全转录组分析△S2 LF-PRLR对人类乳腺癌细胞（MCF-7）基因表达的影响

发布时间：2019-06-29 19:39

【摘要】：催乳素受体(prolactin receptor,PRLR)是I型细胞因子受体超家族的成员之一,为单次跨膜受体。PRLR在介导催乳素(prolactin,PRL)的生物学效应中起着关键的作用。在PRL的作用下,PRLR发生二聚化,从而启动细胞信号转导并实现其生物学效应。△S2 LF-PRLR为Tan等人于人类乳腺癌细胞克隆并首次报道的一种PRLR的变体。其主要结构特征是:受体的膜外区S2亚结构域缺失(由100个氨基酸组成,另一个亚结构域为S1)。已有实验表明,△S2 LF-PRLR在没有PRL存在时,亦能发生二聚化,即所谓的自有二聚化(constitutive dimerization),并产生生物学效应如促进细胞增殖等。提示△S2 LF-PRLR在人类乳腺癌的发生中可能具有重要的分子病理意义。PRLR激活后,通过启动其下游的JAK2-STAT5细胞信号转导途径,进而导致STAT5的磷酸化、二聚化及核转移,从而结合在其靶基因的启动子区域而调节相应基因的表达。△S2 LF-PRLR由于其细胞外的S2亚结构的丢失,我们推测这一缺失改变了PRLR的分子特性,从而影响细胞转导途径进而改变其靶基因的表达谱及表达水平,最终改变乳腺癌细胞的生物学行为,例如细胞的过度增殖等。本研究中,我们采用病毒介导的基因转移方法,将携带LF-PRLR(LF-PRLR组)或△S2 LF-PRLR(△S2 LF-PRLR组)c DNA的腺病毒转染人类乳腺癌细胞(MCF-7),对照组(Con)则以不携带任何外源基因的空病毒转染。细胞继续培养48小时后,提取细胞总RNA,然后进行RNA测序(RNA-Seq),测序数据上传至加州大学桑塔克鲁斯分校基因组浏览器网站(genome.ucsc.edu)进行比对。结果表明:(1)△S2 LF-PRLR过表达后,表达变化较大(即Con、LF-PRPR及△S2LF-PRLR三组细胞之间差异较大)的基因,主要集中在染色体11,12,14,16,17,19、20,而在染色体4、13、18,21差异则相对较小;(2)受其影响的基因不但有编码基因,还包括非编码基因。Con、LF-PRPR及△S2 LF-PRLR三组细胞测得的表达基因数(减去RPKM值最低的5%后)分别为19648(编码基因17594非编码基因2054)、20247(编码基因18093,非编码基因2154)及19500(编码基因17472,非编码基因2028)。(3)全转录组中有192个基因仅在△S2 LF-PRLR MCF-7细胞中表达(其中编码基因有118个,非编码基因有74个);483个基因在△S2 LF-PRLR MCF-7细胞中不表达,而在LF-PRLR及Con组表达(其中编码基因有362个,非编码基因有121个);18924个基因在三组细胞中均有表达(其中编码基因有17053个,非编码基因有1871个);在三组中都有表达但在△S2LF-PRLR组高表达的基因有3个(全为非编码基因);在三组中都有表达但是在△S2 LF-PRLR组低表达的基因有1个(亦为非编码基因);(4)△S2 LF-PRLR过表达引起肿瘤相关基因的表达变化。三组样品的转录组中有30个肿瘤相关基因有表达差异,有27个肿瘤相关基因仅在△S2 LF-PRLR组表达,而在Con组及LF-PRLR组不表达。(5)△S2 LF-PRLR过表达改变部分凋亡相关基因的表达水平。三组中有11个凋亡相关基因表达有差异,其中9个凋亡相关基因仅在△S2LF-PRLR表达,有2个抗凋亡基因在Con及LF-PRLR组均表达,但在△S2 LF-PRLR组不表达。(6)部分微小RNA(micro RNA)亦是△S2 LF-PRLR作用的靶点。三组样品的转录组中有15个Micro RNAs有表达差异,有8个Micro RNAs仅在△S2LF-PRLR组表达,4个Micro RNAs在△S2 LF-PRLR组不表达而在其他2组有表达。(7)△S2 LF-PRLR具有调节sno RNA表达的功能,三组中共有19个sno RNA基因表达有差异,其中13个是box C/D sno RNAs、6个是box H/ACA sno RNAs。其中有10个sno RNA基因在△S2 LF-PRLR不表达,而另外2组有表达,7个sno RNA基因仅在△S2 LF-PRLR表达,其他2组不表达。根据实验结果,本研究主要结论如次:(1)△S2 LF-PRLR过表达,能够广泛影响人类乳腺癌细胞(MCF-7)基因的表达。受其影响的包括编码基因及非编码基因。(2)对人类乳腺癌细胞(MCF)基因转录的影响,△S2 LF-PRLR显然不同于全长的受体LF-PRLR。(3)△S2 LF-PRLR通过调控肿瘤相关基因的表达影响乳腺癌细胞的生物学行为如细胞增殖等。(4)△S2 LF-PRLR下调部分凋亡基因的表达,从而促进乳腺癌细胞增殖。(5)△S2 LF-PRLR影响微小RNA(Micro RNAs)的表达。(6)△S2 LF-PRLR调控sno RNA表达。本研究对于理解△S2 PRLR在人类乳腺癌的分子机理具有重要意义,为进一步探讨△S2 PRLR的作用奠定了基础。
[Abstract]:Prolactin receptor (PRLR) is one of the members of the type I cytokine receptor superfamily and is a single transmembrane receptor. PRLR plays a key role in the biological effects of prolactin (PRL). Under the effect of PRL, PRLR is dimerized, thus initiating cell signal transduction and achieving its biological effect. The preS2 LF-PRLR is a variant of a PRLR that is cloned and first reported by Tan et al. in human breast cancer cells. The main structural feature is that the subdomain of the extracellular domain S2 of the receptor is deleted (consisting of 100 amino acids and the other subdomain is S1). It has been shown that, in the absence of PRL, the TS2 LF-PRLR can also dimerize, that is, the so-called self-dimerization, and to produce biological effects such as promoting cell proliferation and the like. It is suggested that SS2 LF-PRLR may have important molecular and pathological significance in the occurrence of human breast cancer. After the activation of the PRLR, the expression of the corresponding gene is adjusted by activating the JAK2-STAT5 cell signal transduction pathway downstream of it, which in turn results in phosphorylation, dimerization and nuclear transfer of the STAT5, thereby binding to the promoter region of its target gene. If the S2 LF-PRLR is lost due to the S2 substructure outside its cell, we speculate that this deletion has changed the molecular nature of the PRLR, thus affecting the cell transduction pathway and then changing the expression profile and the expression level of the target gene, and finally changing the biological behavior of the breast cancer cell, For example, excessive proliferation of cells, and the like. In this study, we used the virus-mediated gene transfer method to transfect the human breast cancer cell (MCF-7) with the adenovirus carrying LF-PRLR (LF-PRLR group) or the wild-type S2 LF-PRLR (FS2 LF-PRLR group) c DNA, and the control group (Con) was transfected with an empty virus that did not carry any foreign gene. After the cells continue to be cultured for 48 hours, the total RNA of the cells is extracted and then the RNA-Seq is performed and the sequencing data is uploaded to the genome browser website (genome.ucsc.edu) of the University of California, Santa Cruz. The results showed that: (1) After the overexpression of VS2 LF-PRLR, the expression of the gene was larger (that is, the difference between the three groups of Con, LF-PRPR and FS2LF-PRLR was large), mainly in the chromosome 11,12,14,16,17,19,20, while the difference in the chromosome 4,13,18,21 was relatively small; (2) the gene affected by it not only had the coding gene, The non-coding gene is also included. The number of expression genes (5% of the lowest RPKM value) of the three groups of Con, LF-PRPR and FES2 LF-PRLR were 19648 (coding gene 17594 non-coding gene 2054),20247 (coding gene 18093, non-coding gene 2154) and 19500 (encoding gene 17472, non-coding gene 2028), respectively. (3)192 genes in the whole transcriptome were expressed only in the FES2 LF-PRLR MCF-7 cells (in which there were 118 of the coding genes and 74 non-coding genes), and the 483 genes were not expressed in the FS2 LF-PRLR MCF-7 cells, while in the LF-PRLR and Con group (where the coding gene had 362 and the non-coding gene was 121); The 18924 genes were expressed in three groups (1753 of the coding genes and 1871 non-coding genes), and there were 3 (all non-coding genes) of the genes expressed in the SS2LF-PRLR group. In the three groups, there were 1 (also non-coding gene) of the low-expression genes in the FS2 LF-PRLR group, and (4) the overexpressing of the FS2 LF-PRLR resulted in the change of the expression of the tumor-related genes. There were 30 tumor-related genes in the transcriptome of three groups, and 27 tumor-related genes were expressed only in the QS2 LF-PRLR group, but not in the Con group and the LF-PRLR group. (5) The expression level of the apoptosis-related gene was altered by the overexpression of VS2 LF-PRLR. There were differences in the expression of 11 apoptosis-related genes in the three groups, of which 9 of the apoptotic genes were expressed in both the Con and LF-PRLR groups, but the expression of the two anti-apoptotic genes in both the Con and LF-PRLR groups was not expressed. (6) Partial microRNA (micro-RNA) is also the target of the action of TS2 LF-PRLR. There were 15 microRNAs in the transcriptome of three groups, and 8 Micro RNAs were expressed only in the FS2LF-PRLR group and the 4 Micro RNAs were not expressed in the other two groups. (7) The PS2LF-PRLR has the function of regulating the expression of sno RNA, and there are 19 difference in the expression of 19 sno RNA in the three groups,13 of which are box C/ D sno RNAs and 6 are box H/ ACA sno RNAs. Of these,10 of the sno RNA genes were not expressed in the other two groups, while the other two groups were not expressed in the other two groups, while the other two groups were not expressed. According to the results of the experiment, the main conclusions of this study are as follows: (1) The overexpression of VS2 LF-PRLR can affect the expression of human breast cancer cells (MCF-7). It is affected by the coding gene and the non-coding gene. (2) The effect of the transcription of the human breast cancer cell (MCF) gene is clearly different from that of the full-length receptor LF-PRLR. (3) The expression of TS2 LF-PRLR in the control of tumor-related genes affects the biological behavior of breast cancer cells, such as cell proliferation, and so on. (4) The expression of the apoptosis gene is down-regulated by the anti-S2 LF-PRLR, thereby promoting the proliferation of the breast cancer cells. (5) The expression of the micro-RNA (Micro-RNAs) is affected by the FS2 LF-PRLR. (6) The expression of sno RNA was regulated by the control of SS2 LF-PRLR. This study is of great significance to understand the molecular mechanism of TS2 PRLR in human breast cancer.
【学位授予单位】：吉首大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9;Q78

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