Small interfering RNA inhibits glucose regulated protein 94 expression in transplantable models of human ovarian carcinoma in nude mice

doi:10.3969/j.issn.2095-4344.2014.18.019

Abstract

Abstract:

BACKGROUND: After glucose regulated protein 94 (GRP94) was knockout in model mice of transplanted tumor, cellular adhesion is terminated, thus stimulating the proliferation of liver-derived cells and promoting the development of liver cancer. We speculate that GRP94 plays a protective role against liver cancer.
OBJECTIVE: To investigate the expression of endoplasmic reticulum molecular chaperone GRP94 mRNA and protein with small interfering RNA technique in nude mice model of transplantable human ovarian carcinoma, and to explore the effect of GRP94 mRNA and protein expression on the growth of transplanted tumor.
METHODS: The gene sequences of human GRP94 were obtained from Gene Bank. psiSTRIKETM/GRP94 was constructed, which is eukaryotic expression vector controlled by the U6 promoter of human RNA polymerase Ⅲ. The transplantable model of human ovarian carcinoma in nude mice was established using human ovarian cancer HO-8910 cell line. The eukaryotic expression vector was transfected into the transplanted tumors in nude mice, and the growth of the tumor was observed. The nude mice models were divided into three groups, specific small interfering RNA group, non-specific small interfering RNA group and saline control group. The volumes of the subcutaneous tumor were determined. RT-PCR and immunohistochemistry were used to detect the mRNA and protein expression of GRP94 respectively.
RESULTS AND CONCLUSION: The recombinant plasmid of RNA interference specific for GRP94 was successfully constructed. The subcutaneous tumors appeared in all the nude mice 5 days after transplantation. The diameter of subcutaneous tumors was 7-10 mm 14 days after transplantation. The growth of subcutaneous tumors in nude mice with interference specific for GRP94 treatment was significantly decreased as compared with non-specific small interfering RNA group and control group (P < 0.05). The proliferation activity was inhibited by 65.1%. The expression of GRP94 mRNA and protein was significantly down-regulated after treatment of psiSTRIKETM/GRP94 (P < 0.01). The transfection of psiSTRIKETM/GRP94 could significantly induce inhibitory effects on the growth of ovarian carcinoma in nude mice, and the underlying mechanism is associated with the down-regulated expression of GRP94 mRNA and protein.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

Key words: RNA, small interfering, ovarian neoplasms, neoplasm transplantation, oncogenes, plant tumor-inducing plasmids

CLC Number:

R318

Zhang Li-ying, Xu Ai-li, Li Pei-ling, Ai Li-min . Small interfering RNA inhibits glucose regulated protein 94 expression in transplantable models of human ovarian carcinoma in nude mice [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(18): 2897-2902.

Figures/Tables 5

References

[1] 张新晨,杨维良.葡萄糖调节蛋白在肿瘤细胞中的表达及意义[J]. 中华普通外科杂志, 2003,18(8):501-502.
[2] Lee AS. The glucose-regulated proteins: stress induction and clinical applications.Trends Biochem Sci. 2001;26(8):504-511.
[3] Fu Y, Lee AS. Glucose regulated proteins in cancer progression, drug resistance and immunotherapy. Cancer Biol Ther. 2006;5(7):741-744.
[4] 崔照琼,张彦,李艳琼. RNA干扰技术应用新进展[J].安徽医药, 2012,16(3):283-285.
[5] 孙平,赵微,赵毅玲. RNA干扰技术的原理与应用[J].医学综述, 2011,17(2):164-167.
[6] Chiou JF, Tai CJ, Huang MT, et al. Glucose-regulated protein 78 is a novel contributor to acquisition of resistance to sorafenib in hepatocellular carcinoma. Ann Surg Oncol. 2010; 17(2):603-612.
[7] Cabanes D, Sousa S, Cebriá A, et al. Gp96 is a receptor for a novel Listeria monocytogenes virulence factor, Vip, a surface protein. EMBO J. 2005;24(15):2827-2838.
[8] Parmiani G, Testori A, Maio M, et al. Heat shock proteins and their use as anticancer vaccines. Clin Cancer Res. 2004; 10(24): 8142-8146.
[9] Wang XH, Qin Y, Hu MH, et al. Dendritic cells pulsed with gp96-peptide complexes derived from human hepatocellular carcinoma (HCC) induce specific cytotoxic T lymphocytes. Cancer Immunol Immunother. 2005;54(10):971-980.
[10] Binder RJ, Srivastava PK. Peptides chaperoned by heat-shock proteins are a necessary and sufficient source of antigen in the cross-priming of CD8+ T cells. Nat Immunol. 2005;6(6):593-599.
[11] Nicchitta CV, Carrick DM, Baker-Lepain JC. The messenger and the message: gp96 (GRP94)-peptide interactions in cellular immunity. Cell Stress Chaperones. 2004;9(4):325- 331.
[12] Demine R, Walden P. Testing the role of gp96 as peptide chaperone in antigen processing. J Biol Chem. 2005;280 (18):17573-17578.
[13] Liu S, Wang H, Yang Z, et al. Enhancement of cancer radiation therapy by use of adenovirus-mediated secretable glucose-regulated protein 94/gp96 expression. Cancer Res. 2005;65(20):9126-9131.
[14] Di Paolo NC, Tuve S, Ni S, Hellström KE, et al. Effect of adenovirus-mediated heat shock protein expression and oncolysis in combination with low-dose cyclophosphamide treatment on antitumor immune responses. Cancer Res. 2006;66(2):960-969.
[15] Chen WT, Tseng CC, Pfaffenbach K, et al. Liver-specific knockout of GRP94 in mice disrupts cell adhesion, activates liver progenitor cells, and accelerates liver tumorigenesis. Hepatology. 2014;59(3):947-957.
[16] Langer R, Feith M, Siewert JR, et al. Expression and clinical significance of glucose regulated proteins GRP78 (BiP) and GRP94 (GP96) in human adenocarcinomas of the esophagus. BMC Cancer. 2008;8:70.
[17] Suriano R, Ghosh SK, Ashok BT, et al. Differences in glycosylation patterns of heat shock protein, gp96: implications for prostate cancer prevention. Cancer Res. 2005;65(14):6466-6475.
[18] Pan Z, Erkan M, Streit S, et al. Silencing of GRP94 expression promotes apoptosis in pancreatic cancer cells. Int J Oncol.2009;35(4):823-828.
[19] Chavany C, Mimnaugh E, Miller P, et al. p185erbB2 binds to GRP94 in vivo. Dissociation of the p185erbB2/GRP94 heterocomplex by benzoquinone ansamycins precedes depletion of p185erbB2. J Biol Chem. 1996;271(9): 4974-4977.
[20] Chen X, Ding Y, Liu CG, et al. Overexpression of glucose-regulated protein 94 (Grp94) in esophageal adenocarcinomas of a rat surgical model and humans. Carcinogenesis. 2002;23(1):123-130.
[21] 张超,庞全海. siRNA 与miRNA 在生物体基因调控中沉默机制的比较[J].中国生物化学与分子生物学报, 2012,28(5):393-398.
[22] Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science. 2002;296 (5567):550-553. PMID: 11910072
[23] Semizarov D, Frost L, Sarthy A, et al. Specificity of short interfering RNA determined through gene expression signatures. Proc Natl Acad Sci USA. 2003;100 (11):6347-6352.
[24] Jackson AL, Bartz SR, Schelter J, et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol. 2003;21(6):635-637.
[25] Chi JT, Chang HY, Wang NN, et al. Genomewide view of gene silencing by small interfering RNAs. Proc Natl Acad Sci USA. 2003;100 (11): 6343-6346.
[26] Uhlirova M, Foy BD, Beaty BJ, et al. Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis. Proc Natl Acad Sci USA. 2003;100(26):15607-15612.
[27] Van der Giessen K, Di-Marco S, Clair E, et al. RNAi-mediated HuR depletion leads to the inhibition of muscle cell differentiation. J Biol Chem. 2003;278 (47):47119-47128.
[28] Ashrafi K, Chang FY, Watts JL, et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature. 2003;421(6920):268-272.
[29] Pothof J, van Haaften G, Thijssen K, et al. Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi. Genes Dev.2003;1517 (4):443-448.
[30] Kubota K, Tsuda S, Tamai A, et al. Tomato mosaic virus replication protein suppresses virus targeted posttranscriptional gene silencing. J Virol. 2003;77(20): 11016-11026.
[31] Vanitharani R, Chellappan P, Fauquet CM. Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells. Proc Natl Acad Sci USA. 2003;100(6):9632-9636.
[32] McCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol. 2003; 21(6): 639-644.
[33] Kao SC, Krichevsky AM, Kosik KS, et al. BACE1 suppression by RNA interference in primary cortical neurons. J Biol Chem. 2004;279(3):1942-1949.