配子生成素结合蛋白（GGNBP2）在乳腺癌增殖和转移中的调控作用及机制研究

发布时间：2018-08-31 15:36

【摘要】：背景:研究人员利用软件分析得出在人类染色体17q12-q23区域内约编码693个基因,其中在BRCA1基因上游存在一个大小约为6.3MB的基因,称为配子生成素结合蛋白(gametogenetin binding protein 2,GGNBP2)。现阶段对于GGNBP2在恶性肿瘤中所发挥的作用以及其潜在的作用机制的研究仍处于起步阶段。本课题组拟通过临床病例资料、体内、体外实验等不同层面研究阐明GGNBP2在乳腺癌细胞增殖及转移中的作用和机制,了解GGNBP2基因在肿瘤发生发展中的功能,为乳腺癌的治疗研究提供新的靶点。方法:1.利用多组织阵列分析的方法分别检测人九种正常组织中GGNBP2的差异表达情况,同时比较正常乳腺腺体和乳腺癌组织中GGNBP2不同表达情况,且比较了转移灶与未转移灶乳腺癌组织中GGNBP2的表达情况。2.利用RT-PCR实验检测Wnt1转基因小鼠中正常乳腺组织和乳腺癌组织标本中GGNBP2的差异表达情况。3.在不同ERα状态细胞中,构建过表达GGNBP2细胞系,绘制细胞的生长曲线,并利用雌二醇刺激不同GGNBP2水平细胞,检测其对雌二醇刺激的敏感性;并利用三维培养,比较母代T47D细胞和T47D-GGNBP2细胞的细胞团形态和数量改变。4.采用划痕实验、Transwell实验、软琼脂克隆实验,分别检测T47D-His C细胞和T47D-GGNBP2细胞的不同移动能力、侵袭能力以及体外成瘤能力,观察过表达GGNBP2对乳腺癌细胞生物学行为的影响。5.利用多组氨酸Pull-down实验检测ERα、ERβ与GGNBP2蛋白相互结合的情况,并利用雌二醇进行刺激比较其共沉淀的改变。6.将GGNBP2过表达载体(pc DNA3-GGNBP2)或者空载体(pcDNA3-His C)和雌激素反应单元(ERE)的荧光素酶表达载体共转染入T47D细胞,并进行荧光素酶报告基因检测。利用Western blot实验检测在T47D-His C细胞和T47D-GGNBP2细胞中CCND1和TFF1蛋白的不同表达情况。7.构建过表达T47D-GGNBP2细胞的体内移植瘤模型,检测改变GGNBP2表达后对乳腺癌体内的生物学行为的影响。同时利用RT-PCR检测移植瘤中GGNBP2的m RNA转录情况。结果:1.多组织表达阵列分析发现在乳腺、卵巢、子宫、胃、肾、结肠、直肠、甲状腺及前列腺癌等九种组织中,GGNBP2均有基础表达,其中在乳腺组织中的表达量最高,在肾脏组织中表达最低。利用探针阵列分析了50对人体正常乳腺腺体和乳腺肿瘤组织的配对组织中GGNBP2的表达情况,发现:乳腺癌组织GGNBP2的表达水平明显降低,且在同一患者的转移灶组织中,GGNBP2的表达也有所下调。2.在Wnt1转基因小鼠自发产生的乳腺肿瘤组织中,GGNBP2的m RNA水平较正常组织有了显著的降低。3.在人乳腺癌细胞系或者正常乳腺细胞系T47D、MCF-7、MCF-10A和MCF-10F中,构建稳定过表达GGNBP2的细胞系。培养了4天后,相较于空质粒转染的乳腺癌细胞系,仅在ERα阳性的T47D和MCF-7中GGNBP2过表达的细胞系的细胞数量显著降低。4.稳定过表达外源性GGNBP2的T47D细胞系的增殖较对照组(T47D-His C组)显著降低。而且发现相较于T47D-His C细胞,T47D-GGNBP2 clone15细胞对雌二醇的刺激不敏感。三维培养空间发现T47D-GGNBP2细胞的细胞团较小且数量显著降低。5.划痕和Transwell实验显示,相较于母代T47D和T47D-His C细胞,T47D-GGNBP2细胞的迁移和侵袭能力下降;软琼脂克隆检测发现:T47D-GGNBP2细胞克隆形成能力显著下调。6.组氨酸Pull-down实验检测发现,仅ERα可与His标记的GGNBP2共沉淀,而ERβ与GGNBP2无关,且1μM雌二醇刺激下更多的ERα同GGNBP2共沉淀。7.荧光素酶报告基因结果表明,在T47D-His C细胞中,雌二醇可激活ERE的转录活性。然而在T47D-GGNBP2细胞中,雌二醇诱导ERE的转录活性显著降低8.构建裸鼠移植瘤模型,对体内移植瘤体积进行检测发现:相较于母代T47D的移植瘤,T47D-GGNBP2体外移植瘤的增长能力显著下降。结论:1.临床数据及小鼠实验结果显示:GGNBP2在正常乳腺组织存在表达,且在乳腺癌组织中GGNBP2表达下调,说明GGNBP2与乳腺癌的发生及发展密切相关。2.GGNBP2在乳腺癌中可能发挥着抑癌基因的作用,影响ERα阳性乳腺癌体内、体外的生长增殖能力,并抑制ERα阳性乳腺癌细胞的侵袭转移。3.GGNBP2可作为ERα的共阻抑蛋白来调控其靶基因CCND1和TFF1的表达或者活性,最终调控乳腺癌细胞增殖。
[Abstract]:BACKGROUND: Researchers used software to analyze 693 genes encoded in the 17q12-q23 region of human chromosome, including a 6.3 MB gene called gametogenetin binding protein 2 (GGNBP2) upstream of BRCA1 gene. Our Research Group intends to elucidate the role and mechanism of GGNBP2 in the proliferation and metastasis of breast cancer cells through clinical data, in vivo and in vitro experiments, and to understand the function of GGNBP2 gene in the development of breast cancer. Target: Methods: 1. Differential expression of GGNBP2 in nine normal human tissues was detected by multi-tissue array analysis, and the expression of GGNBP2 in normal breast and breast cancer tissues was compared. The expression of GGNBP2 in metastatic breast cancer and non-metastatic breast cancer tissues was compared. 2. Wnt1 was detected by RT-PCR assay. Differential expression of GGNBP2 in normal breast tissues and breast cancer tissues of transgenic mice. 3. Overexpression of GGNBP2 was constructed in different ERalpha state cells, and cell growth curves were drawn. Estradiol was used to stimulate cells with different GGNBP2 levels to detect their sensitivity to estradiol stimulation. Morphological and quantitative changes of maternal T47D cells and T47D-GGNBP2 cells. 4. Scratch test, Transwell test and soft agar cloning test were used to detect the different mobility, invasiveness and tumorigenicity of T47D-His C cells and T47D-GNBP2 cells, respectively. The effects of overexpression of GGNBP2 on the biological behavior of breast cancer cells were observed. 5. Polyhistidine Pull-down assay was used to detect the binding of ERa, ERbeta and GGNBP2 protein, and estradiol was used to stimulate and compare the changes of co-precipitation. 6. Luciferase expression vectors of GGNBP2 overexpression vector (pc DNA 3-GNBP2) or empty vector (pcDNA3-His C) and estrogen response unit (ERE) were co-transfected into T47D cells. The expression of CCND1 and TFF1 proteins in T47D-His C cells and T47D-GGNBP2 cells was detected by Western blot. 7. An in vivo transplanted tumor model was established to detect the effect of GGNBP2 expression on the biological behavior of breast cancer. The expression of GGNBP2 was detected in breast, ovary, uterus, stomach, kidney, colon, rectum, thyroid and prostate cancer. The expression of GGNBP2 was the highest in breast tissue and the lowest in kidney tissue. The expression of GGNBP 2 in 50 paired tissues of human normal breast gland and breast tumor was studied. It was found that the expression of GGNBP 2 in breast cancer was significantly decreased, and the expression of GGNBP 2 was also down-regulated in the metastatic tissues of the same patient. 2. In the spontaneously generated breast tumor tissues of Wnt1 transgenic mice, the expression of GGNBP 2 in M RNA water was observed. In human breast cancer cell lines or normal breast cell lines T47D, MCF-7, MCF-10A and MCF-10F, stable GGNBP2 overexpression cell lines were constructed. After 4 days of culture, compared with empty plasmid transfected breast cancer cell lines, only ER-positive T47D and MCF-7 GGNBP2 overexpression cell lines were constructed. The proliferation of T47D cells stably overexpressing exogenous GGNBP2 was significantly lower than that of the control group (T47D-His C group). Compared with T47D-His C cells, T47D-GGNBP2 clone 15 cells were not sensitive to estradiol stimulation. The cell mass of T47D-GGNBP2 cells was smaller and the number of T47D-GNBP2 cells was significantly lower in three-dimensional culture space. Compared with T47D and T47D-His C cells, the migration and invasion of T47D-GGNBP2 cells were decreased, and the cloning ability of T47D-GGNBP2 cells was significantly down-regulated by soft agar cloning assay. 6. Histidine Pull-down assay showed that only ERa could co-precipitate with His-labeled GGGGNBP2, but ERbeta was not related to GGNBP2, and 1 mu was found. The results of luciferase reporter gene showed that estradiol could activate the transcriptional activity of ERE in T47D-His C cells. However, in T47D-GGNBP2 cells, the transcriptional activity of ERE induced by estradiol decreased significantly 8. The nude mice transplanted tumor model was constructed and the tumor volume in vivo was detected. Compared with maternal T47D transplanted tumor, the growth ability of T47D-GGNBP2 in vitro transplanted tumor decreased significantly. Conclusion: 1. Clinical data and mouse experimental results showed that GGNBP2 was expressed in normal breast tissue, and GGNBP2 was down-regulated in breast cancer tissue, indicating that GGNBP2 is closely related to the occurrence and development of breast cancer. 2. GGGNBP2 can be found in breast cancer. GGNBP2 can act as a co-inhibitory protein of ER-alpha to regulate the expression or activity of its target genes CCND1 and TFF1, and ultimately regulate the proliferation of breast cancer cells.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

【相似文献】