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Inhibition of Renal Cancer Growth in Nude Mice by siRNA Targeting on Survivin

Author: LiuFu
Tutor: HuZiLi
School: Chongqing Medical University
Course: Department of Urology
Keywords: survivin 786-0 cells Kidney Cancer Plasmid siRNA Interference Transplanted tumors Apoptosis RNA interference
CLC: R737.11
Type: Master's thesis
Year: 2011
Downloads: 48
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Abstract

Kidney cancer, also known as renal cell carcinoma (Renal cell carcinoma, RCC), renal adenocarcinoma, which originated in tubular epithelial malignancies. Its incidence of tumors of the urinary tract after bladder cancer in renal cell carcinoma, clear cell carcinoma is the most common. The treatment of kidney cancer is still surgical excision is not sensitive to radiotherapy, although the advent of of existing kidney cancer targeted drugs, but its resistance to the presence of factors such as expensive, and therefore very necessary to seek a more effective means of treatment. In recent years, research on the application of gene therapy is becoming the hotspot of kidney cancer, especially in advanced kidney cancer therapy. Survivin is an apoptosis inhibitory protein discovered in recent years (inhibitor of apoptosis proteins, IAP) family members, in most malignant tissues express the rich, but not in normal adult tissues express or rarely expressed, this selective expression characteristics so that it may become the markers and gene therapy of cancer diagnosis, a new target. In our previous study, using RNAi technology to construct the recombinant plasmid and transfected into 786-0 cells, significantly inhibited the expression of survivin induced apoptosis survivin as a target for RNAi therapy from in vitro studies kidney cancer provides a reliable laboratories basis. The experiments confirmed 786-0 cell lines expressed survivin gene expression by RNAi in vivo inhibition of survivin on the basis of further laboratory evidence for kidney cancer gene therapy. The first part of survivin gene in human renal cell carcinoma 786-0 cells express the meaning of purpose: the detection of human 786-0 cells whether survivin gene expression, to explore the significance of survivin gene expression in human 786-0 cells. Method: 1. 786-0 cells cultured human. 2.RT-PCR detection of survivin mRNA in human renal cell carcinoma 786-0 cells transcriptional level. 3.Western-blot detection of survivin protein expression levels in human 786-0 cells. Results: 1.RT-PCR showed survivin mRNA in human renal cell carcinoma 786-0 cells. 2.Western-blot human renal cell carcinoma 786-0 cells survivin protein expression. Conclusion: In human renal cell carcinoma 786-0 cells survivin gene are expressed at the mRNA and protein levels, suggesting that survivin gene expression and the development of kidney cancer is closely related to, and laid the foundation for human renal cancer gene therapy targeting survivin is . The second part of the purpose of target the specific survivin gene siRNA recombinant plasmid construction and amplification: the survivin gene sequences in GenBank, as well as in our previous study on the basis of building-specific siRNA recombinant interference plasmid and amplification and identification. Methods: 1. Survivin gene in the GeneBank sequence and our previous research to build two sets of of positive siRNA and a negative control, transcription template with pGenesil1.1 of connection and transformed into E. coli strain DH5α. Pick kanamycin-resistant colonies and amplified. 3 on the amplification of the recombinant plasmids identified. Results: The two sets of positive siRNA (survivin1 5'-GGACCACCGCATCTCTACA-3'166; survivin2 5'-GGCTGGCTTCATCCACTGC-3'241) and a negative control (HK 5'-GACTTCATAAGGCGCATGC-3 '). Conclusion: The in vitro synthesis of siRNA targeting survivin provides the basis for further inhibit the 786-0 cell transplantation of survivin expression. Inhibition purpose of the third part of siRNA against survivin in vivo human renal cell carcinoma xenografts in nude mice: BALB / c nude mice injected subcutaneously 786-0 cell suspension after the establishment of the growth of human renal cell carcinoma xenograft model, in tumor intravitreal injection of survivin siRNA, observed survivin siRNA in vivo tumor growth status. Method: 1. Establish the growth of human renal cell carcinoma xenograft model. 2 5:00 intratumoral injection, intratumoral injection of survivin siRNA, depending on the injection of survivin siRNA, the mice were randomly divided into 8 groups [A (survivin1 siRNA liposome pure RPMI 1640); (survivin2 siRNA lipid pure RPMI1640); C (HK siRNA liposome pure RPMI1640);-D (survivin1 siRNA pure RPMI1640); E (survivin2 siRNA pure RPMI1640); pure F (HK siRNA RPMI1640); G the (liposomal pure RPMI1640); H (saline)]. With a vernier caliper measurement and calculation of tumor volumes, tumor growth curve and final stripping tumor weighing. 4.HE staining groups xenografts in nude mice, liver and kidney tissue. Using RT-PCR, Western-blot detection after injection of survivin mRNA and protein expression in tumor tissues. 6.TUNEL method to detect transplanted tumor cell apoptosis. Results: 1. Successfully established the growth of human renal cell carcinoma xenograft model. Xenograft tumor growth changes: calculated by measuring the volume of the transplanted tumor transplanted tumor growth curves. Found that starting from the first 19 days after tumor inoculation until 28 days after inoculation, the A, B group tumor volume was significantly less than C, D, E, F, G, and H group, P LT; 0.05. 28 days after inoculation stripping tumor weighing A, group B tumor tissue weight was significantly lighter than the C, D, E, F, G, H group, P lt; 0.05 3.HE staining observed by light microscopy transplanted tumors are renal transparent cell carcinoma, each group of kidney, liver, there was no significant histopathological abnormalities. 4.RT-PCR amplified: A B group transplanted survivin mRNA expression in the tumor tissue C, D, E, F, G, H group was significantly lower than P lt; 0.05 5.Western-blot detection transplantation of survivin protein expression in tumor tissue: results of the A, B group transplanted tumor tissue of survivin protein expression was significantly weaker than the C, D, E, F, G, H group, P lt; 0.05 6. transplanted tumor cell apoptosis: TUNEL Act detect the transplant tumor cell apoptosis index, A, B group transplanted tumor tissue apoptosis Ming higher than the C, D, E, F, G, H group, P lt; 0.01. Conclusion: In vitro synthesis of siRNA targeting survivin in vivo inhibition of the 786-0 cells transplanted tumor tissue of survivin mRNA and protein expression, and promote apoptosis of transplanted tumors and inhibit tumor growth.

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