WDR7 up-regulation upon knocking down of neighboring non-coding RNA using siRNAs encapsulated in polyamidoamine dendrimers

Document Type: Original Article


1 Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran

2 Medical Cellular and Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran

3 Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Gorgan, Iran

4 Department of Modern Sciences and Technologies, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

5 Department of Molecular Medicine, School of Advanced Technologies, Shahrekord University of Medical Sciences, Shahrekord, Iran

6 Department of Medical Genetics, School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Gorgan, Iran


Objective(s): Breast cancer is the second leading cause of cancer death in females. Understanding molecular mechanisms in cancer cells compared with normal cells is crucial for diagnostic and therapeutic strategies. Long intergenic non-protein coding RNA, a regulator of reprogramming (lincRNA-RoR) is a noncoding RNA which initially was detected in induced pluripotent stem cells, and it has an important role in cell reprogramming and highly expressed in breast cancer cells. A key point in successful gene silencing is the usage of siRNA delivery system that is safe and efficient.
Materials and Methods: In this study, the fifth-generation of PAMAM dendrimer is used as a nanocarrier for entering siRNA molecules for gene silencing of lincRNA-RoR. WDR7 is the gene encoding adjacent of lincRNA-RoR, which has an important role in apoptosis and cell cycle. Gel retardation assay was used to find the best Negative/Positive (N/P) molar charge ratio of siRNA- PAMAM transfected into MDA-MB 231 cells. MTT assay was performed 24 hr after transfection revealed the IC50 value (half maximal inhibitory concentrations) about 100 nanomolar for lincRNA-ROR siRNA.
Results: The lincRNA-RoR and WDR7 gene expression changes were evaluated by real-time PCR after siRNA treatment and showed an increase in the gene expression of WDR7.
Conclusion: This study showed that PAMAM dendrimer G5/ siRNA could be a useful system delivery for future gene therapy approaches.


Main Subjects

1. Siegel RL, Miller KD, Jemal A. Cancer statistics. CA Cancer J Clin2017; 67:7-30.
2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131:861-872.
3. Sahebi R, Malakootian M, Balalaee B, Shahryari A, Khoshnia M, Abbaszadegan MR, et al. Linc-ROR and its spliced variants 2 and 4 are significantly up-regulated in esophageal squamous cell carcinoma. Iran J Basic Med Sci 2016; 19:1131-1135.
4. Wang Y, Xu Z, Jiang J, Xu C, Kang J, Xiao L, et al. Endogenous miRNA sponge lincRNA-RoR regulates Oct4, Nanog, and Sox2 in human embryonic stem cell self-renewal. Dev Cell 2013; 25:69-80.
5. Loewer S, Cabili MN, Guttman M, Loh Y-H, Thomas K, Park IH, et al. Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells. Nat Genetics 2010; 42:1113-1117.
6. Guo L, Zhao Y, Yang S, Zhang H, Wu Q, Chen F. An integrated evolutionary analysis of miRNA–lncRNA in mammals. Mol Biol Rep 2014; 41:201-207.
7. Xu X-D, Li K-R, Li X-M, Yao J, Qin J, Yan B. Long non-coding RNAs: new players in ocular neovascularization. Mol Biol Rep 2014; 41:4493-4505.
8. Rao AKDM, Rajkumar T, Mani S. Perspectives of long non-coding RNAs in cancer. Mol Biol Rep 2017; 44:203-218.
9. Batista PJ, Chang HY. Long noncoding RNAs: cellular address codes in development and disease. Cell 2013; 152:1298-1307.
10. Shi X, Sun M, Liu H, Yao Y, Song Y. Long non-coding RNAs: a new frontier in the study of human diseases. Cancer Lett 2013; 339:159-166.
11. Zhan H-x, Wang Y, Li C, Xu J-w, Zhou B, Zhu J-k, et al. LincRNA-ROR promotes invasion, metastasis and tumor growth in pancreatic cancer through activating ZEB1 pathway. Cancer Lett 2016; 374:261-271.
12. Zhang A, Zhou N, Huang J, Liu Q, Fukuda K, Ma D, et al. The human long non-coding RNA-RoR is a p53 repressor in response to DNA damage. Cell Res 2013; 23:340-350.
13. Guttman M, Donaghey J, Carey BW, Garber M, Grenier JK, Munson G, et al. lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 2011; 477:295-300.
14. Yang P, Yang Y, An W, Xu J, Zhang G, Jie J, et al. The long non‐coding RNA‐ROR promotes the resistance of radiotherapy for human colorectal cancer cells by targeting the P53/miR‐145 pathway. J Gastroenterol Hepato 2017; 32:837-845.
15. Eades G, Wolfson B, Zhang Y, Li Q, Yao Y, Zhou Q. lincRNA-RoR and miR-145 regulate invasion in triple-negative breast cancer via targeting ARF6. Molecular Cancer Res 2015; 13:330-338.
16. Chen Y-M, Liu Y, Wei H-Y, Lv K-Z, Fu P. Linc-ROR induces epithelial-mesenchymal transition and contributes to drug resistance and invasion of breast cancer cells. Tumor Biol 2016; 37:10861-10870.
17. Hou P, Zhao Y, Li Z, Yao R, Ma M, Gao Y, et al. LincRNA-ROR induces epithelial-to-mesenchymal transition and contributes to breast cancer tumorigenesis and metastasis. Cell Death Dis 2014; 5:e1287.
18. Takahashi K, Yan IK, Haga H, Patel T. Modulation of hypoxia-signaling pathways by extracellular linc-RoR. J Cell Sci 2014; 127:1585-1594.
19. Takahashi K, Yan IK, Kogure T, Haga H, Patel T. Extracellular vesicle‐mediated transfer of long non‐coding RNA ROR modulates chemosensitivity in human hepatocellular cancer. FEBS Open Bio 2014; 4:458-467.
20. Zhou X, Gao Q, Wang J, Zhang X, Liu K, Duan Z. Linc-RNA-RoR acts as a “sponge” against mediation of the differentiation of endometrial cancer stem cells by microRNA-145. Gynecol oncol 2014; 133:333-339.
21. Rezaei M, Emadi-Baygi M, Hoffmann MJ, Schulz WA, Nikpour P. Altered expression of LINC-ROR in cancer cell lines and tissues. Tumor Biol 2016; 37:1763-1769.
22. Liu Xx, Rocchi P, Qu Fq, Zheng Sq, Liang Zc, Gleave M, et al. PAMAM dendrimers mediate siRNA delivery to target Hsp27 and produce potent antiproliferative effects on prostate cancer cells. Chem Med Chem 2009; 4:1302-1310.
23. Perez A, Romero E, Morilla M. Ethylendiamine core PAMAM dendrimers/siRNA complexes as in vitro silencing agents. Int J Pharm 2009; 380:189-200.
24. Merkulova M, Păunescu TG, Azroyan A, Marshansky V, Breton S, Brown D. Mapping the H (+)(V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation. Sci Rep 2014; 5:14827-14827.
25. Sanders S, Keck-Waggoner C, Zimonjic D, Popescu N, Thorgeirsson S. Assignment1 of WDR7 (alias TRAG, TGF-β resistance associated gene) to orthologous regions of human chromosome 18q21. 1→ q22 and mouse chromosome 18D. 1–E. 3 by fluorescence in situ hybridization. Cytogenet Genome Res 2000; 88:324-325.
26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 2001; 25:402-408.
27. Kreso A, Dick JE. Evolution of the cancer stem cell model. Cell stem cell 2014; 14:275-291.
28. Korkaya H, Liu S, Wicha MS. Breast cancer stem cells, cytokine networks, and the tumor microenvironment. J Clin Invest 2011; 121:3804-3809.
29. Nagano T, Fraser P. No-nonsense functions for long noncoding RNAs. Cell 2011; 145:178-181.
30. Wu J, Huang W, He Z. Dendrimers as carriers for siRNA delivery and gene silencing: a review. Sci World J 2013; 2013.
31. Gallas A, Alexander C, Davies MC, Puri S, Allen S. Chemistry and formulations for siRNA therapeutics. Chem Soc Rev 2013; 42:7983-7997.
32. LiD R. WD-repeatproteins: Structurecharacteristics, biologicalfunction, and their involvement in human diseases. Cell Mol Life Sci 2001; 58:2085-2097.
33. Villegas VE, Zaphiropoulos PG. Neighboring gene regulation by antisense long non-coding RNAs. Int J Mol Sci 2015; 16:3251-3266.