应用小RNA下调GRP78表达与上调KLF4表达抑制前列腺癌细胞恶性生物学行为的实验研究

发布时间：2018-06-22 08:31

本文选题：前列腺癌 + 葡萄糖调节蛋白78KD　；参考：《华中科技大学》2014年博士论文

【摘要】：背景前列腺癌是泌尿外科常见的肿瘤,随着医学界对这一疾病的认识不断深入,针对前列腺癌的治疗手段亦趋于多样化,但是对于去势抵抗性前列腺癌(CRPC)仍缺乏有效治疗手段。近年来,针对CRPC的基因治疗逐渐成为研究热点,而其中寻找到能够发挥抗前列腺癌效应基因靶点显得尤为重要。葡萄糖调节蛋白78KD (GRP78)是一种在肿瘤细胞生存和进展过程中发挥重要作用的蛋白,近年的研究发现下调多种类型肿瘤细胞中的GRP78表达可发挥抗肿瘤效应。然而,目前尚缺少通过下调GRP78观察其能否在前列腺癌中发挥抗肿瘤效应的研究。此外,近期的部分研究表明非对称小干扰RNA (asiRNA)可比传统的对称性小干扰RNA更加有效且持久的下调靶基因的表达。因此在本研究中,我们针对GRP78的mRNA序列设计了一系列的非对称小干扰RNA,以观察其对前列腺癌细胞生物学行为的效应。方法通过RT-PCR和Western Blotting方法评估了三种不同的前列腺细胞系中GRP78的表达水平；针对GRP78的mRNA序列设计并合成了一系列非对称小干扰RNA；通过流式细胞术筛选合适的转染试剂和转染浓度；对去势抵抗性前列腺癌PC-3细胞进行转染后,观察非对称小干扰RNA下调细胞内GRP78表达的效应,并与对称性小干扰RNA进行比较；随后观察特异性下调GRP78对PC-3细胞生长率,凋亡及迁移的影响；通过检测caspase信号途径相关蛋白和细胞迁移相关蛋白探讨非对称小干扰RNA下调细胞内GRP78抗前列腺癌效应的可能机制。结果相对于传统的对称性小干扰RNA,针对GRP78mRNA设计的15bp非对称小干扰RNA (asiGRP78-3)表现出了更强的下调PC-3细胞GRP78表达水平的效应。通过asiGRP78-3下调GRP78在PC-3中的表达,可抑制PC-3细胞生长率,诱导其凋亡并抑制其迁移能力。进一步研究表明,GRP78下调抑制了PC-3细胞的AKT磷酸化并下调pro-caspase9和pro-caspase3表达,可能是asiGRP78-3促进PC-3细胞凋亡的机制；下调GRP78的同时PC-3细胞中波形蛋白(vimentin)的表达也下调,这可能是asiGRP78-3抑制PC-3迁移的机制。结论相比于对称性小分子干扰RNA,非对称小干扰RNA可更有效的下调前列腺癌细胞内GRP78表达水平。非对称小干扰RNA下调GRP78表达后可抑制前列腺癌细胞的增殖,诱导其凋亡并抑制其迁移,因此GRP78具有作为去势抵抗性前列腺癌的基因治疗靶点的价值,而非对称小干扰RNA在下调前列腺癌GRP78表达方面具有一定的应用价值。背景前列腺癌已成为泌尿系统最常见的恶性肿瘤之一。虽然目前针对前列腺癌的治疗手段非常多样,但去势抵抗性前列腺癌(CRPC)仍然是前列腺癌的治疗难点,因此针对CRPC的基因治疗逐渐成为治疗热点。Kruppel样因子4(KLF4)是细胞内一类重要的转录因子,其表达下调可促进肿瘤增殖和迁移。近年的研究显示,小RNA不仅可以下调细胞内特定基因表达,也可以特异性上调某些基因表达。与人工合成的双链小RNA相比,通过质粒载体表达的短发卡小、RNA(shRNA)作用持续性更佳且可以与靶向分子等材料偶联。本研究拟针对KLF4启动子中特定序列设计重组质粒pENTR/CMV-EGFP-U6-saKLF4-496真核表达载体,并将其导入前列腺癌细胞PC-3中,观察其上调KLF4表达的效应,并检测其对前列腺癌细胞肿瘤生物学行为的影响。方法本研究根据shRNA设计原则,针对KLF4启动子-496序列设计并人工合成saKLF4-496激活序列,使用T4DNA ligase连接酶将合成后的saKLF4-496连接入重组载体pENTR/CMV-EGFP-U6构建重组质粒pENTR/CMV-EGFP-U6-saKLF4-496真核表达载体；重组质粒经扩增、提取后测序鉴定；使用阳离子聚合物将pENTR/CMV-EGFP-U6-saKLF4-496转染入PC-3细胞,优化转染效率后,以RT-PCR方法和Western Blotting方法检测PC-3细胞内KLF4表达情况；采用transwell方法,检测转染了pENTR/CMV-EGFP-U6-saKLF4-496的PC-3细胞迁移能力的变化。结果成功构建的针对KLF4启动子-496序列设计的重组质粒pENTR/CMV-EGFP-U6-saKLF4-496真核表达载体；转染此载体后96小时,RT-PCR检测到PC-3细胞内KLF4mRNA表达上调,转染后96小时Western Blotting检测到PC-3细胞内KLF4蛋白表达上调；Transwell检测表明,转染pENTR/CMV-EGFP-U6-saKLF4-496载体的PC-3细胞迁移能力较对照组下调。结论可成功构建pENTR/CMV-EGFP-U6-saKLF4-496真核表达载体,此表达载体可上调前列腺癌细胞内KLF4mRNA和蛋白水平；转染pENTR/CMV-EGFP-U6-saKLF4-496真核表达载体后可前列腺癌细胞的迁移能力。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

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