Lentivirus-Mediated Small Interfering RNA Targeting VEGF-C Inhibited Tumor Lymphangiogenesis and Growth in Breast Carcinoma.код для вставкиСкачать
THE ANATOMICAL RECORD 292:633–639 (2009) Lentivirus-Mediated Small Interfering RNA Targeting VEGF-C Inhibited Tumor Lymphangiogenesis and Growth in Breast Carcinoma BAOLIANG GUO,1 YAFANG ZHANG,2 GUOQING LUO,1 LICHUN LI,2 AND JIANGUO ZHANG1* 1 Department of General Surgery, The Second Clinical Hospital, Harbin Medical University, Harbin, China 2 Department of Anatomy, Harbin Medical University, Harbin, China ABSTRACT Lymph node metastasis is a major prognostic factor for patients with breast cancer. The activation of vascular endothelial growth factor (VEGF)-C plays a key role in lymph node metastasis through promoting lymphangiogenesis. Thus, we attempted to elucidate whether small interfering RNAs (siRNA) targeting VEGF-C could suppress lymphangiogenesis and lymph node metastasis in vivo. A lentivirus-based VEGF-C siRNA vector was infected into breast cancer cells and a xenograft model. The expression of VEGF-C mRNA and protein were quantiﬁed by quantitative real-time polymerase chain reaction (QRT-PCR), immunohistochemistry, and western blot analysis. The effect of VEGF-C siRNA on breast cancer cells was investigated by an invasion assay. Lymphangiogenesis was analyzed with anti-LYVE-1 and anti-D2-40 by immunohistochemical analysis. Lentivirus-mediated VEGF-C siRNA stably reduced VEGF-C mRNA and protein expression. VEGF-C siRNA inhibited the invasive ability of breast cancer cells in vitro. Five weeks after intratumoral injection, the tumor volume was signiﬁcantly smaller in the VEGF-C siRNA group than in the control scramble siRNA group in the MDA-MB-231 cell xenograft model. The numbers of LYVE-1 and D2-40 positive vessels per microscopic ﬁeld were signiﬁcantly decreased in the VEGF-C siRNA group, which indicates that VEGF-C siRNA inhibited lymphangiogenesis. Moreover, lymph node metastasis was signiﬁcantly suppressed by VEGF-C siRNA in vivo. In conclusion, these results indicate that lentivirus-mediated VEGF-C siRNA offers a new approach for therapeutic intervention to prevent tumor growth and lymphatic metastasis of breast cancer. Anat Rec, C 2009 Wiley-Liss, Inc. 292:633–639, 2009. V Key words: VEGF-C; RNA interference; lymph node metastasis; breast cancer Drs. Baoliang Guo and Yafang Zhang contributed equally to this work. Grant sponsor: National Natural Science Foundation of China; Grant number: 30672420; Grant sponsor: Department of Education of Heilongjiang Province Foundation of China; Grant number: No.1053G019. *Correspondence to: Jianguo Zhang, M.D., Department of General Surgery, The Second Clinical Hospital, Harbin Medical C 2009 WILEY-LISS, INC. V University, 246 Xuefu Road, Harbin 150081, China. Fax: þ86-451-86605079. E-mail: email@example.com Received 5 December 2008; Accepted 29 January 2009 DOI 10.1002/ar.20893 Published online in Wiley InterScience (www.interscience.wiley. com). 634 GUO ET AL. The spread of tumor cells to regional lymph nodes through the lymphatic system plays a central role in the dissemination of various human cancers such as breast cancer (Achen et al., 2006). Experimental tumor models and human clinicopathologic data indicate that lymphangiogenesis and lymph node metastasis are important prognostic indicators for the spread of cancer (Eccles et al., 2007; Miyahara et al., 2007). Moreover, lymphangiogenesis induced by breast cancer correlates with lymph node metastasis and promotes metastasis (Skobe et al., 2001). Lymphangiogenic factors have been shown to stimulate tumor lymphangiogenesis and correlate with lymph node metastasis (Stacker et al., 2002; Mohammed et al., 2007). Among them, VEGF-C appears to be the most important factor that contributes to lymphangiogenesis and acts via the cognate receptor tyrosine kinase VEGF receptor-3 (VEGFR-3) located on lymphatic endothelial cells (Achen and Stacker, 2008). Increased VEGF-C expression can promote the formation of tumor lymphangiogenesis and increase the metastatic spread of tumor cells to lymph nodes in numerous cancers including breast cancer (Mattila et al., 2002; Tamura and Ohta, 2003; Liu et al., 2008). Transgenic and knock-out mice tumor models indicate that VEGF-C is a vital factor in tumor lymphangiogenesis and the subsequent formation of lymph node metastasis (Mandriota et al., 2001; Karkkainen et al., 2004). Furthermore, overexpression of VEGF-C in the tumor microenvironment has been reported to be associated with poor prognosis and lymph node metastasis in human breast cancer patients (Nakamura et al., 2003; Mylona et al., 2007). In addition, tumor-secreted VEGF-C has recently been shown to act systemically by inducing lymphangiogenesis in the sentinel lymph node even prior to tumor cell invasion (Hirakawa et al., 2007). Small RNA interference (siRNA) technology has very rapidly emerged as a revolutionary tool for elucidating gene functions and offers a potential therapeutic strategy for various diseases including cancers (Dykxhoorn and Lieberman, 2005; Izquierdo, 2005). In this study, lentiviral-mediated siRNA targeting VEGF-C vector was used to maintain high-transfection efﬁciency. We investigated the effect of VEGF-C siRNA on the invasive ability of breast cancer cells in vitro and on lymphangiogenesis and lymph node metastasis in vivo. Moreover, tumor growth was examined in a xenograft model with subcutaneous implantation of MDA-MB-231 cells. Our results indicate that lentivirus-mediated VEGF-C siRNA decreased tumor invasion in vitro and inhibited lymphangiogenesis and lymph node metastasis in vivo. Lentivirus-mediated VEGF-C siRNA also inhibited growth of the primary tumors in vivo. These results indicate that gene therapy targeting VEGF-C by siRNA provides a novel approach for the treatment of metastatic breast cancer. 100 U/mL penicillin. Cells were cultured in a humidiﬁed 37 C incubator with 5% CO2, fed every 2 days with complete medium containing 10% FBS, and then subcultured when conﬂuence was reached. Construction of lentiviral vectors and transfection. Lentivirus vectors for human VEGF-C small hairpin RNA (shRNA) encoding a green ﬂuorescent protein (GFP) sequence was constructed by GENECHEM (Shanghai, China). The target shRNA sequence is 50 GGAGGCTGGCAACATAACA-30 (human VEGF-C gene GenBank accession no. NM_005429), and four RNAi candidate target sequences to human VEGF-C were designed. The lentivirus vectors containing VEGF-C shRNA were constructed by ligating the Xho I/Sac II digests of pEGFP-N1 and the VEGF-C shRNA PCR product and were conﬁrmed by sequencing. After testing knockdown efﬁciencies in 293T cells by western blotting, the lentivirus vectors with the best interference efﬁciency were selected to knockdown the endogenous VEGF-C in breast cancer cells. Negative control shRNA was provided by GENECHEM (Shanghai, China). Lentivirus-encoded shRNA against VEGF-C and control were prepared and titered to 2 108 (TU/mL) as previously described (Li and Rossi, 2008). Cells (1 104 cells/well) were seeded in six-well plates overnight before transfection. The virus (0.1 mL) was mixed with 0.1 mL complete medium containing polybrene (8 mg/mL) and added to cells and incubated for 1 hr at 37 C. Then, the cells were incubated in fresh complete medium containing polybrene for 24 hr, followed by incubation for 48 hr in complete RPMI-1640 medium. The cells were then harvested for subsequent studies. MATERIALS AND METHODS Quantitative real-time PCR. Total RNA was isolated using Trizol reagent (Promega) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed into cDNA with Moloney murine leukemia virus RT (Promega). Human GAPDH RNA was used as an internal control. Primers for VEGF-C and GAPDH are as follows: VEGF-C: forward: 50 -GGGGAAGGAGTTTGGAGT-30 , reverse: 50 -GCTCGTG CTGGTGTTCAT-30 ; GAPDH: forward: 50 -CCATCACCAT CTTCCAGG-30 , reverse: 50 -ATGAGTCCTTCCACGATAC30 . Gene expression levels were evaluated by real-time quantitative PCR kinetics with SYBR Master Mix (Takara, Japan). Real-time PCR was performed with 1.0 lL of appropriate diluted cDNA, 0.5 lL (5 lM) of forward and reverse primers speciﬁc for human VEGF-C and GAPDH, 10 lL of SYBR PremixEx taq, and 8.0 lL of water. The cycling conditions were as follows: 95 C for 15 sec; 45 cycles of 95 C for 5 sec, and 60 C for 30 sec. At the end of the cycles, the temperature was raised to 95 C for 1 min. The melting curve was achieved by ﬁrst cooling samples to 55 C for 1 min, and then followed by 81 cycles (30 sec/cycle), in which the temperature was raised by 0.5 C per cycle to a maximum temperature of 95 C. Cell culture. The human breast cancer cell line MDA-MB-231 was obtained from the American Type Culture Collection. Cells were maintained in RPMI1640 (Solarbio, Beijing, China) supplemented with 10% fetal bovine serum (FBS), 100 lg/mL streptomycin, and Western blot analysis. Cells were lysed in ice-cold lysis buffer containing 100 mM Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate (SDS), 20% glycerol, and 2% hydroxyethanal. Equal amounts of cell lysate protein (20 lg of protein per lane) were subjected to SDS- In Vitro VEGF-C siRNA INHIBIT TUMOR LYMPHATIC METASTASIS polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinyl diﬂuoride (PVDF) membranes. For immunoblot analysis, the membranes were probed with speciﬁc primary antibodies: anti- VEGF-C, anti-Bcl-2, anti-Bax, and anti-GAPDH antibody (Santa Cruz). The bound primary antibodies were then incubated with horseradish peroxidase-conjugated anti-goat or antimouse (Santa Cruz) secondary antibodies, and then developed with an enhanced chemiluminescence detection system according to the manufacturer’s instructions. Invasion assay. The in vitro cell invasive assay was done in modiﬁed Boyden chambers. The top and bottom of the cell invasion chamber were separated by a polycarbonate ﬁlter with pores of 8-lm, which was coated with Matrigel. Approximately 1 104 cells were suspended in 100 lL serum-free supplement and were seeded onto the top chamber, and 1 mL of culture medium with 10% FBS was added to the lower chamber. Forty-eight hours later, the cells that had migrated to the lower chamber were ﬁxed in methanol and stained with hematoxylin. The invasive activity of cancer cells was determined by counting the cells with a microscope at 200 magniﬁcation. Five random visual ﬁelds were counted for each well, and the average was determined. In Vivo Animals and tumor production. Four- to 5-weekold female nu/nu nude mice were purchased from Beijing Vital River Experimental Animals Co. (Beijing, China) and housed in the Experimental Center of the Harbin Medical University. All animal protocols used for this study were approved by the Institutional Animal Care and Use Committee. To produce tumors, MDA-MB-231 cells (1 106 cells) in 0.1 mL HBSS were implanted into the left inguinal mammary fat pads of mice. Mice were randomized into three groups (six mice/group). After 1 week, one group of nude mice was intratumorally injected with PBS, the second group with lentivirus scramble siRNA, and the third group with lentivirus VEGF-C siRNA. Mice in all groups were injected weekly until the completion of experiments. Tumor growth was monitored weekly and measured in two dimensions. Tumor volume was calculated using the formula V ¼ W2 L/2, where W and L are the shortest and longest diameters, respectively. After 5 weeks, mice were killed and tumors were ﬁxed in 10% formalin solution and processed for immunohistochemical analysis of lymphangiogenesis and lymph node metastasis. Immunohistochemical analysis. Immunohistochemical staining was performed using the streptavidinperoxidase conjugate method. Brieﬂy, serial 4-lm-thick sections were cut from formalin-ﬁxed and parafﬁnembedded tumor blocks, dewaxed in xylene, rehydrated through sequential changes of alcohol, and then incubated with fresh 3% hydrogen peroxide for 15 min at room temperature. After washing with phosphate-buffered saline (PBS), the tissue sections were antigenretrieved in 0.01 M citrate buffer, pH 6.0, in a pressure steamer for 10 min. Sections were blocked with appropriate normal serum in PBS. After washing with PBS, slides were incubated overnight at 4 C with VEGF-C polyclonal rabbit anti-human antibody (Zhongshan Bio- 635 technology, Beijing, China), LYVE-1 monoclonal antimouse LYVE-1 antibody (R&D Systems, Minneapolis, MN) or D2-40 monoclonal anti-mouse antibody (Santa Cruz Biotechnology). After washing with PBS, slides were incubated with polyHRP goat anti-rabbit IgG (Zhongshan Biotechnology, Beijing, China) for 25 min at room temperature. After washing with PBS, slides were stained with fresh 3,30 -diaminobenzidine (DAB; Zhongshan Biotechnology, Beijing, China), counterstained in hematoxylin, dehydrated, and mounted. Sections of axillary lymph nodes were stained with anti-pan-cytokeratin antibody (Santa Cruz Biotechnology). The number of positive vessels was counted at 400 magniﬁcation in the area with highest intensity. Six different ﬁelds were quantiﬁed for each sample and the average value was calculated. Each section was examined by two independent observers. Statistical analysis. Data are expressed as means SE. The signiﬁcance of the data was determined by Student’s t test (two-tailed) and ANOVA analysis for all in vitro studies and the Mann-Whitney U test for in vivo studies. A P-value < 0.05 was considered statistically signiﬁcant. All statistical analyses were done using SPSS software. RESULTS Effect of VEGF-C siRNA on VEGF-C Expression Multiple siRNA sequences were designed, and the lentivirus siRNA vector with the greatest inhibitory activity was chosen. As shown in Fig. 1A, the real-time RT-PCR results indicate that endogenous VEGF-C mRNA expression was signiﬁcantly inhibited at 24 hr after infection in MDA-MB-231 cells. Compared with the control siRNA group, the VEGF-C siRNA group showed relatively lower quantities of VEGF-C mRNA; mRNA expression was decreased by nearly 70% (Fig. 1A). In accordance with this, Western blotting showed that VEGF-C protein expression was suppressed in the VEGF-C siRNA group (by 48%) compared with the control siRNA group in MDA-MB-231 cells (Fig. 1B). Infection success was also demonstrated by visual inspection for green ﬂuorescence. The transfection rate was calculated by the percentage of ﬂuorescent cells in the total cells and was over 70% in each visual ﬁeld (Fig. 1C). Effect of VEGF-C siRNA on Cell Invasion and Bcl-2/Bax Protein Expression To determine the function of lentivirus-mediated VEGF-C siRNA, MDA-MB-231 cells, a highly metastatic breast cancer cell line, were used. Using a Boyden chamber, we determined changes in cell invasion after 48 hr. The cells were ﬁxed and stained with crystal violet to determine the number of cells that invaded across the membrane. Compared with the control siRNA infected cells, VEGF-C siRNA infected cells showed a signiﬁcant decrease in invasion (Fig. 2A). Colorimetric analysis of the crystal violet-stained migratory cells indicated a 52% decrease in the number of invasive VEGF-C siRNA infected cells compared with control siRNA infected cells. Next, we also observed the effect of VEGF-C siRNA on Bcl-2 and Bax protein expression. Results showed 636 GUO ET AL. Fig. 1. Downregulating VEGF-C expression by lentivirus-mediated VEGF-C siRNA in MDA-MB-231 cells. MDA-MB-231 cells were infected with lentivirus-based VEGF-C siRNA and control scrambled siRNA. VEGF-C mRNA (A) and protein (B) expression were signiﬁcantly suppressed in the VEGF-C siRNA group compared with the control scramble siRNA group. Representative images were taken by ﬂuorescence microscope at 24 hr after infection (C). The green ﬂuorescence (GFP) demonstrates successful infection. A representative image for one of three independent experiments is shown. * Signiﬁcantly different from scramble siRNA (P < 0.05). that VEGF-C siRNA decreased the ratio of Bcl2/Bax (Fig. 2B). was observed in the mouse experiments, as assessed by changes in behavior, appearance, or weight. Effect of VEGF-C siRNA on Primary Tumor Growth Effect of VEGF-C siRNA on Tumor Lymphangiogenesis We next examined the effect of lentivirus-mediated VEGF-C siRNA on murine mammary tumor growth. As shown in Fig. 3, at day 35, the xenograft tumor sizes are as follows (mean SE): 915 186 mm3 in the PBS group, 869 165 mm3 in the scramble siRNA group, and 422 104 mm3 in the VEGF-C siRNA group. ANOVA analysis revealed that the inhibitory effect exerted by siRNA infection in the xenografts was signiﬁcant compared with that for scramble siRNA or for PBS (P < 0.05). Compared with the starting volume, delayed tumor growth was signiﬁcant in the VEGF-C siRNA group and was evident from the day after starting therapy until the day the mice were sacriﬁced. No toxicity Positive immunostaining for VEGF-C was observed in the cytoplasm of the tumor cells and the expression level was signiﬁcantly decreased in the VEGF-C siRNA group compared with the scramble siRNA and PBS groups (Fig. 4A). Next, the effect of VEGF-C siRNA on lymphangiogenesis was determined by immunohistochemical analysis using anti-LYVE-1 and anti-D2-40 antibodies. Representative results and quantitative data from the microlymphatic density analysis are shown in Fig. 4B,C, respectively. The numbers of LYVE-1 and D2-40 positive cells per microscopic ﬁeld were 3.78 1.1 and 3.76 1.0 in VEGF-C siRNA group, compared with 7.48 1.0 and 7.48 0.9 per microscopic ﬁeld in the scramble siRNA VEGF-C siRNA INHIBIT TUMOR LYMPHATIC METASTASIS Fig. 2. Inhibiton of breast cancer cell invasive ability by lentivirusmediated VEGF-C siRNA. A cell invasion assay of breast cancer cells was performed using a cell invasion assay kit. MDA-MB-231 cells were infected with VEGF-C siRNA or control scramble siRNA. The rate of cell invasion was evaluated in a modiﬁed Boyden chamber assay as described in the Materials and Methods. Histograms show the percentage of invading cells, with the invasion of cells in the scramble siRNA group designated as 100%. Data are representative of three independent experiments (A). The ratio of Bcl-2 and Bax protein (B) expression were signiﬁcantly suppressed in the VEGF-C siRNA group compared with the scramble siRNA group. * Signiﬁcantly different from scramble siRNA (P < 0.05). group and 7.55 0.9 and 7.62 0.9 per microscopic ﬁeld in the PBS group, respectively (Fig. 4). These data suggest that inhibition of VEGF-C in primary tumors by VEGF-C siRNA decreases lymphangiogenesis. Effect of VEGF-C siRNA on Local Lymph Node Metastasis Finally, we investigated the effect of VEGF-C siRNA on local lymph node metastasis. We found that nearly all of the control mice treated without VEGF-C siRNA developed metastasis in the lymph nodes, whereas some mice in VEGF-C siRNA group did not have lymph node metastases. The incidence of lymph node metastasis was signiﬁcantly decreased to 42% in the VEGF-C siRNA group compared with 81% in the scramble siRNA group and 83% in PBS group. These data suggest that inhibition of VEGF-C in primary tumors by VEGF-C siRNA suppressed local lymph node metastasis. In addition, the frequency of pan-cytokeratin-positive metastatic cells in lymph nodes was signiﬁcantly lower in the VEGF-CsiRNA group compared with the control groups (Fig. 5). DISCUSSION In this study, VEGF-C mRNA and protein expression was suppressed by lentivirus-mediated VEGF-C siRNA. VEGF-C siRNA inhibited the tumor invasion ability and the ratio of Bcl-2/Bax in vitro. Lentivirus-mediated VEGF-C siRNA inhibited lymphangiogenesis and spontaneous lymph node metastasis in the MDA-MB-231 cell xenograft model. Furthermore, lentivirus-mediated VEGF-C siRNA inhibited primary tumor growth of breast cancer in vivo. Breast cancer is the most common malignant tumor and metastasis of tumor cells is the second leading cause of cancer death affecting women worldwide (Stewart and Kleihues, 2003). Although tumor cell dissemination is mediated by a number of mechanisms, the most common 637 Fig. 3. Suppression of tumor growth by lentivirus-mediated VEGFC siRNA in MDA-MB-231 cell xenografts. MDA-MB-231 cell xenograft mice were intratumorally injected with lentivirus-mediated VEGF-C siRNA, scramble siRNA, or PBS. The size of the primary tumors was measured every week. Mice were sacriﬁced after 5 weeks. Points, mean tumor volume in each group; bars, SE. * Signiﬁcantly different from scramble siRNA (P < 0.05). pathway of initial dissemination is through the lymphatic system to the regional lymph nodes (Pepper et al., 2000). The presence of metastases in regional lymph nodes is a strong indicator of low-patient survival rate, poor patient prognosis in many types of cancer (Skobe et al., 2001; Miyahara et al., 2007; Liu et al., 2008). Despite advances in the treatment of breast cancer, morbidity and mortality from breast cancer remain unacceptably high. Nevertheless, more novel targets for therapeutic development have been identiﬁed and are being explored. Recent studies have demonstrated that VEGF-C overexpression is associated with lymph node metastasis by enhancing intratumoral lymphangiogenesis in breast carcinoma (Skobe et al., 2001; Mattila et al., 2002). Experiments with transgenic mice have shown that overexpression of VEGF-C results in lymphatic endothelial proliferation and vessel enlargement (Jeltsch et al., 1997). Likewise, the clinical signiﬁcance of VEGF-C expression in relation to lymph node metastasis and patient outcome has been reported in cancers of the esophagus, stomach, and colorectum (Liu et al., 2008). Downregulation of VEGF-C or blockade of VEGFR-3 signaling inhibits lymphangiogenesis and lymph node metastasis in experimental animal cancer models (Chen et al., 2005; Lin et al., 2005; Burton et al., 2008). These results indicate that VEGF-C could be an important therapeutic target to suppress lymphangiogenesis and lymphatic metastasis. Consists with previous study, our results showed VEGF-C siRNA might be exerted its function by decreasing the ratio of Bcl-2/Bax protein expression. SiRNA technology provides a novel strategy for investigating gene expression and function (Bantounas et al., 2004). Compared with antisense oligonucleotides and ribozymes, siRNA allows us to target any gene with greater speciﬁcity and efﬁciency, and it can be developed in various ways allowing for numerous in vitro and in vivo applications (Elbashir et al., 2001; Chen et al., 638 GUO ET AL. Fig. 4. Suppression of tumor lymphangiogenesis by lentivirus-mediated VEGF-C siRNA in MDA-MB-231 cell xenografts. Tumors were harvested and immunostained with VEGF-C, LYVE-1 and D2-40 antibody to assess lymphangiogenesis, as described in the Materials and Methods. VEGF-C expression in tumor cells (A), LYVE-1-positive microlymphatic vessel (B), and D2-40-positive microlymphatics (C) were decreased in the VEGF-C-siRNA group compared with the control and scramble siRNA groups (original magniﬁcation 400). Scale bar of immunostain results was shown in Fig.4D. * Signiﬁcantly different from scramble siRNA. 2003; Song et al., 2003). In addition, siRNA is becoming an important tool for the study of biological processes and has the potential for therapeutic applications in human cancer diseases (Dykxhoorn et al., 2006; Takeshita and Ochiya, 2006). Thus, we used siRNA to assess the effect of suppressing the VEGF-C gene in lymphatic metastasis in breast cancer. Effective delivery of siRNA to target cells is still a major issue. The use of siRNA/shRNA for gene knockout requires an efﬁcient stable transfection or transduction process. To improve efﬁciency, a lentivirus vector-based siRNA was constructed instead of synthetic RNA oligonucleotides or plasmid-encoding shRNAs (An et al., 2003). In this study, lentivirus vector delivery produced high-transfection rate in vitro and in vivo. Compared with the other vectors, the lentivirus-based gene vectors had advantages over the other viral and nonviral vectors in that they can be delivered to and integrate with genetic material in cells that are dividing or not (Cockrell and Kafri, 2007). Thus lentivirus-mediated siRNA targeting of VEGF-C appears to be particularly efﬁcacious to ensure gene silencing in vitro and in vivo. Fig. 5. Suppression of local lymph node metastasis by lentivirusmediated VEGF-C siRNA in MDA-MB-231 cell xenografts. Mice were sacriﬁced after 5 weeks and spontaneous metastatic lymph nodes were ﬁxed. Lymph node metastases were examined by immunohistochemistry with pan-cytokeratin staining. Columns represent the incidence and frequency of pan-cytokeratin-positive cells in the lymph nodes. * Signiﬁcantly different from scramble siRNA (P < 0.05). 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