本文選題：前列腺癌 + 葡萄糖調(diào)節(jié)蛋白78KD��； 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：背景 前列腺癌是泌尿外科常見的腫瘤,隨著醫(yī)學(xué)界對這一疾病的認(rèn)識不斷深入,針對前列腺癌的治療手段亦趨于多樣化,但是對于去勢抵抗性前列腺癌(CRPC)仍缺乏有效治療手段。近年來,針對CRPC的基因治療逐漸成為研究熱點,而其中尋找到能夠發(fā)揮抗前列腺癌效應(yīng)基因靶點顯得尤為重要。葡萄糖調(diào)節(jié)蛋白78KD (GRP78)是一種在腫瘤細(xì)胞生存和進(jìn)展過程中發(fā)揮重要作用的蛋白,近年的研究發(fā)現(xiàn)下調(diào)多種類型腫瘤細(xì)胞中的GRP78表達(dá)可發(fā)揮抗腫瘤效應(yīng)。然而,目前尚缺少通過下調(diào)GRP78觀察其能否在前列腺癌中發(fā)揮抗腫瘤效應(yīng)的研究。此外,近期的部分研究表明非對稱小干擾RNA (asiRNA)可比傳統(tǒng)的對稱性小干擾RNA更加有效且持久的下調(diào)靶基因的表達(dá)。因此在本研究中,我們針對GRP78的mRNA序列設(shè)計了一系列的非對稱小干擾RNA,以觀察其對前列腺癌細(xì)胞生物學(xué)行為的效應(yīng)。 方法 通過RT-PCR和Western Blotting方法評估了三種不同的前列腺細(xì)胞系中GRP78的表達(dá)水平；針對GRP78的mRNA序列設(shè)計并合成了一系列非對稱小干擾RNA；通過流式細(xì)胞術(shù)篩選合適的轉(zhuǎn)染試劑和轉(zhuǎn)染濃度；對去勢抵抗性前列腺癌PC-3細(xì)胞進(jìn)行轉(zhuǎn)染后,觀察非對稱小干擾RNA下調(diào)細(xì)胞內(nèi)GRP78表達(dá)的效應(yīng),并與對稱性小干擾RNA進(jìn)行比較；隨后觀察特異性下調(diào)GRP78對PC-3細(xì)胞生長率,凋亡及遷移的影響；通過檢測caspase信號途徑相關(guān)蛋白和細(xì)胞遷移相關(guān)蛋白探討非對稱小干擾RNA下調(diào)細(xì)胞內(nèi)GRP78抗前列腺癌效應(yīng)的可能機(jī)制。 結(jié)果 相對于傳統(tǒng)的對稱性小干擾RNA,針對GRP78mRNA設(shè)計的15bp非對稱小干擾RNA (asiGRP78-3)表現(xiàn)出了更強(qiáng)的下調(diào)PC-3細(xì)胞GRP78表達(dá)水平的效應(yīng)。通過asiGRP78-3下調(diào)GRP78在PC-3中的表達(dá),可抑制PC-3細(xì)胞生長率,誘導(dǎo)其凋亡并抑制其遷移能力。進(jìn)一步研究表明,GRP78下調(diào)抑制了PC-3細(xì)胞的AKT磷酸化并下調(diào)pro-caspase9和pro-caspase3表達(dá),可能是asiGRP78-3促進(jìn)PC-3細(xì)胞凋亡的機(jī)制；下調(diào)GRP78的同時PC-3細(xì)胞中波形蛋白(vimentin)的表達(dá)也下調(diào),這可能是asiGRP78-3抑制PC-3遷移的機(jī)制。 結(jié)論 相比于對稱性小分子干擾RNA,非對稱小干擾RNA可更有效的下調(diào)前列腺癌細(xì)胞內(nèi)GRP78表達(dá)水平。非對稱小干擾RNA下調(diào)GRP78表達(dá)后可抑制前列腺癌細(xì)胞的增殖,誘導(dǎo)其凋亡并抑制其遷移,因此GRP78具有作為去勢抵抗性前列腺癌的基因治療靶點的價值,而非對稱小干擾RNA在下調(diào)前列腺癌GRP78表達(dá)方面具有一定的應(yīng)用價值。 背景 前列腺癌已成為泌尿系統(tǒng)最常見的惡性腫瘤之一。雖然目前針對前列腺癌的治療手段非常多樣,但去勢抵抗性前列腺癌(CRPC)仍然是前列腺癌的治療難點,因此針對CRPC的基因治療逐漸成為治療熱點。Kruppel樣因子4(KLF4)是細(xì)胞內(nèi)一類重要的轉(zhuǎn)錄因子,其表達(dá)下調(diào)可促進(jìn)腫瘤增殖和遷移。近年的研究顯示,小RNA不僅可以下調(diào)細(xì)胞內(nèi)特定基因表達(dá),也可以特異性上調(diào)某些基因表達(dá)。與人工合成的雙鏈小RNA相比,通過質(zhì)粒載體表達(dá)的短發(fā)卡小、RNA(shRNA)作用持續(xù)性更佳且可以與靶向分子等材料偶聯(lián)。本研究擬針對KLF4啟動子中特定序列設(shè)計重組質(zhì)粒pENTR/CMV-EGFP-U6-saKLF4-496真核表達(dá)載體,并將其導(dǎo)入前列腺癌細(xì)胞PC-3中,觀察其上調(diào)KLF4表達(dá)的效應(yīng),并檢測其對前列腺癌細(xì)胞腫瘤生物學(xué)行為的影響。 方法 本研究根據(jù)shRNA設(shè)計原則,針對KLF4啟動子-496序列設(shè)計并人工合成saKLF4-496激活序列,使用T4DNA ligase連接酶將合成后的saKLF4-496連接入重組載體pENTR/CMV-EGFP-U6構(gòu)建重組質(zhì)粒pENTR/CMV-EGFP-U6-saKLF4-496真核表達(dá)載體；重組質(zhì)粒經(jīng)擴(kuò)增、提取后測序鑒定；使用陽離子聚合物將pENTR/CMV-EGFP-U6-saKLF4-496轉(zhuǎn)染入PC-3細(xì)胞,優(yōu)化轉(zhuǎn)染效率后,以RT-PCR方法和Western Blotting方法檢測PC-3細(xì)胞內(nèi)KLF4表達(dá)情況；采用transwell方法,檢測轉(zhuǎn)染了pENTR/CMV-EGFP-U6-saKLF4-496的PC-3細(xì)胞遷移能力的變化。 結(jié)果 成功構(gòu)建的針對KLF4啟動子-496序列設(shè)計的重組質(zhì)粒pENTR/CMV-EGFP-U6-saKLF4-496真核表達(dá)載體；轉(zhuǎn)染此載體后96小時,RT-PCR檢測到PC-3細(xì)胞內(nèi)KLF4mRNA表達(dá)上調(diào),轉(zhuǎn)染后96小時Western Blotting檢測到PC-3細(xì)胞內(nèi)KLF4蛋白表達(dá)上調(diào)；Transwell檢測表明,轉(zhuǎn)染pENTR/CMV-EGFP-U6-saKLF4-496載體的PC-3細(xì)胞遷移能力較對照組下調(diào)。 結(jié)論 可成功構(gòu)建pENTR/CMV-EGFP-U6-saKLF4-496真核表達(dá)載體,此表達(dá)載體可上調(diào)前列腺癌細(xì)胞內(nèi)KLF4mRNA和蛋白水平；轉(zhuǎn)染pENTR/CMV-EGFP-U6-saKLF4-496真核表達(dá)載體后可前列腺癌細(xì)胞的遷移能力。
[Abstract]:background
Prostate cancer is a common tumor in the Department of urology. With the deepening understanding of this disease in the medical field, the treatment methods for prostate cancer are also diversified, but there is still a lack of effective treatment for the castration resistant prostate cancer (CRPC). In recent years, the gene therapy for CRPC has gradually become a hot spot of research. 78KD (GRP78) is a protein that plays an important role in the survival and progression of tumor cells. In recent years, it has been found that down regulation of GRP78 expression in various types of tumor cells can be used as an anti tumor effect. In addition, some recent studies have shown that asymmetric small interference RNA (asiRNA) can be more effective and more persistent than the traditional symmetrical small interference RNA to reduce the expression of target genes. In this study, we have designed a series of non - mRNA sequences in this study on the mRNA sequence of GRP78. Symmetrical small interference RNA was used to observe its effect on the biological behavior of prostate cancer cells.
Method
The expression level of GRP78 in three different prostate cell lines was evaluated by RT-PCR and Western Blotting methods. A series of asymmetric small interference RNA were designed and synthesized for mRNA sequence of GRP78, and suitable transfection reagents and transfection concentration were screened by flow cytometry. The transfection of castrated prostate cancer PC-3 cells was carried out. After that, the effect of asymmetric small interference (RNA) on the expression of GRP78 in cells was downregulated and compared with the symmetrical small interference RNA, and the effects of specific down-regulation of GRP78 on the growth rate, apoptosis and migration of PC-3 cells were observed, and the downregulation of asymmetric small interference RNA was explored by detecting caspase signaling pathway related proteins and cell migration related proteins. The possible mechanism of intracytoplasmic GRP78 on the effect of prostate cancer.
Result
Compared with the traditional symmetrical small interference RNA, the 15bp asymmetric small interference RNA (asiGRP78-3) designed for GRP78mRNA shows the effect of decreasing the GRP78 expression level of PC-3 cells. Down regulation of GRP78 in PC-3 can inhibit the growth rate of PC-3 cells, induce its apoptosis and inhibit its migration ability. The results showed that the downregulation of GRP78 inhibited the phosphorylation of AKT in PC-3 cells and down regulation of pro-caspase9 and pro-caspase3 expression, which may be the mechanism of asiGRP78-3 to promote apoptosis of PC-3 cells, and down regulated the expression of vimentin (vimentin) in PC-3 cells as well, which may be the mechanism of asiGRP78-3 inhibition of PC-3 migration.
conclusion
Compared with symmetrical small molecular interference RNA, asymmetric small interference RNA can reduce the level of GRP78 expression in prostate cancer cells more effectively. Asymmetric small interference RNA down regulation of GRP78 can inhibit the proliferation of prostate cancer cells, induce apoptosis and inhibit its migration. Therefore, GRP78 has a gene therapy target as a castrated prostatic cancer. The value of dots, rather than the small interfering RNA, has a certain application value in downregulating the expression of GRP78 in prostate cancer.
background
Prostate cancer has become one of the most common malignant tumors of the urinary system. Although the treatment of prostate cancer is very diverse, CRPC is still a difficult point for the treatment of prostate cancer. Therefore, the gene therapy for CRPC is gradually becoming a hot point.Kruppel like factor 4 (KLF4), which is one of the most important cells in the cell. In recent years, small RNA can not only reduce the specific gene expression in cells, but also specifically regulate some gene expression. Compared with the artificial double strand small RNA, the short hairpin expressed by plasmid carrier, RNA (shRNA) has a better and better effect. This study aims to design recombinant plasmid pENTR/CMV-EGFP-U6-saKLF4-496 eukaryotic expression vector for the specific sequence of KLF4 promoter, and introduce it into the prostate cancer cell PC-3, to observe the effect of its up regulation of KLF4 expression and to detect its effect on the biological behavior of the prostate cancer cell tumor.
Method
According to the design principle of shRNA, the study designed and synthesized the saKLF4-496 activation sequence according to the -496 sequence of the KLF4 promoter, and used the T4DNA ligase ligase to connect the synthesized saKLF4-496 into the recombinant vector pENTR/CMV-EGFP-U6 to construct the recombinant plasmid pENTR/CMV-EGFP-U6-saKLF4-496 eukaryotic expression vector; the recombinant plasmid was amplified and extracted after the amplification. Sequence identification; using cationic polymers to transfect pENTR/CMV-EGFP-U6-saKLF4-496 into PC-3 cells and optimize the transfection efficiency, the expression of KLF4 in PC-3 cells was detected by RT-PCR method and Western Blotting method, and Transwell method was used to detect the change in the migration ability of PC-3 cells transfected with pENTR/ CMV-EGFP-U6-saKLF4-496.
Result
The recombinant plasmid pENTR/CMV-EGFP-U6-saKLF4-496 eukaryotic expression vector designed for the KLF4 promoter -496 sequence was successfully constructed. After 96 hours transfection of the vector, RT-PCR detected the up regulation of KLF4mRNA expression in PC-3 cells, and the expression of KLF4 protein expression in PC-3 cells was up regulated by Western Blotting at 96 hours after transfection; Transwell detection showed transfection P. The PC-3 migration ability of ENTR/CMV-EGFP-U6-saKLF4-496 vector was lower than that of control group.
conclusion
The pENTR/CMV-EGFP-U6-saKLF4-496 eukaryotic expression vector can be successfully constructed. The expression vector can up regulate the level of KLF4mRNA and protein in prostate cancer cells, and the transfection of pENTR/CMV-EGFP-U6-saKLF4-496 eukaryotic expression vector can lead to the migration of prostate cancer cells.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.25

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