慢病毒RNA干扰GRP78对肾癌细胞增殖和侵袭力的影响

发布时间：2018-06-14 15:22

本文选题：慢病毒 + GRP78　；参考：《广西医科大学》2016年硕士论文

【摘要】：肾细胞癌(renal cell carcinoma, RCC)又简称肾癌,起源于肾实质肾小管上皮细胞,是泌尿生殖系统中最为常见恶性肿瘤之一,在肾癌中最常见病理类型为肾透明细胞癌(CRCC)。CRCC治疗以根治性切除术(radicalnephrectomy, RN)为主,由于早期临床症状不明显,临床上以晚期患者为多。并且部分肾癌患者存在术后肿瘤复发或转移以及多耐药性,对放、化疗治疗不敏感,疗效不佳。免疫治疗由于长期疗效尚不能确定和总缓解率不高限制其广泛推广。因此深入研究肾细胞癌的增殖和侵袭的分子机制对提高早期诊断率和后续治疗疗效十分关键。葡萄糖调节蛋白78 (glucose regulated protein78, GRP78) 是 GRP家族中的重要成员,与热休克蛋白(heat shock protein70, HSP70)具有较高的同源性,属于HSP70家族成员之一。 GRP78在肿瘤细胞株和肿瘤组织如肝癌、结肠癌及肺癌、神经胶质细胞瘤等高表达,并可能与肿瘤的凋亡、耐药密切相关。但GRP78在肾细胞癌中的癌细胞增殖和侵袭等方面所起的作用尚未清楚。本研究在证实GRP78基因在肾癌细胞中呈明显高表达的基础上,初步提示了GRP78基因可能与肾细胞癌的发生发展存在相关。本实验首先成功构建好的小干扰RNA(small interference RNA, siRNA),然后转染进入到表达GRP78基因的肾癌786-0细胞中。确认siRNA-GRP78在基因和蛋白水平可以有效抑制GRP78的表达。接着研究干扰GRP78基因后对于肾癌细胞增殖和细胞侵袭力的影响。通过RNA干扰法探讨GRP78基因后对肾癌786-0细胞的细胞增殖和侵袭力的影响,为今后肾癌的基因治疗提供一定的理论和实验基础。第一部分：慢病毒RNA干扰GRP78基因载体的构建及筛选目的探讨慢病毒RNA干扰GPR78基因载体的构建及筛选。方法构建慢病毒靶向siRNA-GPR78干扰载体并转染进入肾癌786-0细胞株。转染48h后利用Realtime-PCR和Western blot分别检沏G RP78mRNA和蛋白表达。筛选出一条抑制率最高的siRNA-GPR78进入下一步研究。结果Realtime-PCR结果显示与空白对照组(1.85±0.08)和阴：性对照组(1.79±0.15)比较,GRP78mRNA的表达水平在siRNAl(0.32±0.09)、siRNA2(0.83±0.07)以及siRNA3组(0.79±0.11)均降低,差异有统计学意义(P0.01)；Western blot结果显示与空白对照组(1±0.03)或阴性对照组(1±0.07)比较,GRP78蛋白表达水平在siRNAI组(0.21±0.03)、siRNA2组(0.79±0.03)以及siRNA3组(0.86±0.04)均降低,差异有统计学意义(P0.0 1)；空白对照组与阴性对照组的GRP78mRNA和蛋白的表达水平差异无统计学意义(P0.05)。siRNA1抑制GRP78蛋白效率最佳。结论构建成功的siRNA-GRP78慢病毒载体可有效抑制肾癌786-0细胞中的GRP78基因及蛋白表达,以siRNAl抑制GRP78蛋白效率最佳,选择siRNA1进下一步研究。第二部分：RNA干扰GRP78基因对肾癌细胞增殖和侵袭力的影响目的探讨RNA干扰GPR78基因后对肾癌细胞的增殖和侵袭能力的影响。方法将siRNAl-GRP78干扰载体转染进入肾癌786-0细胞中。利用MTT法和Transwell法分别检测出肾癌细胞的增殖和侵袭力。结果MTT法检测结果显示与空白对照组和阴性对照组相比,siRNAl组肾癌细胞生长抑制明显,差异有统计学意义(P0.01)。Transwell法检测结果显示与空白对照组和阴性对照组相比,siRNAl组肾癌细胞生长抑制明显,差异有统计学意义(P0.01)。而阴性对照组与空白对照组比较,细胞的生长抑制不明显,差异无统计学意义(P0.05)。结论RNA干扰GRP78基因后,下调GRP78的表达,同时影响肾癌细胞的增殖和降低细胞的侵袭力。GRP78在促进肾癌细胞发生发展、细胞的增殖,以及维持细胞侵袭力等方面中可能起到重要作用。本研究也为以GRP78作为靶点的肿瘤基因治疗提供可靠的理论和实验基础。
[Abstract]:Renal cell carcinoma (RCC), also referred to as renal carcinoma, is derived from renal tubular epithelial cells. It is one of the most common malignant tumors in the genitourinary system. The most common pathological type of renal cell carcinoma is renal clear cell carcinoma (CRCC).CRCC treatment with radical resection (radicalnephrectomy, RN), due to early clinical symptoms. It is not obvious that the patients with advanced stage are more clinically, and some of the patients with renal cancer have recurrence or metastasis of postoperative tumor and multidrug-resistant. Chemotherapy is not sensitive to chemotherapy, and the curative effect is poor. Molecular mechanism is crucial to improving early diagnostic rate and follow-up treatment. Glucose regulatory protein 78 (glucose regulated protein78, GRP78) is an important member of the GRP family. It has a high homology with the heat shock protein (heat shock protein70, HSP70), one of the members of the HSP70 family. GRP78 in tumor cell lines and tumors. Tissues such as liver cancer, colon cancer, lung cancer, glioma and so on are highly expressed, and may be closely related to the apoptosis and resistance of the tumor. But the role of GRP78 in the proliferation and invasion of the cancer cells in renal cell carcinoma is not clear. This study has suggested that the GRP78 gene is highly expressed in the cell cell of renal cell carcinoma. GRP78 gene may be associated with the development of renal cell carcinoma. First, a successful small interference RNA (small interference RNA, siRNA) was successfully constructed and then transfected into 786-0 cells expressing the GRP78 gene. It was confirmed that siRNA-GRP78 at the gene and protein level could effectively inhibit the expression of GRP78. Then, the interference of GRP78 base was studied. The effect of RNA interference on the proliferation and invasiveness of 786-0 cells in renal carcinoma after GRP78 gene interference, and to provide a theoretical and experimental basis for the gene therapy of renal cancer in the future. Part 1: Construction and screening of GRP78 gene vector of lentivirus RNA interference The construction and screening of the lentivirus RNA interference GPR78 gene carrier. Methods the lentivirus targeted siRNA-GPR78 interference carrier was constructed and transfected into 786-0 cell lines of renal carcinoma. 48h and Western blot were used to detect G RP78mRNA and protein expression respectively after transfection of Realtime-PCR and Western blot. A new siRNA-GPR78 with the highest inhibition rate was selected for the next step of study. Ealtime-PCR results showed that the expression level of GRP78mRNA was in siRNAl (0.32 + 0.09), siRNA2 (0.83 + 0.07) and siRNA3 group (0.79 + 0.11) in the blank control group (1.85 + 0.08) and the sex control group (1.79 + 0.15), and the difference was statistically significant (P0.01). The Western blot results showed that the control group was (1 + 0.03) or negative control group (1). The expression level of GRP78 protein in siRNAI group was (0.21 + 0.03), siRNA2 group (0.79 + 0.03) and siRNA3 group (0.86 + 0.04) decreased, the difference was statistically significant (P0.0 1), and there was no statistical difference between the blank control group and the negative control group in the expression level of GRP78mRNA and protein (P0.05) and the conclusion that the efficiency of the inhibition of GRP78 protein was the best. The construction of a successful siRNA-GRP78 lentivirus vector can effectively inhibit the GRP78 gene and protein expression in 786-0 cells of renal cell carcinoma, with siRNAl to inhibit the efficiency of GRP78 protein, and select siRNA1 for the next step. The second part: RNA interference of GRP78 gene to the proliferation and invasiveness of renal cell carcinoma cells to investigate the effect of RNA interfering with GPR78 gene on renal cancer The effect of cell proliferation and invasion. Methods the siRNAl-GRP78 interference carrier was transfected into 786-0 cells of renal carcinoma. The proliferation and invasiveness of renal cell carcinoma cells were detected by MTT and Transwell. Results the results of MTT assay showed that the growth inhibition of renal cell carcinoma cells in siRNAl group was significantly lower than that in the blank control group and the negative control group. The results of different statistical significance (P0.01).Transwell assay showed that the growth inhibition of renal cell carcinoma cells in siRNAl group was significantly higher than that in the blank control group and the negative control group, and the difference was statistically significant (P0.01). Compared with the blank control group, the cell growth inhibition was not obvious, and the difference was not statistically significant (P0.05). Conclusion RNA interfered with GRP7. After the 8 gene, the expression of GRP78, the proliferation of renal cell carcinoma cells and the reduction of cell invasiveness.GRP78 may play an important role in promoting the development of renal cell carcinoma cells, cell proliferation, and maintaining cell invasiveness. This study also provides a reliable theory and experiment for the tumor gene therapy with GRP78 as a target. Basics.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.11

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