【摘要】：催乳素受體(prolactin receptor,PRLR)是I型細(xì)胞因子受體超家族的成員之一,為單次跨膜受體。PRLR在介導(dǎo)催乳素(prolactin,PRL)的生物學(xué)效應(yīng)中起著關(guān)鍵的作用。在PRL的作用下,PRLR發(fā)生二聚化,從而啟動細(xì)胞信號轉(zhuǎn)導(dǎo)并實(shí)現(xiàn)其生物學(xué)效應(yīng)。△S2 LF-PRLR為Tan等人于人類乳腺癌細(xì)胞克隆并首次報道的一種PRLR的變體。其主要結(jié)構(gòu)特征是:受體的膜外區(qū)S2亞結(jié)構(gòu)域缺失(由100個氨基酸組成,另一個亞結(jié)構(gòu)域?yàn)镾1)。已有實(shí)驗(yàn)表明,△S2 LF-PRLR在沒有PRL存在時,亦能發(fā)生二聚化,即所謂的自有二聚化(constitutive dimerization),并產(chǎn)生生物學(xué)效應(yīng)如促進(jìn)細(xì)胞增殖等。提示△S2 LF-PRLR在人類乳腺癌的發(fā)生中可能具有重要的分子病理意義。PRLR激活后,通過啟動其下游的JAK2-STAT5細(xì)胞信號轉(zhuǎn)導(dǎo)途徑,進(jìn)而導(dǎo)致STAT5的磷酸化、二聚化及核轉(zhuǎn)移,從而結(jié)合在其靶基因的啟動子區(qū)域而調(diào)節(jié)相應(yīng)基因的表達(dá)。△S2 LF-PRLR由于其細(xì)胞外的S2亞結(jié)構(gòu)的丟失,我們推測這一缺失改變了PRLR的分子特性,從而影響細(xì)胞轉(zhuǎn)導(dǎo)途徑進(jìn)而改變其靶基因的表達(dá)譜及表達(dá)水平,最終改變?nèi)橄侔┘?xì)胞的生物學(xué)行為,例如細(xì)胞的過度增殖等。本研究中,我們采用病毒介導(dǎo)的基因轉(zhuǎn)移方法,將攜帶LF-PRLR(LF-PRLR組)或△S2 LF-PRLR(△S2 LF-PRLR組)c DNA的腺病毒轉(zhuǎn)染人類乳腺癌細(xì)胞(MCF-7),對照組(Con)則以不攜帶任何外源基因的空病毒轉(zhuǎn)染。細(xì)胞繼續(xù)培養(yǎng)48小時后,提取細(xì)胞總RNA,然后進(jìn)行RNA測序(RNA-Seq),測序數(shù)據(jù)上傳至加州大學(xué)桑塔克魯斯分�；蚪M瀏覽器網(wǎng)站(genome.ucsc.edu)進(jìn)行比對。結(jié)果表明:(1)△S2 LF-PRLR過表達(dá)后,表達(dá)變化較大(即Con、LF-PRPR及△S2LF-PRLR三組細(xì)胞之間差異較大)的基因,主要集中在染色體11,12,14,16,17,19、20,而在染色體4、13、18,21差異則相對較小;(2)受其影響的基因不但有編碼基因,還包括非編碼基因。Con、LF-PRPR及△S2 LF-PRLR三組細(xì)胞測得的表達(dá)基因數(shù)(減去RPKM值最低的5%后)分別為19648(編碼基因17594非編碼基因2054)、20247(編碼基因18093,非編碼基因2154)及19500(編碼基因17472,非編碼基因2028)。(3)全轉(zhuǎn)錄組中有192個基因僅在△S2 LF-PRLR MCF-7細(xì)胞中表達(dá)(其中編碼基因有118個,非編碼基因有74個);483個基因在△S2 LF-PRLR MCF-7細(xì)胞中不表達(dá),而在LF-PRLR及Con組表達(dá)(其中編碼基因有362個,非編碼基因有121個);18924個基因在三組細(xì)胞中均有表達(dá)(其中編碼基因有17053個,非編碼基因有1871個);在三組中都有表達(dá)但在△S2LF-PRLR組高表達(dá)的基因有3個(全為非編碼基因);在三組中都有表達(dá)但是在△S2 LF-PRLR組低表達(dá)的基因有1個(亦為非編碼基因);(4)△S2 LF-PRLR過表達(dá)引起腫瘤相關(guān)基因的表達(dá)變化。三組樣品的轉(zhuǎn)錄組中有30個腫瘤相關(guān)基因有表達(dá)差異,有27個腫瘤相關(guān)基因僅在△S2 LF-PRLR組表達(dá),而在Con組及LF-PRLR組不表達(dá)。(5)△S2 LF-PRLR過表達(dá)改變部分凋亡相關(guān)基因的表達(dá)水平。三組中有11個凋亡相關(guān)基因表達(dá)有差異,其中9個凋亡相關(guān)基因僅在△S2LF-PRLR表達(dá),有2個抗凋亡基因在Con及LF-PRLR組均表達(dá),但在△S2 LF-PRLR組不表達(dá)。(6)部分微小RNA(micro RNA)亦是△S2 LF-PRLR作用的靶點(diǎn)。三組樣品的轉(zhuǎn)錄組中有15個Micro RNAs有表達(dá)差異,有8個Micro RNAs僅在△S2LF-PRLR組表達(dá),4個Micro RNAs在△S2 LF-PRLR組不表達(dá)而在其他2組有表達(dá)。(7)△S2 LF-PRLR具有調(diào)節(jié)sno RNA表達(dá)的功能,三組中共有19個sno RNA基因表達(dá)有差異,其中13個是box C/D sno RNAs、6個是box H/ACA sno RNAs。其中有10個sno RNA基因在△S2 LF-PRLR不表達(dá),而另外2組有表達(dá),7個sno RNA基因僅在△S2 LF-PRLR表達(dá),其他2組不表達(dá)。根據(jù)實(shí)驗(yàn)結(jié)果,本研究主要結(jié)論如次:(1)△S2 LF-PRLR過表達(dá),能夠廣泛影響人類乳腺癌細(xì)胞(MCF-7)基因的表達(dá)。受其影響的包括編碼基因及非編碼基因。(2)對人類乳腺癌細(xì)胞(MCF)基因轉(zhuǎn)錄的影響,△S2 LF-PRLR顯然不同于全長的受體LF-PRLR。(3)△S2 LF-PRLR通過調(diào)控腫瘤相關(guān)基因的表達(dá)影響乳腺癌細(xì)胞的生物學(xué)行為如細(xì)胞增殖等。(4)△S2 LF-PRLR下調(diào)部分凋亡基因的表達(dá),從而促進(jìn)乳腺癌細(xì)胞增殖。(5)△S2 LF-PRLR影響微小RNA(Micro RNAs)的表達(dá)。(6)△S2 LF-PRLR調(diào)控sno RNA表達(dá)。本研究對于理解△S2 PRLR在人類乳腺癌的分子機(jī)理具有重要意義,為進(jìn)一步探討△S2 PRLR的作用奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉首大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R737.9;Q78

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