乳腺癌中RNPC1通过mRNA稳定性调节孕激素受体功能的机制研究

发布时间：2018-05-02 00:28

本文选题：RNA结合蛋白 + RNPC1　；参考：《南京医科大学》2016年硕士论文

【摘要】：目的：研究乳腺癌中RNA结合蛋白RNPC1通过调节mRNA稳定性进而调节PR的具体机制,并研究RNPC1对于PR功能的影响。材料与方法：在乳腺癌组织中运用免疫组化方法检测RNPC1和PR的表达情况,并在乳腺癌细胞中用免疫荧光检测RNPC1和PR的表达定位。通过慢病毒转染,在乳腺癌细胞株MCF7, BT474, SUM1315, MDA-MB-231中构建RNPC1和PR的过表达及干扰的细胞模型。实时荧光定量PCR (qRT-PCR)及Western-blot分别用来检测相关PR及其下游靶基因mRNA和蛋白的表达水平。同时鉴于ER对于PR也有调节作用,首先通过转染ER干扰慢病毒,进行ER敲除,进而进行RNPC1过表达及干扰细胞系构建,并检测PR的mRNA和蛋白的表达水平。放线菌素D用以处理RNPC1(RNPC1)过表达及其对照(NC)细胞株,RNPC1干扰(shRNPC1)及其相应对照(SCR)细胞株,并分别于不同时间点用qRT-PCR检测PR的mRNA水平。RNA免疫共沉淀(RIP)用以验证RNPC1蛋白与PR mRNA的结合反应。RNA电泳迁移率(REMSA)实验用以检测RNPC1蛋白与PR具体结合位点。双荧光素酶实验用以进一步证实RNPC1蛋白与PR结合的位点并验证其功能。CCK8和平板克隆实验用以检测RNPC1过表达及敲除后的细胞株对孕激素处理的功能变化。通过RNPC1及ER的双敲除细胞株中PR的检测以确定ER在RNPC1调节PR过程中的作用。通过在NOD-SCID小鼠体内构建人源性乳腺移植模型,提高乳腺癌细胞成瘤效率,并通过皮下包埋孕激素缓释片检测过表达RNPC1对于孕激素处理的体内成瘤效果的影响。结果：在乳腺癌组织中RNPC1与PR表达具有正相关性(P0.05)。RNPC1在乳腺癌细胞中的定位是在细胞核及细胞质中,PR主要定位于细胞核。乳腺癌细胞MCF-7级BT474细胞中,过表达RNPC1, PR表达上调,敲除RNPC1, PR表达降低,同时检测PR下游靶基因Wnt4和β-catemin均受到RNPC1调节。而在敲除ER的细胞系中,RNPC1对于PR仍有调节作用,说明RNPC1对于PR的调节是独立的过程。过表达RNPC1后,PR的mRNA稳定性增强,而敲除RNPC1则降低PR的mRNA稳定性。RNPC1可以和PR的3’非翻译区的AU-富含元件结合进而增强其稳定性。功能实验也表明,RNPC1可以增强通过PR而发挥促进增殖作用的孕激素的功能。结论：乳腺癌中RNPC1与PR具有相关性,过表达RNPC1可以促进PR表达,干扰RNPC1降低PR表达。RNPC1可以和PR的mRNA 3'非翻译区结合,增强mRNA稳定性。RNPC1可以增强通过PR发挥作用的孕激素的促增殖作用。
[Abstract]:Aim: to study the mechanism of RNA binding protein RNPC1 regulating PR by regulating the stability of mRNA in breast cancer and the effect of RNPC1 on PR function. Materials and methods: immunohistochemical method was used to detect the expression of RNPC1 and PR in breast cancer, and immunofluorescence was used to detect the expression of RNPC1 and PR in breast cancer cells. The overexpression and interference of RNPC1 and PR in breast cancer cell lines MCF7, BT474, SUM1315, MDA-MB-231 were established by lentivirus transfection. Real-time quantitative PCR qRT-PCR and Western-blot were used to detect the expression level of mRNA and protein in PR and its downstream target genes, respectively. At the same time, ER can also regulate PR. Firstly, ER interferes with lentivirus, carries out ER knockout, then overexpresses RNPC1 and interferes cell line construction, and detects the expression level of mRNA and protein of PR. Actinomycin D was used to treat the overexpression of RNPC1 / RNPC1 and its control cell line (RNPC1), which interfered with shRNPC1) and its corresponding control cell line (SCR). At different time points, qRT-PCR was used to detect the mRNA level of PR. The RIPs were used to verify the binding reaction between RNPC1 protein and PR mRNA. The RMSAs were used to detect the specific binding sites of RNPC1 protein to PR. Double luciferase assay was used to further confirm the binding site of RNPC1 protein to PR, and to verify its function. CCK8 and plate cloning assay were used to detect the changes of progesterone treatment in RNPC1 overexpression and knockout cell lines. The role of ER in the regulation of PR by RNPC1 was determined by the detection of PR in RNPC1 and ER double knockout cell lines. Human mammary gland transplantation model was established in NOD-SCID mice to improve the tumorigenic efficiency of breast cancer cells. The effect of overexpression of RNPC1 on the tumorigenesis of breast cancer cells treated with progesterone was detected by subcutaneous embedding of progesterone sustained-release tablets. Results: there was a positive correlation between the expression of RNPC1 and PR in breast cancer cells. The localization of RNPC1 in breast cancer cells was mainly located in the nucleus and cytoplasm. Overexpression of RNPC1, up-regulation of PR expression, down-regulation of knockout RNPC1 and decrease of PR expression were observed in BT474 cells of MCF-7 grade. Wnt4 and 尾 -catemin, the downstream target genes of PR, were all regulated by RNPC1. However, RNPC1 still regulates PR in the cell line that knockout ER, indicating that the regulation of PR by RNPC1 is an independent process. The mRNA stability of PR was enhanced after overexpression of RNPC1, while knockout RNPC1 decreased the mRNA stability of PR. RNPC1 could bind to AU-rich elements in 3'untranslated region of PR and enhance its stability. Functional experiments also showed that RNPC1 could enhance the progesterone function which promoted proliferation through PR. Conclusion: there is a correlation between RNPC1 and PR in breast cancer. Overexpression of RNPC1 can promote the expression of PR and interfere with the decrease of PR expression. RNPC1 can bind to the mRNA 3 'untranslated region of PR. Enhancing the stability of mRNA. RNPC1 could enhance the proliferation of progesterone acting through PR.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9

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