Knockdown of Stat3 expression using RNAi inhibits growth of laryngeal tumors in vivo

Introduction

Signal transducers and activators of transcription (STATs) were originally identified as key components of the cytokine signaling pathways that regulate gene expression^[1,2]. Recent studies suggest that they have potential as novel molecular targets for control of the development and survival of laryngeal carcinomas. In mammals, there are 7 members in the stat family. Constitutive activation of one stat family member, stat3, has been shown to play a key role in promoting proliferation, differentiation, anti-apoptosis and cell cycle progression. Constitutive activation of stat3 occurs in a variety of tumor cell lines^[3–7], thus suggesting that stat3 is an important molecular target for tumor therapy. Constitutive stat3 signaling represents one of the key molecular events in the multistep process leading to carcinogenesis.

Several recent reports demonstrate that blockade of stat3 expression in human cancer cells suppresses proliferation in vitro and tumorigenicity in vivo. Attempts to block stat3 expression have been made using tyrosine kinase inhibitors^[8,9], antisense oligonucleotides^[5], decoy oligonucleotides^[10], dominant-negative stat3 protein^[11,12] and RNA interference (RNAi)^[13,14]. In vitro studies have shown that inhibition of stat3 activity in human tumor cells induced apoptosis and/or growth arrest. In human head and neck squamous carcinoma cells, blocking of stat3 signaling by decoy oligonucleotides or antisense oligonucleotides abrogates transforming growth factor and suppresses the oncogenic growth of these cells^[15,16].

In the RNAi approach, sequence-specific post-transcriptional gene silencing is achieved by small interfering RNA (siRNA): short double-stranded RNA molecules in which the antisense strand is complementary to the target mRNA of a given gene^[17,18]. RNAi technology is currently being used not only as a powerful tool for analyzing gene function, but also for developing highly specific therapeutics. Our previous studies have demonstrated that blockade of stat3 expression by siRNA in Hep2 human laryngeal tumor cells suppresses proliferation and induces apoptosis in vitro^[19]. However, it has not been determined whether blocking stat3 signaling with siRNA is sufficient to inhibit tumor growth in vivo.

In the present study, we used a DNA-vector-based stat3-specific RNAi approach to block stat3 signaling and to evaluate the biological consequences of stat3 downmodulation on tumor growth in a mouse model. The results indicate that blockade of stat3 expression using a specific RNAi approach can significantly reduce laryngeal tumor growth and induce apoptosis in vivo.

Material and methods

Plasmid construction pSilencer1.0-U6 (Ambion, Austin, TX, USA) was used for DNA vector-based siRNA synthesis under the control of the U6 promoter in vivo. The vector was constructed by first synthesizing the double-stranded DNA template encoding the siRNA oligonucleotides (GenBank accession number for human stat3: NM003150), which contained a sense strand of 19 nucleotides followed by a short space (TTCAAGAGA), then the reverse complement of the sense strand, followed by five thymidines as a RNA polymerase III transcriptional stop signal. The sequences were: forward 5'-GCAGCAGCTGAACAAC ATGTTCAAGAGA-CATGTTGTTCAGCTGCTGCTTTTTT-3' and reverse 5'-AATTAAAAAAGCAGCAGCTGAACAACATGTCTCTTGAA-CATGTTGTTCAGCT GCTGCGGCC-3' (located in the SH2 domain). The oligonucleotides were annealed in a buffer [100 mmol/L potassium acetate, 30 mmol/L N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-KOH (pH 7.4), and magnesium acetate 2 mmol/L] and the mixture was incubated at 90 ºC for 3 min and then at 37 ºC for 1 h. The double-stranded oligonucleotides were cloned into a ApaI-EcoRI site in the pSilencer 1.0-U6 vector (Ambion), in which short hairpin RNAs (shRNA) were expressed under the control of the U6 promoter. A negative control scrambled siRNA (Ambion), which had no significant homology to mouse or human gene sequences, was designed to detect any non-specific effects.

Cell culture and establishment of animal model Hep2 cells (2×10⁶/150 µL) were inoculated subcutaneously into the right flanks of nude mice, and establishment of palpable tumors was confirmed. The tumor volume (m₁²×m₂×0.5236, where m₁ represents the short axis, and m₂ the longer axis) was measured every 2–3 d. When tumors reached an average volume of ~50.69±11.25 mm³, 3 experimental groups (5 mice per group) were tested: (1) mock transfection (phosphate-buffered saline [PBS] buffer alone); (2) scrambled siRNA control (20 µg/mouse); and (3) pSilencer1.0-U6-STAT3-3 siRNA (20 µg/mouse). The samples were diluted in 50 µL of PBS buffer, and injected percutaneously into the tumor by using a syringe with a 27-gauge needle. Immediately after injection, tumors were pulsed with an electroporation generator (ECM 830, BTX Holliston, MA, USA). Pulses were delivered at a frequency of 1/s 150 V/cm for a duration of 50 ms. This process was repeated on day 20. Mice were killed on d 27, the tumors treated with either scrambled siRNA or STAT3 siRNA were excised for hematoxylin and eosin (HE) staining, and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and fluorescence-activated cell sorting (FACS) assays.

HE staining and TUNEL assays Serial sections of tumor tissue excised from animals were fixed in formalin, stained with HE, and processed for routine histological examination. The TUNEL assay was performed by using the in situ Cell Death Detection Kit (Roche), which relies on fluorescent labeling of DNA strand breaks. Three-micrometer sections from paraffin-embedded tissues were dewaxed and hydrated according to the standard protocol. After incubation with proteinase K (200 µg/mL) for 30 min at 21 °C, the TUNEL reaction mix containing BrdUTP, terminal deoxynucleotidyl transferase, and reaction buffer was added to the slides, and they were incubated in a humidified chamber for 60 s at 37 °C, followed by washing and incubation with a fluorescein isothiocyanate-labeled anti-BrdU monoclonal antibody for 30 min at room temperature. The reaction was visualized by fluorescence microscopy. TUNEL-positive cells exhibited green fluorescence.

Western blot analysis Anti-stat3, anti-phospho-Tyr705-stat3 (p-stat3), anti-cyclin D1, anti-survivin and anti-β-actin antibodies were obtained from Santa Cruz Biotech. Anti-Bcl-2 antibody was obtained from Dako Biotech. For Western blot analyses, 100 mg tumor tissue as described earlier was lysed with lysis buffer [5 mmol/L ethylenediamine tetraacetic acid (EDTA), 300 mmol/L NaCl, 0.1% Igepal, 0.5 mmol/L NaF, 0.5 mmol/L Na₃VO₄, 0.5 mmol/L phenylmethyl-sulfonyl fluoride, and 10 µg/mL each of aprotinin, pepstatin, and leupeptin; Sigma]. After centrifugation at 15 000×g for 30 min, the supernatant was analyzed for protein content using Bradford reagent (Bio-Rad, USA). For the analysis of stat3, 50 µg of total protein was electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred onto a PVDF membrane (Milli-pore, Bedford, MA, USA), and incubated with anti-STAT3 or anti-p-stat3 antibody as indicated earlier. For Bcl-2, 50 µg of total protein was resolved on a 12% SDS-PAGE gel, transferred onto PVDF membranes and then probed with anti-Bcl-2 antibody. For cyclin D1, 50 µg of total protein was resolved on a 10% SDS-PAGE gel, transferred onto PVDF membranes and then probed with anti-cyclin D1 antibody. For survivin, 50 µg of total protein was resolved on a 12% SDS-PAGE gel, transferred onto PVDF membranes and then probed with anti-survivin antibody. The immunoblots were visualized by using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, USA). The optical density of each band in these western blots was measured by using densitometry and the results are given as the relative expression of tumors versus normal tissue.

Northern blot analysis Total RNA was extracted from tissue with the Trizol reagent (Invitrogen) following the manufacturer’s instructions. For Northern blot analysis, 20 µg of total RNA was electrophoresed on a 1.2% agarose-formaldehyde gel, and blotted onto Hybond-N⁺ membranes (Amersham Pharmacia Biotech). Hybridization was performed using the express Hyb buffer (BD Clontech) with ³²P-labeled cDNA of survivin and actin as probes. Blots were exposed to Kodak MS film and then quantitated using a Molecular Dynamics PhosphorImager.

Statistical analysis A χ² -analysis was performed to evaluate the significance of differences between the experimental groups. For a single comparison of two groups, Student’s t-test was used. Two-way ANOVA using the Student-Newman-Keuls method was used for comparisons of tumor size in mice after different treatments. For all analyses, the level of significance was set at P<0.05. All statistical calculations were performed using the SigmaStat statistical software package (SPSS, Chicago, IL). Data are presented as the Mean±SD.

Results

Antitumor activity of stat3 siRNA In order to evaluate the effects of the stat3 siRNA vector on laryngeal tumor growth in vivo, we examined its antitumor efficacy using a nude mouse model. Mice were subcutaneously inoculated with 2×10⁶ Hep2 cells into their right flank. By d 13, palpable tumors had developed at the sites of injection (mean volume 50.69±11.25 mm³, n=5). The mice were divided into 3 groups of 5 mice each and injected intratumorally with TE Tris EDTA buffer, scrambled-siRNA control or pSilencer1.0-U6-STAT3 siRNA. This process was repeated on d 20. Animals were killed on d 27 and tumor sizes were determined. The mean tumor volume in mice treated with buffer alone was 918.12±89.03 mm³ on d 27. The mean tumor volume in mice treated with scrambled siRNA control was 896.42±92.23 mm³, and that in the group treated with stat3-siRNA was 306.24±28.13 mm³. The difference in tumor size between the mice treated with buffer and those treated with siRNA did not achieve statistical significance (P>0.05), whereas the group treated with stat3 siRNA showed markedly suppressed tumor growth compared with the controls (Figure 1A, 1B; P<0.01). To determine the mechanism of tumor growth inhibition in vivo, Hep2 tumors treated with either scrambled siRNA control or stat3 siRNA were excised for HE staining and analyzed by using TUNEL assay. The results from both experiments showed that stat3 siRNA-treated tumors had undergone massive apoptosis compared with the controls (Figure 1C–1H). These data suggest that stat3 siRNA injection into the tumor can exert significant antitumor effects.

Figure 1 Intratumoral electroinjection of STAT3 siRNA resulted in significant inhibition of tumor growth and induced apoptosis in tumor cells in vivo. (A) Mice treated with a scrambled vector control had visible tumors, whereas mice treated with 20 µg of the STAT3 siRNA vector had reduced tumor volumes. (B) Growth curves of Hep2 tumors treated with STAT3 siRNA. Mice were inoculated subcutaneously with Hep2 cells, and by d 13, the mean volume of the palpable tumors reached ~50.69±11.25 mm³ (n=5) at the sites of injection. At this time, the mice were divided into 3 groups and injected intratumorally with buffer, STAT3 siRNA or a scrambled vector control. This injection was repeated on d 20, and the tumor sizes were determined on days 0, 5, 13, 20, and 27. Mean±SEM. n=5. ^cP<0.01. (C–E) HE staining at 60× magnification, and (F–H) TUNEL assay at 400×magnification of in vivo Hep2 tumors treated with buffer (C,F), scrambled siRNA (D,G), or STAT3 siRNA (E,H). TUNEL-positive cells are green.

Reduction of stat3 expression siRNA-specific to the stat3 gene can significantly suppress stat3 protein expression and inhibit the growth of cells in vitro^[19]. To further study the molecular mechanism of growth arrest of the tumor in vivo, stat3 and p-stat3 expression in the tumors were analyzed by Western blotting, and the results indicate that stat3 and p-stat3 expression are markedly reduced in tumors treated with siRNA stat3, whereas scrambled and buffer groups had high levels of stat3 and p-stat3 expression (P<0.01; Figure 2A, 2B).

Figure 2 Western blot analyses for STAT3, p-STAT3, Bcl-2, cyclin D1 and survivin expression in Hep2 tumors transfected with STAT3 siRNAs. (A) STAT3 protein level in Hep2 tumors transfected with STAT3 siRNAs and scrambled siRNA revealed by Western blotting. (B) p-STAT3 protein level in Hep2 tumors after transfection with STAT3-siRNAs revealed by western blot analysis. (C) Cyclin D1 protein level in Hep2 tumors after transfection with STAT3-siRNAs revealed by western blot analysis. (D) Bcl-2 protein level in Hep2 tumors after transfection with STAT3-siRNAs revealed by Western blot analysis. (E) Survivin protein level in Hep2 tumors after transfection with STAT3-siRNAs revealed by western blot analysis. (F) Amounts of STAT3, p-STAT3, cyclin D1, Bcl-2 and survivin protein in Hep2 tumors transfected with STAT3 siRNAs, Mean±SEM in 3 separate experiments, ^cP<0.01 vs untreated.

Effects of downregulation of stat3 expression Recent studies^[20–27] indicate that a constitutively active stat3 induces the expression of anti-apoptotic genes such as Bcl-2, cyclin D1, and survivin. In order to determine if stat3 downregulation results in the suppression of these genes, Western blot and Northern blot analyses were performed on the extracts from tumors transfected with stat3 siRNA. Western blotting showed that the intracellular Bcl-2, cyclin D1, and survivin levels were significantly decreased in stat3 siRNA-transfected tumors compared with controls (Figure 2C–E). Northern blot analysis showed that intracellular survivin mRNA was significantly decreased in the tumors (Figure 3). Thus, we concluded that stat3 siRNA treatment downregulated the expression of Bcl-2, cyclin D1, and survivin.

Figure 3 Northern blot analysis of survivin mRNA in Hep2 tumors. (A) Hep2 tumors were transfected with either 20 µg of pSilencer1.0-U6 vector STAT3 siRNAs or scrambled siRNA. Twenty micrograms of total RNA extract was used for Northern blot analysis. (B) STAT3 mRNA expression as mean±SEM of 3 separate experiments, normalized to the expression level of β-actin. ^bP<0.05 vs buffer and scrambled siRNA.

Discussion

Laryngeal carcinoma, especially late-stage laryngeal carcinoma, is associated with high morbidity and poor long-term survival because of the absence of effective treatment methods. Current therapies for advanced laryngeal cancer are only marginally effective. Thus, better understanding of the molecular mechanisms underlying proliferation, differentiation and survival of laryngeal carcinoma is critical for the development of optimal therapeutic methodologies. Elevated stat3 activities have been detected in the primary tissues and cell lines of laryngeal tumors. Stat3 activates several genes whose products promote cell cycle progres-sion, for example cyclin D1, and c-Myc^[20–22], and prevent apoptosis, for example Bcl-2 and Bcl-XL^[23–27]. Our previous studies showed that stat3 plays a key role in promoting laryngeal tumor proliferation in vitro. In the present study, we further demonstrated that STAT3 played a key role in promoting laryngeal tumor proliferation in vivo.

Western blot analysis with anti-STAT3 or anti-phospho-STAT3 antibodies showed that stat3 siRNAs suppress stat3 expression in laryngeal tumors in vivo. The expression of p-stat3 in laryngeal tumors treated with stat3 siRNAs declined approximately 90%, indicating a good silencing efficiency (Figure 2B). More importantly, direct inhibition of stat3 signaling was accompanied by growth inhibition and induction of apoptosis in laryngeal tumors. We observed that Bcl-2, cyclin D1, and survivin expression were greatly diminished in tumors transfected with stat3 siRNA (Figures 2B, 2C, 3). Additionally, massive apoptosis of the tumor cells were detected by TUNEL and HE assays. The results of our study are consistent with those of two recent reports, in which stat3 siRNA was also used for the study of astrocytomas and human prostate cancer^[13,28]. The results of all 3 studies support the hypothesis that stat3 participates in oncogenesis is by inhibiting apoptosis through the induction of anti-apoptotic genes. Konnikova et al reported that stat3 was required for the survival of the anti-apoptotic genes survivin and Bcl-xL (a member of the Bcl-2 family of proteins) in astrocytoma cells^[14]. Likewise, Lee et al also showed that inhibition of stat3 gene expression by siRNA induces apo-ptosis in human prostate cancer^[13]. Moreover, emerging evidence suggests that constitutive activation of stat3 appears to be ubiquitous in tumors, which renders tumor cells resistant to apoptotic death by unbalancing the expression levels of anti-apoptotic and apoptotic genes^[13,28].

Chemical synthesis of siRNAs is not cost-effective for large-scale screening projects, and simple synthetic siRNAs are unstable in mammalian cells, especially for use in in vivo studies. Fortunately, this problem has been addressed by using plasmid expression vectors as a delivery tool^[11,29,30]. These vector systems produce stable amounts of siRNA by utilizing the cellular machinery^[31]. Mammalian expression vectors synthesizing siRNA-like transcripts are able to cause gene knockdown^[32]. In order to evaluate the effects of stat3 siRNA on in vivo laryngeal tumor growth, we examined the antitumor efficacy of STAT3 siRNA in a nude mouse tumor model. We discovered that inhibition of stat3 by administration of appropriate vector-based siRNAs into the tumor was an effective and feasible approach for laryngeal cancer therapy. In this study, we used DNA injection as a tool for suppressing stat3 and tumor growth. Further efforts to evaluate the therapeutic value of this promising approach should be followed.

In conclusion, the data presented here show that the blockade of stat3 signaling using the RNAi approach significantly suppressed stat3 expression in vivo, suggesting that stat3 signaling is a potential molecular target for laryngeal cancer therapy. Plasmid-based siRNA therapy for tumor suppression may offer an effective and inexpensive approach for the treatment of laryngeal tumors.

Acknowledgement

We thank Dr Su-qin PAN for providing valuable technical support.

References

1. Darnell JE Jr. STATs and gene regulation. Science 1997;277:1630-35.
2. Bromberg J, Darnell JE Jr. The role of STATs in transcriptional control and their impact on cellular function. Oncogene 2000;19:2468-73.
3. Garcia R, Yu CL, Hudnall A, Catlett R, Nelson KL, Smithgall T, et al. Constitutive activation of STAT3 in fibroblasts transformed by diverse oncoproteins and in breast carcinoma cells. Cell Growth Differ 1997;8:1267-76.
4. Garcia R, Bowman TL, Niu G, Yu H, Minton S, Muro-Cacho CA, et al. Constitutive activation of STAT3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells. Oncogene 2001;20:2499-513.
5. Grandis JR, Drenning SD, Chakraborty A, Zhou MY, Zeng Q, Pitt AS, et al. Requirement of STAT3 but not stat1 activation for epidermal growth factor receptor-mediated cell growth in vitro. J Clin Invest 1998;102:1385-92.
6. Takemoto S, Mulloy JC, Cereseto A, Migone TS, Patel BK, Matsuoka M, et al. Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins. Proc Natl Acad Sci USA 1997;94:13897-902.
7. Gouilleux-Gruart V, Gouilleux F, Desaint C, Claisse JF, Capiod JC, Delobel J, et al. STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients. Blood 1996;87:1692-7.
8. Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, et al. Inhibition of acute lymphoblastic leukaemia by a jak-2 inhibitor. Nature 1996;379:645-48.
9. Blaskovich MA, Sun J, Cantor A, Turkson J, Jove R, Sebti SM, et al. Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice. Cancer Res 2003;63:1270-9.
10. Leong PL, Andrews GA, Johnson DE, Dyer KF, Xi S, Mai JC, et al. Targeted inhibition of STAT3 with a decoy oligonucleotide abrogates head and neck cancer cell growth. Proc Natl Acad Sci USA 2003;100:4138-43.
11. Ni Z, Lou W, Leman ES, Gao AC. Inhibition of constitutively activated STAT3 signaling pathway suppresses growth of prostate cancer cells. Cancer Res 2000;60:1225-8.
12. Nakajima K, Yamanaka Y, Nakae K, Kojima H, Ichiba M, Kiuchi N, et al. A central role for STAT3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells. Embo J 1996;15:3651-8.
13. Lee SO, Lou W, Qureshi KM, Mehraein-Ghomi F, Trump DL, Gao AC. RNA interference targeting STAT3 inhibits growth and induces apoptosis of human prostate cancer cells. Prostate 2004;60:303-9.
14. Konnikova L, Kotecki M, Kruger MM, Cochran BH. Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells. BMC Cancer 2003;3:23.
15. Leong PL, Andrews GA, Johnson DE, Dyer KF, Xi SC, Mai JC, et al. Targeted inhibition of STAT3 with a decoy oligonucleotide abrogates head and neck cancer cell growth. Proc Natl Acad Sci USA 2003;100:4138-43.
16. Mora LB, Buettner R, Seigne J, Diaz J, Ahmad N, Garcia R, et al. Constitutive activation of STAT3 in human prostate tumors and cell lines: direct inhibition of STAT3 signaling induces apoptosis of prostate cancer cells. Cancer Res 2002;62:6659-66.
17. Fuchs B, Zhang K, Schabel A, Bolander ME, Sarkar G. Identification of twenty-two candidate markers for human osteogenic sarcoma. Gene 2001;278:245-52.
18. Grandis JR, Zheng Q, Drenning SD. Epidermal growth factor receptor-mediated STAT3 signaling blocks apoptosis in head and neck cancer. Laryngoscope 2000;110:868-74.
19. Gao LF, Xu DQ, Wen LJ, Zhang XY, Shao YT, Zhao XJ. Inhibition of STAT3 expression by siRNA suppresses growth and induces apoptosis in laryngeal cancer cells. Acta Pharmacol Sin 2005;26:377-383.
20. Masuda M, Suzui M, Yasumatu R, Nakashima T, Kuratomi Y, Azuma K, et al. Constitutive activation of signal transducers and activators of transcription 3 correlates with cyclin D1 overexpression and may provide a novel prognostic marker in head and neck squamous cell carcinoma. Cancer Res 2002;62:3351-5.
21. Sinibaldi D, Wharton W, Turkson J, Bowman T, Pledger WJ, Jove R. Induction of Survivin WAF1/CIP1 and cyclin D1 expression by the Src oncoprotein in mouse fibroblasts: role of activated STAT3 signaling. Oncogene 2000;19:5419-27.
22. Hannon GJ. RNA interference. Nature 2002;418:244-51.
23. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C, et al. STAT3 as an oncogene. Cell 1999;98:295-303.
24. Alas S, Bonavida B. Rituximab inactivates signal transducer and activation of transcription 3 (STAT3) activity in B-non-Hodgkin’s lymphoma through inhibition of the interleukin 10 autocrine/paracrine loop and results in down-regulation of Bcl-2 and sensitization to cytotoxic drugs. Cancer Res 2001;61:5137-44.
25. Puthier D, Bataille R, Amiot M. IL-6 up-regulates mcl-1 in human myeloma cells through JAK/STAT rather than ras/MAP kinase pathway. Eur J Immunol 1999;29:3945-50.
26. Aoki Y, Feldman GM, Tosato G. Inhibition of STAT3 signaling induces apoptosis and decreases surviving expression in primary effusion lymphoma. Blood 2003;101:1535-42.
27. Tsujimoto Y, Shimizu S. Bcl-2 family: life-or-death switch. FEBS Lett 2000;466:6-10.
28. Scott S, Higdon R, Beckett L, Shi XB, deVere White RW, Earle JD, et al. Bcl 2 antisense reduces prostate cancer cell survival following irradiation. Cancer Biother Radiopharm 2002;17:647-56.
29. Far KR, Sczakiel G. The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucleic Acids Res 2003;31:4417-24.
30. Elbashir SM, Harborth J, Weber K, Tuschl T, et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 2002;26:199-213.
31. Dechow TN, Pedranzini L, Leitch A, Leslie K, Gerald WL, Linkov I, et al. Requirement of matrix metalloproteinase-9 for the transformation of human mammary epithelial cells by STAT3-C. Proc Natl Acad Sci USA 2004;101:10602-7.
32. Lou KQ, Chang DC. The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region. Biochem Biophys Res Commun 2004;318:303-10.