發(fā)布時間：2018-04-16 00:25

  本文選題：CD151 + RNAi��； 參考：《吉林大學(xué)》2015年博士論文

【摘要】：乳腺癌是來源于乳腺上皮組織的惡性腫瘤,嚴(yán)重威脅女性的健康和生命。由于乳腺癌細(xì)胞具有高度異質(zhì)性,單純病理分型已不能滿足臨床診斷和治療的需要。近年來國內(nèi)外專家越來越重視乳腺癌的分子分型,并且取得重大進(jìn)展。乳腺癌的分子分型強(qiáng)調(diào)的是腫瘤個體的分子特征,有助于臨床實施個體化治療和準(zhǔn)確判斷預(yù)后。四次跨膜蛋白(Tetraspanins,TM4SF)是一個特殊的細(xì)胞膜糖蛋白家族,它的成員廣泛分布于細(xì)胞和組織中,對細(xì)胞的生長和增殖、黏附和遷移、浸潤和轉(zhuǎn)移以及血管形成有調(diào)節(jié)作用。編碼四次跨膜蛋白的基因絕大多數(shù)是抑癌基因,CD151被認(rèn)為是為數(shù)不多的癌基因,它在多種腫瘤組織中高表達(dá),與預(yù)后負(fù)相關(guān)。CD151對HER2過表達(dá)型乳腺癌的影響及相關(guān)分子調(diào)控機(jī)制有報道,但對Luminal型和Basal-like型乳腺癌的影響及相關(guān)分子調(diào)控機(jī)制未見報道,為此本研究在檢測乳腺癌CD151m RNA和蛋白表達(dá)的基礎(chǔ)上,以代表Luminal型和Basal-like型的兩種乳腺癌細(xì)胞為研究對象,通過慢病毒介導(dǎo)的RNA干擾和RNA-seq方法,探討CD151對兩種類型乳腺癌細(xì)胞生物學(xué)行為的影響并探討可能的分子調(diào)控機(jī)制,為乳腺癌的分子靶向治療提供實驗依據(jù)和理論支持。方法:1.檢測CD151在乳腺癌組織中的表達(dá):用q PCR和IHC分別檢測乳腺癌組織中CD151的m RNA和蛋白表達(dá),并探討其與臨床病理參數(shù)的相關(guān)性。2.檢測CD151在乳腺癌細(xì)胞中的表達(dá):用免疫熒光和Western Blot分別檢測乳腺癌細(xì)胞MCF-7和MDA-MB-468及正常乳腺上皮細(xì)胞HBL-100中CD151蛋白表達(dá)。3.構(gòu)建CD151-sh RNA慢病毒表達(dá)載體并轉(zhuǎn)染靶細(xì)胞:構(gòu)建重組慢病毒載體,轉(zhuǎn)染靶細(xì)胞,q PCR和Western Blot檢測沉默效果。4.檢測CD151-sh RNA對乳腺癌細(xì)胞生物學(xué)行為的影響:CCK8實驗檢測細(xì)胞增殖,流式技術(shù)檢測細(xì)胞周期和細(xì)胞凋亡,劃痕實驗和Transwell實驗檢測細(xì)胞的遷移和侵襲能力。裸鼠成瘤實驗檢測細(xì)胞成瘤及生長情況。5.CD151-sh RNA對乳腺癌細(xì)胞基因表達(dá)的機(jī)制研究:用RNA-seq技術(shù)檢測沉默CD151基因前后的MCF-7和MDA-MB-468兩組乳腺癌細(xì)胞的轉(zhuǎn)錄組,比較兩組細(xì)胞轉(zhuǎn)錄組譜的差異,應(yīng)用生物信息學(xué)方法(GO、KEGG富集分析)對全部DEGs進(jìn)行功能注釋和信號通路網(wǎng)絡(luò)分析。q PCR驗證具有明顯差異的候選基因。結(jié)果:1.乳腺癌組織中CD151m RNA和蛋白的表達(dá):q PCR和IHC結(jié)果顯示CD151在乳腺癌組織高表達(dá),與癌旁組織相比差異顯著,具有統(tǒng)計學(xué)意義。CD151表達(dá)與淋巴結(jié)轉(zhuǎn)移、高增殖指數(shù)正相關(guān)。2.乳腺癌細(xì)胞中CD151蛋白的表達(dá):免疫熒光和Western Blot結(jié)果顯示CD151蛋白在MCF-7和MDA-MB-468細(xì)胞中呈強(qiáng)陽性表達(dá)。3.慢病毒表達(dá)載體構(gòu)建及沉默效率測定:構(gòu)建4個慢病毒表達(dá)載體侵染靶細(xì)胞,q PCR和Western Blot檢測CD151沉默效率,其中sh RNA-CD151-Lv3463載體轉(zhuǎn)染的MCF-7和MDA-MB-468細(xì)胞內(nèi)CD151的m RNA和蛋白經(jīng)72h和96h測定,m RNA的含量下降80%(P均0.01),蛋白表達(dá)也明顯下降80%以上。陰性病毒對照組和空白細(xì)胞組內(nèi)CD151m RNA和蛋白表達(dá)無明顯差別。上述結(jié)果表明我們設(shè)計的sh RNA-CD151-Lv3463可以明顯抑制CD151基因在兩株乳腺癌細(xì)胞內(nèi)表達(dá)。4.CD151基因沉默抑制MCF-7和MDA-MB-468細(xì)胞的生長和增殖:使用CCK8法測定CD151基因沉默對細(xì)胞增殖的影響。隨著作用時間增加對CD151基因表達(dá)的抑制作用增強(qiáng),細(xì)胞增殖活力在第4d后較空白對照組和陰性對照組顯著降低(P均0.05)。結(jié)果表明沉默CD151明顯抑制乳腺癌MCF-7和MDA-MB-468細(xì)胞的增殖能力。5.CD151基因沉默干擾MCF-7和MDA-MB-468細(xì)胞的細(xì)胞周期并促進(jìn)凋亡:FCM檢測顯示,sh RNA-CD151-3463可使MCF-7和MDA-MB-468細(xì)胞中G0/G1期細(xì)胞百分比明顯增加,S期細(xì)胞百分比顯著降低,上述結(jié)果表明沉默CD151導(dǎo)致MCF-7細(xì)胞和MDA-MB-468細(xì)胞G0/G1期阻滯。凋亡檢測發(fā)現(xiàn)沉默CD151后兩株細(xì)胞發(fā)生早期凋亡、晚期凋亡的比例明顯增加。6.CD151基因沉默抑制MCF-7和MDA-MB468細(xì)胞的遷移和侵襲能力:劃痕實驗和Transwell法檢測了對照組、實驗組及空載組乳腺癌細(xì)胞的遷移和侵襲能力,實驗組細(xì)胞遷移能力和侵襲能力明顯降低。7.CD151基因沉默抑制裸鼠移植瘤形成。8.CD151調(diào)控乳腺癌的分子機(jī)制:用RNA-seq技術(shù)檢測了CD151基因沉默前后的MCF-7和MDA-MB-468兩組乳腺癌細(xì)胞的轉(zhuǎn)錄組,比較兩組細(xì)胞轉(zhuǎn)錄組譜的差異,應(yīng)用生物信息學(xué)方法(GO、KEGG富集分析)對全部DEGs進(jìn)行功能注釋和信號通路網(wǎng)絡(luò)分析,q PCR驗證具有明顯差異的候選基因。兩株細(xì)胞都顯示P53信號通路中的基因高度富集,調(diào)控細(xì)胞周期的基因高度一致,q PCR驗證結(jié)果與RNA-Seq一致。MDA-MB-468細(xì)胞的有關(guān)Wnt信號通路基因富集,Wnt5a和Wnt6在沉默CD151后顯著上調(diào),PKC的表達(dá)與Wnt5a和Wnt6的表達(dá)高度一致。結(jié)論:1.CD151調(diào)控Luminal型和Basal-like型乳腺癌細(xì)胞的生物學(xué)行為,可作為乳腺癌靶向治療的潛在靶點(diǎn)。2.CD151調(diào)控Luminal型和Basal-like乳腺癌細(xì)胞的增殖與凋亡可能與P53信號通路有關(guān)。3.CD151調(diào)控Basal-like型乳腺癌細(xì)胞的侵襲和轉(zhuǎn)移可能與非經(jīng)典Wnt信號通路有關(guān)。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.9

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1 杜文英;乳腺癌分子亞型的臨床與病理特點(diǎn)[D];鄭州大學(xué);2011年

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5 葛廣哲;樹，

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