RNAa-mediated overexpression of WT1 induces apoptosis in HepG2 cells

World Journal of Surgical Oncology

Zheng-Yan Zhao¹  Shou-Jiang Huang¹  Kai Yang²  Qi-Qi Mao²  Hong Chen²  Xiang-Yi Zheng²  Yi-Wei Lin²  Qi Qin¹ 

[1] Department of General Surgery, Children Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China;Department of Urology, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, China

关键词: Apoptosis;    HepG2 cell;    Hepatocellular carcinoma;    dsRNA;    Small activating RNA;    WT1;   

Others  :  828078
DOI  :  10.1186/1477-7819-10-11

 received in 2011-10-02, accepted in 2012-01-13,  发布年份 2012

【 摘 要 】

Aim

Recent studies have reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a process termed RNA activation. The present study was conducted to evaluate the potential of WT1 induction by small activating RNA targeting the WT1 promoter (dsWT1) in the treatment of hepatocellular carcinoma.

Methods

The human hepatocellular carcinoma cell line HepG2 was transfected with dsRNA by liposomes. The expression of mRNA and protein in cells were investigated using real-time reverse real-time quantitative PCR and Western blot, respectively. Cell viability and clonogenicity were determined by MTT assay and clonogenicity assay, respectively. Cell apoptosis was evaluated by flow-cytometric analysis.

Results

Expressions of WT1 mRNA and protein in dsWT1 treated HepG2 cells were significantly elevated. Inhibition of cell viability by dsWT1 was dose-dependent and time-dependent. Reduction of the number and size of colonies formed were found in dsWT1 treated cells. dsWT1 induced significant apoptosis in HepG2 cells. The decreased anti-apoptotic protein Bcl-2 and elevated pro-apoptotic protein Bak expression were detected in dsWT1 treated cells. The level of pro-caspase-3 remarkably decreased and cleaved caspase-3 and PARP fragment were also detected in dsWT1 treated cells.

Conclusion

These data show that RNAa-mediated overexpression of WT1 may have therapeutic potential in the treatment of hepatocellular carcinoma.

【 授权许可】

   
2012 Qin et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]El-Serag HB: Hepatocellular carcinoma: an epidemiologic view. J Clin Gastroenterol 2002, 35(5 Suppl 2):S72-78.
• [2]Parkin DM, Pisani P, Ferlay J: Global cancer statistics. CA Cancer J Clin 1999, 49(1):33-64. 31
• [3]Bruix J, Llovet JM: Prognostic prediction and treatment strategy in hepatocellular carcinoma. Hepatology 2002, 35(3):519-524.
• [4]Li LC, Okino ST, Zhao H, Pookot D, Place RF, Urakami S, Enokida H, Dahiya R: Small dsRNAs induce transcriptional activation in human cells. Proc Natl Acad Sci USA 2006, 103(46):17337-17342.
• [5]Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, Corey DR: Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nat Chem Biol 2007, 3(3):166-173.
• [6]Wang J, Place RF, Huang V, Wang X, Noonan EJ, Magyar CE, Huang J, Li LC: Prognostic value and function of KLF4 in prostate cancer: RNAa and vector-mediated overexpression identify KLF4 as an inhibitor of tumor cell growth and migration. Cancer Res 2010, 70(24):10182-10191.
• [7]Yang L, Han Y, Suarez Saiz F, Minden MD: A tumor suppressor and oncogene: the WT1 story. Leukemia 2007, 21(5):868-876.
• [8]Hohenstein P, Hastie ND: The many facets of the Wilms' tumour gene, WT1. Hum Mol Genet 2006, 15(Spec No 2):R196-201.
• [9]Tatsumi N, Oji Y, Tsuji N, Tsuda A, Higashio M, Aoyagi S, Fukuda I, Ito K, Nakamura J, Takashima S, et al.: Wilms' tumor gene WT1-shRNA as a potent apoptosis-inducing agent for solid tumors. Int J Oncol 2008, 32(3):701-711.
• [10]Menke AL, Shvarts A, Riteco N, van Ham RC, van der Eb AJ, Jochemsen AG: Wilms' tumor 1-KTS isoforms induce p53-independent apoptosis that can be partially rescued by expression of the epidermal growth factor receptor or the insulin receptor. Cancer Res 1997, 57(7):1353-1363.
• [11]Perugorria MJ, Castillo J, Latasa MU, Goni S, Segura V, Sangro B, Prieto J, Avila MA, Berasain C: Wilms' tumor 1 gene expression in hepatocellular carcinoma promotes cell dedifferentiation and resistance to chemotherapy. Cancer Res 2009, 69(4):1358-1367.
• [12]Huang V, Qin Y, Wang J, Wang X, Place RF, Lin G, Lue TF, Li LC: RNAa is conserved in mammalian cells. PLoS One 2010, 5(1):e8848.
• [13]Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R: MicroRNA-373 induces expression of genes with complementary promoter sequences. Proc Natl Acad Sci USA 2008, 105(5):1608-1613.
• [14]Kuwabara T, Hsieh J, Nakashima K, Taira K, Gage FH: A small modulatory dsRNA specifies the fate of adult neural stem cells. Cell 2004, 116(6):779-793.
• [15]Morris KV, Santoso S, Turner AM, Pastori C, Hawkins PG: Bidirectional transcription directs both transcriptional gene activation and suppression in human cells. PLoS Genet 2008, 4(11):e1000258.
• [16]Schwartz JC, Younger ST, Nguyen NB, Hardy DB, Monia BP, Corey DR, Janowski BA: Antisense transcripts are targets for activating small RNAs. Nat Struct Mol Biol 2008, 15(8):842-848.
• [17]Clark AJ, Archibald AL, McClenaghan M, Simons JP, Wallace R, Whitelaw CB: Enhancing the efficiency of transgene expression. Philos Trans R Soc Lond B Biol Sci 1993, 339(1288):225-232.
• [18]Hartkamp J, Carpenter B, Roberts SG: The Wilms' tumor suppressor protein WT1 is processed by the serine protease HtrA2/Omi. Mol Cell 2010, 37(2):159-171.
• [19]McMaster ML, Gessler M, Stanbridge EJ, Weissman BE: WT1 expression alters tumorigenicity of the G401 kidney-derived cell line. Cell Growth Differ 1995, 6(12):1609-1617.
• [20]Englert C, Hou X, Maheswaran S, Bennett P, Ngwu C, Re GG, Garvin AJ, Rosner MR, Haber DA: WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis. EMBO J 1995, 14(19):4662-4675.
• [21]Sera T, Hiasa Y, Mashiba T, Tokumoto Y, Hirooka M, Konishi I, Matsuura B, Michitaka K, Udaka K, Onji M: Wilms' tumour 1 gene expression is increased in hepatocellular carcinoma and associated with poor prognosis. Eur J Cancer 2008, 44(4):600-608.
• [22]Morrison DJ, English MA, Licht JD: WT1 induces apoptosis through transcriptional regulation of the proapoptotic Bcl-2 family member Bak. Cancer Res 2005, 65(18):8174-8182.
• [23]Bouillet P, Strasser A: BH3-only proteins - evolutionarily conserved proapoptotic Bcl-2 family members essential for initiating programmed cell death. J Cell Sci 2002, 115(Pt 8):1567-1574.
• [24]Salvesen GS, Dixit VM: Caspase activation: the induced-proximity model. Proc Natl Acad Sci USA 1999, 96(20):10964-10967.
• [25]Germain M, Affar EB, D'Amours D, Dixit VM, Salvesen GS, Poirier GG: Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7. J Biol Chem 1999, 274(40):28379-28384.
• [26]Ivana Scovassi A, Diederich M: Modulation of poly(ADP-ribosylation) in apoptotic cells. Biochem Pharmacol 2004, 68(6):1041-1047.