慢病毒介导的shRNA沉默CD151对Luminal和Basal-like型乳腺癌细胞生物学行为的影响及相关调控机制的研

发布时间：2018-04-16 00:25

本文选题：CD151 + RNAi　；参考：《吉林大学》2015年博士论文

【摘要】：乳腺癌是来源于乳腺上皮组织的恶性肿瘤,严重威胁女性的健康和生命。由于乳腺癌细胞具有高度异质性,单纯病理分型已不能满足临床诊断和治疗的需要。近年来国内外专家越来越重视乳腺癌的分子分型,并且取得重大进展。乳腺癌的分子分型强调的是肿瘤个体的分子特征,有助于临床实施个体化治疗和准确判断预后。四次跨膜蛋白(Tetraspanins,TM4SF)是一个特殊的细胞膜糖蛋白家族,它的成员广泛分布于细胞和组织中,对细胞的生长和增殖、黏附和迁移、浸润和转移以及血管形成有调节作用。编码四次跨膜蛋白的基因绝大多数是抑癌基因,CD151被认为是为数不多的癌基因,它在多种肿瘤组织中高表达,与预后负相关。CD151对HER2过表达型乳腺癌的影响及相关分子调控机制有报道,但对Luminal型和Basal-like型乳腺癌的影响及相关分子调控机制未见报道,为此本研究在检测乳腺癌CD151m RNA和蛋白表达的基础上,以代表Luminal型和Basal-like型的两种乳腺癌细胞为研究对象,通过慢病毒介导的RNA干扰和RNA-seq方法,探讨CD151对两种类型乳腺癌细胞生物学行为的影响并探讨可能的分子调控机制,为乳腺癌的分子靶向治疗提供实验依据和理论支持。方法:1.检测CD151在乳腺癌组织中的表达:用q PCR和IHC分别检测乳腺癌组织中CD151的m RNA和蛋白表达,并探讨其与临床病理参数的相关性。2.检测CD151在乳腺癌细胞中的表达:用免疫荧光和Western Blot分别检测乳腺癌细胞MCF-7和MDA-MB-468及正常乳腺上皮细胞HBL-100中CD151蛋白表达。3.构建CD151-sh RNA慢病毒表达载体并转染靶细胞:构建重组慢病毒载体,转染靶细胞,q PCR和Western Blot检测沉默效果。4.检测CD151-sh RNA对乳腺癌细胞生物学行为的影响:CCK8实验检测细胞增殖,流式技术检测细胞周期和细胞凋亡,划痕实验和Transwell实验检测细胞的迁移和侵袭能力。裸鼠成瘤实验检测细胞成瘤及生长情况。5.CD151-sh RNA对乳腺癌细胞基因表达的机制研究:用RNA-seq技术检测沉默CD151基因前后的MCF-7和MDA-MB-468两组乳腺癌细胞的转录组,比较两组细胞转录组谱的差异,应用生物信息学方法(GO、KEGG富集分析)对全部DEGs进行功能注释和信号通路网络分析。q PCR验证具有明显差异的候选基因。结果:1.乳腺癌组织中CD151m RNA和蛋白的表达:q PCR和IHC结果显示CD151在乳腺癌组织高表达,与癌旁组织相比差异显著,具有统计学意义。CD151表达与淋巴结转移、高增殖指数正相关。2.乳腺癌细胞中CD151蛋白的表达:免疫荧光和Western Blot结果显示CD151蛋白在MCF-7和MDA-MB-468细胞中呈强阳性表达。3.慢病毒表达载体构建及沉默效率测定:构建4个慢病毒表达载体侵染靶细胞,q PCR和Western Blot检测CD151沉默效率,其中sh RNA-CD151-Lv3463载体转染的MCF-7和MDA-MB-468细胞内CD151的m RNA和蛋白经72h和96h测定,m RNA的含量下降80%(P均0.01),蛋白表达也明显下降80%以上。阴性病毒对照组和空白细胞组内CD151m RNA和蛋白表达无明显差别。上述结果表明我们设计的sh RNA-CD151-Lv3463可以明显抑制CD151基因在两株乳腺癌细胞内表达。4.CD151基因沉默抑制MCF-7和MDA-MB-468细胞的生长和增殖:使用CCK8法测定CD151基因沉默对细胞增殖的影响。随着作用时间增加对CD151基因表达的抑制作用增强,细胞增殖活力在第4d后较空白对照组和阴性对照组显著降低(P均0.05)。结果表明沉默CD151明显抑制乳腺癌MCF-7和MDA-MB-468细胞的增殖能力。5.CD151基因沉默干扰MCF-7和MDA-MB-468细胞的细胞周期并促进凋亡:FCM检测显示,sh RNA-CD151-3463可使MCF-7和MDA-MB-468细胞中G0/G1期细胞百分比明显增加,S期细胞百分比显著降低,上述结果表明沉默CD151导致MCF-7细胞和MDA-MB-468细胞G0/G1期阻滞。凋亡检测发现沉默CD151后两株细胞发生早期凋亡、晚期凋亡的比例明显增加。6.CD151基因沉默抑制MCF-7和MDA-MB468细胞的迁移和侵袭能力:划痕实验和Transwell法检测了对照组、实验组及空载组乳腺癌细胞的迁移和侵袭能力,实验组细胞迁移能力和侵袭能力明显降低。7.CD151基因沉默抑制裸鼠移植瘤形成。8.CD151调控乳腺癌的分子机制:用RNA-seq技术检测了CD151基因沉默前后的MCF-7和MDA-MB-468两组乳腺癌细胞的转录组,比较两组细胞转录组谱的差异,应用生物信息学方法(GO、KEGG富集分析)对全部DEGs进行功能注释和信号通路网络分析,q PCR验证具有明显差异的候选基因。两株细胞都显示P53信号通路中的基因高度富集,调控细胞周期的基因高度一致,q PCR验证结果与RNA-Seq一致。MDA-MB-468细胞的有关Wnt信号通路基因富集,Wnt5a和Wnt6在沉默CD151后显著上调,PKC的表达与Wnt5a和Wnt6的表达高度一致。结论:1.CD151调控Luminal型和Basal-like型乳腺癌细胞的生物学行为,可作为乳腺癌靶向治疗的潜在靶点。2.CD151调控Luminal型和Basal-like乳腺癌细胞的增殖与凋亡可能与P53信号通路有关。3.CD151调控Basal-like型乳腺癌细胞的侵袭和转移可能与非经典Wnt信号通路有关。
[Abstract]:Breast cancer from breast epithelial tissue malignant tumor, a serious threat to women's health and life. The breast cancer cells are highly heterogeneous, simple pathological type has been unable to meet the needs of clinical diagnosis and treatment. In recent years, domestic and foreign experts pay more attention to the molecular classification of breast cancer, and made significant progress. Classification of breast cancer that is the molecular characteristics of individual tumors, contribute to the clinical individualized treatment and judging prognosis. Four transmembrane protein (Tetraspanins, TM4SF) is a specific cell membrane glycoprotein family, its members are widely distributed in the cells and tissues, proliferation and growth of cells the adhesion, migration and invasion and metastasis and angiogenesis regulation. Encoding four transmembrane protein gene is the most tumor suppressor gene, CD151 is considered as a cancer number of genes, which in a variety of tumor The high expression in tumor tissue, and prognosis of.CD151 negative correlation effect of overexpression of HER2 type breast cancer and the related molecular mechanisms have been reported, but no influence on the regulation mechanism of Luminal type and Basal-like type of breast cancer and the related molecular basis of reports, the purpose of this study is expressed in the detection of breast cancer CD151m RNA and protein, to two breast cancer cells represent Luminal type and Basal-like type as the research object, RNA interference and RNA-seq by lentivirus mediated, to explore the effects of CD151 on the two types of breast cancer cell biological behavior and to explore the possible molecular regulation and control mechanism, to provide experimental evidence and theoretical support for the molecular targeted therapy in breast cancer treatment methods: 1. expression of CD151 in breast cancer tissues by Q PCR and IHC m were detected RNA and CD151 protein expression in breast carcinoma tissues, and explore its correlation with clinicopathological parameters of.2. To detect the expression of CD151 in breast cancer cells: to detect.3. constructed CD151-sh RNA lentiviral expression vector and transfected into target cells expressing CD151 protein MCF-7 and MDA-MB-468 breast cancer cells and normal breast epithelial cells in HBL-100 by immunofluorescence and Western Blot: to construct the recombinant virus vector transfection slow, target cells, Q PCR and Western Blot effect detection of.4. RNA detection of CD151-sh silencing effect on cell biological behavior of breast cancer cell proliferation: CCK8 assay, flow cytometry to detect the cell cycle and apoptosis, migration and invasion of wound healing and Transwell assay. The nude mice were used to detect cell tumor growth and mechanism of.5.CD151-sh on the expression of RNA gene in breast cancer cells in the transcriptome detected before and after the CD151 gene silencing technology of RNA-seq MCF-7 and MDA-MB-468 two groups of breast cancer cells, compared two groups of fine Different cell transcriptome profiling and bioinformatics methods (GO, KEGG enrichment analysis) candidate gene functional annotation and pathway analysis of.Q network PCR verification has obvious difference for all the DEGs. Results: the expression of CD151m and RNA protein in 1. breast cancer tissues: Q PCR and IHC showed that CD151 in breast cancer the high expression of tissue, tissues and significant differences, with statistical significance of.CD151 expression and lymph node metastasis, the expression of CD151 protein and high proliferation index is positively related to.2. in breast cancer cells: immunofluorescence and Western Blot showed that CD151 protein showed strong positive expression of.3. in MCF-7 and MDA-MB-468 lentiviral expression vector construction and determination of 4 silencing efficiency: to construct a lentiviral vector infection of target cells, Q PCR and Western Blot detection of CD151 silencing efficiency in SH cells transfected with RNA-CD151-Lv3463 MCF-7 and MDA-MB-468 cells. M RNA and CD151 protein by 72h 96h and m RNA assay, content decreased 80% (P 0.01), protein expression also decreased more than 80%. Negative control virus group and blank cell group CD151m RNA and protein expression had no significant difference. The results show that our design of SH RNA-CD151-Lv3463 can significantly inhibit the expression of growth and the proliferation of.4.CD151 gene silencing MCF-7 and MDA-MB-468 cells of the CD151 gene in two strains of breast cancer cells: Determination of CD151 gene silencing by CCK8 on cell proliferation. With the increase of reaction time increase the inhibitory effect on CD151 gene expression and cell proliferation in the 4D compared with the blank control group and negative control group was significantly lower (P 0.05). The results showed that the proliferation ability of.5.CD151 cell cycle gene silencing and MCF-7 interference silenced MDA-MB-468 cells CD151 significantly inhibits breast cancer MCF-7 and MDA-MB-468 cells and promote In apoptosis: FCM analysis showed that sh RNA-CD151-3463 can increase the percentage of G0/G1 phase cells and MCF-7 MDA-MB-468 cells significantly increased the percentage of S phase cells decreased significantly, the results showed that silencing of CD151 resulted in MCF-7 and MDA-MB-468 cells G0/G1 phase arrest. Apoptosis detected silence after CD151 two cell apoptosis, migration and invasion of advanced the apoptosis ratio increased.6.CD151 gene silencing MCF-7 and MDA-MB468 cells: scratch test and Transwell method to detect the migration and invasion of control group, experimental group and vector group of breast cancer cells, the molecular mechanism of the experimental group cell migration and invasion significantly decreased.7.CD151 gene silencing inhibits the formation of.8.CD151 regulation of breast cancer xenografts in nude mice transcription of CD151 gene silencing group before and after MCF-7 and MDA-MB-468 two groups of breast cancer cells were detected by using RNA-seq technology, than The differences of cell transcriptome spectra were compared between the two groups, using bioinformatics method (GO, KEGG enrichment analysis) analysis of functional annotation and signaling networks to all DEGs, candidate gene Q PCR verification has obvious difference. Two cell lines are shown in the P53 signaling pathway genes are highly enriched, regulation of cell cycle gene together, the Wnt signaling pathway gene Q PCR results consistent with RNA-Seq.MDA-MB-468 and Wnt6 Wnt5a cell enrichment, up-regulated in silence after CD151, the expression of PKC and Wnt5a and Wnt6 are highly consistent. Conclusion: the biological behavior of 1.CD151 regulation of Luminal type and Basal-like type of breast cancer cells, breast cancer can be used as a target to.2.CD151 a potential target for the treatment of regulation of type Luminal and Basal-like breast cancer cell proliferation and apoptosis and P53 signaling pathway on regulation of.3.CD151 type Basal-like breast cancer cell invasion and Metastasis may be associated with non classical Wnt signaling pathways.

【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

【相似文献】