因表达还是母源等位基因表达，我们进行了家系追踪筛查，结果表明IRAIN是父源等位基因表达、母源印记的基因。 5．首次发现乳腺癌组织中发生了异常的IRAIN等位基因印记转换（allele-switch）现象，即同一患者的正常组织与乳腺癌组织IRAIN呈现不同父母来源的单等位基因表达，也见到乳腺癌原发部位与转移部位印记转换现象。 6．BSP方法分析富含CpG岛的IRAIN基因启动子区DNA甲基化状态，发现乳腺癌中IRAIN启动子区域甲基化异常，提示DNA甲基化可能参与调控IRAIN基因单等位印记表达及发生印记转换。 7．IRAIN在正常乳腺组织中表达最高，在恶性程度高的三阴性乳腺癌及HER2阳性型乳腺癌显著低表达，提示IRAIN可能作为抑癌基因，调控肿瘤细胞生长。IRAIN表达高低与患者月经状态、肿瘤直径、淋巴结转移情况、组织学分级、脉管癌栓及增殖指数无显著相关性。研究结论： 1．本研究发现全新的位于IGF-1R基因区的长链非编码RNA IRAIN在乳腺癌中广泛表达，IRAIN在正常乳腺组织中表达最高，在恶性程度高的三阴性乳腺癌及HER2阳性型乳腺癌显著低表达，提示IRAIN可能作为抑癌基因，调控肿瘤细胞生长。 2．IRAIN转录方向与IGF-1R mRNA的方向相反，是IGF-1R基因的反义长链非编码RNA。 3．IRAIN在人正常乳腺组织及乳腺癌组织中呈单等位基因表达，单等位基因呈不均衡表达，优势表达其中一条等位基因。是父源等位基因表达、母源印记的基因，与其启动子区DNA甲基化异常有关，是人类基因组为数不多印记基因中新发现的成员之一。 4．首次发现乳腺癌中存在IRAIN等位基因印记转换（allele-switch）现象，这在乳腺癌和其他恶性肿瘤中国内外均未见报道，如果像我们推测的IRAIN作为抑癌基因，那么印记转换很可能是抑癌基因失活的一种全新的方式，正如印记丢失(LOI)在肿瘤中的地位一样，是一种标志性的事件。 5．IRAIN有望成为对乳腺癌有预测、预后价值的生物标记物及个体化的乳腺癌治疗靶点。 6．IRAIN的研究得到了权威机构的认可，在2014年末IRAIN被世界最权威的美国国立生物信息中心基因库（gene bank）验证通过，将它收录入库，Gene ID:104472848。
[Abstract]:Research background:
Breast cancer is a malignant tumor that is a serious threat to women's health, which accounts for 18% of all women's malignant tumors. It has become the first cause of death for women at the age of 35-65. Although the rapid development of individual targeted therapy and other treatment means significantly prolongs the survival period of breast cancer, it is still a target treatment for breast cancer treatment. Drug resistance and three negative breast cancer have no effective drug treatment. This is mainly due to the high heterogeneity of breast cancer itself, the pathogenesis, the evolution of the disease, the treatment response and the tendency of the organ metastasis. To overcome breast cancer thoroughly, it is necessary to study breast cancer in depth, develop related signaling pathways, and seek for the development of related signaling pathways. New individualized therapeutic targets. In view of the importance of the upstream members of the PI3K/AKT/mTOR pathway such as ER, PR and HER2 in breast cancer, and the upstream members of the insulin like growth factor 1 receptor (insulin-like growth factor1receptor, IGF-1R), the great interest of the researchers is that.IGF-1R is an isotetraploid cytoplasmic membrane glycoprotein. A transmembrane tyrosine receptor. After binding to the IGF ligand, the PI3K and MAPK signaling pathways are activated by autophosphorylation to regulate cell proliferation, differentiation and senescence. The high frequency of this pathway in breast cancer is high, and its mediated signal is demonstrated in the growth, evolution, invasion, and angiogenesis of breast cancer. As one of the most promising targets for tumor targeting therapy, the targeting therapy for IGF-1R is also coming out. Early results were very encouraging. Unfortunately, many results were failed in subsequent trials, which indicated that we were not fully aware of the signaling pathways. It makes it necessary for us to further explore its regulatory mechanism, and to further explore biomarkers that can predict the effectiveness of anti IGF-1R therapy, and then select the right target population with potential benefits. Recently, the new study found that the long chain non coded RNA (long non-codingRNA), which was once considered as a "by-product" or "dark matter", was considered to be a gene transcription. LncRNA) can regulate the gene expression of tumor cells through a variety of ways of action, so it is widely involved in the occurrence and metastasis of tumor..LncRNA is the general name of a class of RNA molecules with a length of more than 200 nucleotides. It has become one of the most popular frontiers in molecular biology. Although a number of human genome has been found in the human genome, it has been found in the human genome. Some LncRNA, but it is not clear about its regulation of the genome and its specific mechanism, and there are no reports on the LncRNA regulation of IGF-1R expression in the world. Professor Hu Jifan, the guest tutor of the subject, used his previous study on chromatin spatial structure, published on , to find the regulatory factors involved in the IGF-1R promoter region in I The GF-1R gene region found a new lnRNA, named as IRAIN. based on the IGF-1R pathway in breast cancer and the fact that IRAIN is in the IGF-1R gene region. We speculate that IRAIN plays a role in the development of breast cancer and the development of IRAIN in the field of breast cancer is still completely blank.
The purpose of the study is:
To clarify the expression level, expression of a new long chain non coding RNA IRAIN in normal breast tissue and the subtype of subtype breast cancer, and the correlation with IGF-1R gene expression; further clarify the key molecules of the IGF-1R signaling pathway and its mechanism of action, and reveal the clinicopathological factors and prognosis of IRAIN and breast cancer. It provides a new idea and experimental basis for breast cancer to find biomarkers with prognostic value and individualized target for breast cancer treatment.
Research methods:
1. semi quantitative reverse transcriptase polymerase chain reaction (RT-PCR) determination of the expression of IRAIN mRNA in breast cancer tissues by.2. application real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) method to detect the expression of IRAIN mRNA and IGF-1R mRNA in normal mammary tissues and different molecular subtypes of breast cancer tissues, and to express the direction of.3. application chain. Specific RT-PCR (Strand-Specific RT-PCR, SSRT-PCR) plotted IRAIN transcriptional direction.4. using polymerase chain reaction restriction fragment length polymorphism analysis (PCR-RELP) and DNA sequencing method to analyze the heterozygous state and allele expression characteristics of IRAIN SNP loci (rs8034564) in breast cell lines and frozen human breast cancer tissues. The.5. DNA sequencing method was used to trace the source of the imprinted parent, and to analyze the changes of the normal tissue and breast cancer tissue, the original site of the breast cancer and the imprint of the metastatic site..6. binding heavy sulfite sequencing method (Bisulfite Genomic Sequence, BSP) was used to analyze the DNA methylation status of IRAIN promoter, and the single allele was first explored. The mechanism of imprinting expression and imprinting transformation.7. statistical analysis of the relationship between IRAIN mRNA and IGF-1R mRNA expression and clinicopathological features and prognosis of breast cancer.
The results of the study:
1. semi quantitative analysis showed that IRAIN was widely expressed in breast cancer and the expression level was different. The quantitative analysis of IRAIN mRNA expression in all molecular types of breast cancer and normal breast tissue was analyzed by real-time fluorescence quantitative PCR. The results showed that the expression of IRAIN in the subtypes of breast cancer was lower than that of normal mammary tissues. The expression of three negative breast cancer was the most obvious, and the expression of IRAIN in HER2 positive breast cancer was significantly lower than that of normal breast tissue. The difference was statistically significant (P < 0.05).Luminal type was also lower than that of normal breast tissue, but no statistical significance was found. There was no statistical significance (P > 0.05).IGF-1R at the most prognosis (P > 0.05).IGF-1R in the prognosis. The highest expression in the good Luminal type was close to the normal breast tissue, but the expression of TNB and HER2+ in the poor prognosis was significantly lower than that of the normal breast tissue and the Luminal type (P < 0.05). There was no statistically significant difference between the TNB and the HER2+ type (P > 0.05).
2. select the two different positions of IRAIN gene and determine the IRAIN transcriptional direction by SSRT-PCR method in the tissues of multiple breast cancer patients. The results are all consistent. The IRAIN direction is opposite to the direction of IGF-1R mRNA, and the antisense long chain uncoded RNA. IRAIN is IGF-1R.
The expression of single allele in human normal mammary tissues and single allele are still expressed in breast cancer tissues. It is interesting that the single allele is unevenly expressed and the allele 'A' superiority is expressed. In 18 SNP loci as heterozygotes, 16 alleles are "A", accounting for 89%, and only 2 expression alleles "G" "11%", it is unclear whether the dominant expression of allele "A" is related to the function of this non coding RNA.
4. in order to determine whether the parent source of IRAIN is the parent allele or the mother source allele, we carried out a family tracking screening. The results showed that IRAIN was the parent allele expression and the parent imprinted gene.
5. the abnormal IRAIN allelic imprint conversion (allele-switch) phenomenon was found in the breast cancer tissue for the first time. That is, the normal tissue of the same patient and the breast cancer tissue IRAIN present the single allele expression of different parent sources, and also the original location of the breast cancer and the transfer part of the breast cancer.
The 6.BSP method analyzed the DNA methylation status of the IRAIN gene promoter region of the rich CpG Island, and found the abnormal methylation in the IRAIN promoter region of the breast cancer, suggesting that DNA methylation may be involved in the regulation of the single allelic expression of the IRAIN gene and the alteration of the imprinting of the IRAIN gene.
The expression of 7.IRAIN is the highest in normal breast tissue, which is significantly lower in three negative breast cancer and HER2 positive breast cancer, suggesting that IRAIN may be used as a tumor suppressor gene to regulate the level of.IRAIN expression in tumor cell growth and the patient's menstrual state, tumor diameter, lymph node metastasis, histological grading, vascular tumor thrombus and proliferation. There was no significant correlation between the index and the index.
The conclusions are as follows:
1. we found that the new long chain non coded RNA IRAIN located in the IGF-1R gene region is widely expressed in breast cancer. IRAIN is expressed in the normal breast tissue, and the high level of malignant breast cancer and HER2 positive breast cancer are significantly lower, suggesting that IRAIN may act as a tumor suppressor gene and regulate the growth of tumor cells.
The direction of 2.IRAIN transcription is opposite to that of IGF-1R mRNA, which is an antisense long chain non coding RNA. of IGF-1R gene.
3.IRAIN is one of the single alleles expressed in human normal breast tissue and breast cancer tissue. The single allele is unevenly expressed, and one of the alleles is expressed. It is the expression of the allele of the parent source. The gene of the parent imprint is related to the abnormal DNA methylation in the promoter region. It is a new discovery in the few imprinted genes of the human genome. One of the members.
4. the IRAIN allele imprinted conversion (allele-switch) phenomenon is found in breast cancer for the first time, which is not reported in both breast and other malignant tumors. If we speculate that IRAIN is a tumor suppressor gene, imprint conversion is likely to be a new way of inactivation of the tumor suppressor gene, as the loss of LOI (LOI) is swollen. The same position in the tumor is a landmark event.
5.IRAIN is expected to become a biomarker for predicting and prognostic value of breast cancer and an individualized target for breast cancer therapy.
6.IRAIN's research was approved by authoritative institutions, and at the end of 2014, IRAIN was approved by the world's most authoritative US National bioinformation center gene library (gene bank) to include it in the library, Gene ID:104472848..
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

【共引文献】