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SNCG shRNA suppressed breast cancer cell xenograft formation and growth in nude mice SHEN Peihong1, FAN Qingxia 2,*, LI Yanwei3, ZHANG Wei4, HE Xiaokai2, WANG Zhen4 and ZHANG Yunhan2 1 The First Affiliated Hospital, and Department of Pathology, The Fifth Affiliated Hospital, Zhengzhou University, Zhengzhou 450000, China 2 Department of Oncology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450051, China 3 Department of Pathology, The Fifth Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China 4 Graduate School, Zhengzhou University, Zhengzhou 450001, China *Corresponding author: Prof. Fan Qingxia, Department of Oncology, The First Affiliated Hospital, Zhengzhou University Tel.: +86-0371-66902152; Email: [email protected] Grant support: A Specified Key Research Grant from Henan Province (No. 92102310224). Abstract Background: Overexpression of breast cancer-specific gene 1 (SNCG) is associated with poor prognosis in advanced breast cancer patients. This study aimed to determine the effects of SNCG knockdown in breast cancer cells by using small hairpin (sh)RNA. Methods: Four different SNCG shRNA oligonucleotides were designed and chemically synthesized to construct mammalian expression vectors. These vectors were then stably transfected into a breast cancer MCF-7 cell line to knockdown SNCG expression. After SNCG knockdown was confirmed, the stable cell lines were inoculated into nude mice. SNCG mRNA and protein expressions were analyzed by semi-quantitative RT-PCR and immunohistochemistry, respectively in both the stable cell lines and xenografts. Results: All four SNCG shRNA constructs significantly reduced SNCG mRNA and protein levels in MCF-7 cells, as compared to the unrelated sequence control shRNA and the liposome control mice (P < 0.05). SNCG-knockdown MCF-7 cells formed significantly smaller tumor masses than cells expressing the unrelated sequence control or the liposome control mice (P < 0.05). Conclusions: SNCG shRNA effectively suppressed breast cancer cell formation in vivo and may be a useful clinical strategy to control breast cancer. Keywords: Breast cancer, SNCG, shRNA, MCF-7 cells, xenograft assay; RT-PCR INTRODUCTION Breast cancer is the leading cause of cancer-related death in women between the ages of 35 and 45 years old and represents the second most common type of cancer and the fifth most common cause of cancer death in the world. Although recent diagnostic advances have enabled the identification of breast cancer at very early stages, remarkably improving treatment outcomes, many women still develop metastatic disease and ultimately die[1]. Development of breast cancer is thought to involve multiple genetic alterations, including activation of oncogenes and inactivation of tumor suppressor genes[2]. The oncogenic protein-encoding breast cancer-specific gene 1 (SNCG) has been found to be overexpressed in advanced breast cancers. Subsequent study determined that overexpression of SNCG is an independently predictive biomarker for breast cancer tumor recurrence and metastasis; thus, SNCG has been hypothesized as a potential therapeutic target for breast cancer[3]. SNCG is a member of the synuclein family of proteins that are believed to be involved in pathogenesis of neurodegenerative diseases. Synucleins are small soluble proteins expressed primarily in neural tissue and certain tumors[3]. The three synucleins that comprise the entire protein family (-synuclein, -synuclein, and SNCG, the latter is known as -synuclein) are characterized by a highly conserved alpha-helical lipid-binding motif with similarity to the class-A2 lipid-binding domains of the exchangeable apolipoproteins. However, the normal exact cellular function of -synuclein remains to be defined, although some data have suggested a role in the regulation of membrane stability and/or turnover[3]. A previous study by cancer researchers revealed that SNCG was completely absent in normal mammary gland tissue and in the early stages of breast cancer, but was highly expressed in advanced breast cancer[4]. Another study found that in colorectal cancer tissues aberrant expression and demethylation of SNCG was correlated with disease progression and a sensitive predictor of lymph node involvement[5]. Still other studies have reported that SNCG expression levels are significantly associated with perineural invasion/distant metastasis and are an effective prognostic factor in pancreatic cancer[3-6]. Tobacco exposure was able induce altered expression of SNCG in lung cancer cells through down-regulation of a key methyltransferase gene, DNMT3b. In addition, the gene that encodes the AP-1 transcription factor has been proposed as a key regulator SNCG gene expression, since inhibition of AP1 transactivation led to down-regulated SNCG expression and suppressed the tumor phenotype. Furthermore, -synuclein was implicated in late stage breast and ovarian cancer metastasis by enhancing cell motility through activation of the RHO family small-GTPases and ERK1/2[4]. SNCG was also required for effective ER-alpha signaling in breast cancer[5]. Collectively, these findings suggest that SNCG could be a potential target for tumor treatment[3-5]. In this study, we investigated whether knockdown of SNCG expression using small hairpin (sh)RNA could effectively control breast cancer. MATERIALS AND METHODS Materials A human breast cancer MCF-7 cell line was obtained from Nanjing Kenou Biology Co., Ltd. (Nanjing, China). RPMT-1640 cell growth medium and Hyclone fetal bovine serum (FBS) were purchased from Baoxin Bio-Technology Co., Ltd. (Zhengzhou, China). The RNA isolation reagent was from Shanghai GenePharma Company (Shanghai, China). pGPU6 expression vector carrying SNCG shRNAs, nonsense shRNA, and vector-control and RNAi-Mate transfection reagents were custom-made by Shanghai Jima Medicine and Technology Co., Ltd. (Shanghai, China). Nude mice were purchased from the Institute of Laboratory Animal Slack Co., Ltd. (Shanghai, China). The reverse transcription-polymerase chain reaction (RT-PCR) kit, SNCG primers, and molecular weight markers were purchased from Baosight Biotechnology Co., Ltd. (Zhengzhou, China). Anti-SNCG antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The SP hypersensitivity kit for immunohistochemistry was obtained from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Cell culture MCF-7 cells were grown in RPMT-1640 containing 10% FBS, penicillin (100 u/ml), streptomycin (100 u/ml) in a humidified incubator at 37oC with 5% CO2. Construction of SNCG shRNA and gene transfection pGPU6 vectors carrying one of four different SNCG shRNA constructs were custom-designed by using online software available from the Ambion and TaKaRa SNCG shRNA sequences were: shRNA2, shRNA1, 5'-CCAAGGAGAATGTTGTACAGA-3'; 5'-CAAGACCAAGGAGAATGTTGT-3'; 5'-GCCAAGACCAAGGAGAATGTT-3'; 5'-TGGTGAGCAGCGTCAACACTG-3'. shRNA3, and A nonsense shRNA4, sequence shRNA (5'-GTTCTCCGAACGTGTCACGT-3') was prepared and inserted into the pGPU6 vector for use as a negative control; GAPDH shRNA (5'- GTATGACAACAGCCTCAAG-3') was constructed as a positive control. All plasmids were synthesized and constructed by Shanghai GenePharma Company (China). The resultant plasmids were amplified and confirmed by sequencing prior to use. pGPU6 vector containing the different SNCG shRNA constructs, vector-only, nonsense, and positive control vectors were transfected into MCF-7 cells using a transfection kit from Shanghai Jima Medicine and Technology Co., Ltd. (China) according to the manufacturer’s instructions. Forty-eight hours later, the cells were treated with 800 g/mL of G418. Two weeks later, the G418-resistant clones were selected and expanded in 400 g/mL G418-containing growth medium. Knockdown of SNCG expression was confirmed by RT-PCR and immunocytochemistry. Nude mouse xenograft assay The animal care and usage protocol was carried out with approval from our Institution’s Animal Care and Ethics Committee. SNCG shRNA and control vector-transfected breast cancer MCF-7 cells were grown in monolayer in RPMT-1640 plus 400 g/mL G418 for three days and then subcutaneously injected into nu/nu nude mice (8 weeks of age) in the right flank through a 22-gauge needle with 2 106 tumor cells mixed with 50% Matrigel (BD Biosciences, Bedford, MA, USA) for a total volume of 200 l per mouse. The animals were then monitored for tumor formation and growth. At the end of the experiments, the tumor xenografts were excised for wet-weight determination and analysis. RT-PCR SNCG mRNA levels were analyzed by using a semi-quantitative RT-PCR. Briefly, total cellular RNA was isolated from MCF-7 xenografts using Trizol reagent and then reversely transcribed into cDNA using a reverse transcription kit. cDNA was then amplified in a mixture containing 2 l cDNA, 1 l primer each, 2 l dNTP, 0.5 l Taq, 2.5 10 x buffer under the following conditions: initial denaturation at 94oC for 4 min; 35 cycles of 94oC for 30s, 54oC for 45s, 72oC for 1 min; and a final extension at 72oC for 10 min. The PCR products were separated on a 1.5% agarose gel by electrophoresis and visualized under a UV light. Immunohistochemistry The xenografts taken from the nude mice were embedded in paraffin blocks and sectioned for immunostaining with an hypersensitivity immunohistochemistry kit. Briefly, the sections were de-waxed in xylene twice for 10 min each, rehydrated in a gradient ethanol series (100% down to 50%), and then incubated with 3% H2O2 at 37oC for 10 min and heated at 95oC for 15 min in 0.01M citrate buffer (pH 6.0) for antigen retrieval. After that, the sections were incubated with normal goat serum at 37oC for 10 min and replaced with the first antibody and incubated overnight at 4oC. The next day, the sections were washed with PBS three times for 5 min each and then incubated with a secondary anti-biotin antibody at 37oC for 30 min. Following washing with PBS, the sections were incubated with horseradish peroxidase-labeled streptomycin avidin solution at 37oC for 30 min. For color development, the sections were incubated with 3'-diaminobenzidine/H2O2 solution for up to 10 min. After washing with tap water, the sections were counterstained with hematoxylin solution and then subjected to conventional dehydration in ethanol (50% up to 100%) and “clarification” in xylene and mounting with coverslips. Control sections were incubated with normal goat IgG instead of primary antibodies or with the second antibody only. The stained sections were reviewed and scored using a Nikon microscope to visualize cytoplamic and nuclear staining of SNCG protein. Five high power fields were randomly selected and analyzed by using a high-definition[4]. Statistical analysis All measurements, such as SNCG expression in xenografts, are presented as mean ± standard deviation (SD) and statistically analyzed by using a paired Student’s t-test with SPSS software, version 10.0 (SPSS Inc., Chicago, USA). Statistically significant values were defined as P < 0.05. RESULTS Detection of SNCG shRNA transfection efficiency Transfection of SNCG shRNA vector into MCF-7 cells resulted in about 5-10% efficiency, as assessed under a fluorescent microscope. We, therefore, made stable MCF-7 cell sub-lines for each of the four SNCG shRNA constructs, the vector-only, and the nonsense shRNA vector for subsequent injections into nude mice to generate xenografts. SNCG mRNA expression in mouse xenografts The levels of SNCG mRNA in MCF-7 cells of the SNCG shRNA-transfected MCF-7 cells were assessed by semi-quantitative RT-PCR analysis. Levels of SNCG mRNA were found to be much lower in SNCG shRNA-transfected MCF-7 cells than in the liposome or non-sense control groups (P﹤0.05). There was no significant difference among the levels detected for the four SNCG shRNA-transfected groups (P > 0.05) or between the non-sense control and the liposome control groups (P > 0.05) (Table 1 and Histogram 1). Table 1. Levels of SNCG mRNA expression in MCF-7 cells Transfected construct SNCG mRNA concentration (mean ± SD) SNCG-siRNA-1 0.624 ± 0.010* SNCG-siRNA-2 0.626 ± 0.013* SNCG-siRNA-3 0.634 ± 0.008* SNCG-siRNA-4 0.631 ± 0.010* Nonsense shRNA 0.976 ± 0.076# Liposome control 0.983 ± 0.052# *P < 0.05 vs. control; #P > 0.05 between the two different controls SNCG protein expression in MCF-7 cells SNCG protein expression was assessed in the transfected MCF-7 cells by using immunocytochemical analysis. Expression of SNCG protein was found to be significantly decreased in MCF-7 cells transfected with any of the four SNCG shRNA, as compared to the controls (Fig. 1 and Histogram 2). Quantification of the protein levels in immunocytochemically stained sections (Table 2) revealed SNCG was significantly reduced by SNCG shRNA in all four SNCG shRNA-sublines, as compared to the control groups (P < 0.05). Table 2. Expression of SNCG protein in MCF-7 cells Transfected construct SNCG protein concentration (mean ± SD) SNCG-siRNA-1 4.27 ± 0.12* SNCG-siRNA-2 4.19 ± 0.22* SNCG-siRNA-3 4.15 ± 0.14* SNCG-siRNA-4 4.17 ± 0.13* Nonsense shRNA 7.92 ± 0.22# Liposome control 8.02±0.13# *P < 0.05 vs. control; #P > 0.05 between the two different controls SNCG shRNA-mediated inhibition of xenograft formation and growth in vivo The respective MCF-7 sublines were subcutaneously injected into nude mice (n=5 per group). After 14 days of growth, the tumor masses obtained from the SNCG shRNA-transfected MCF-7 cell xenografts were remarkably smaller than those from the control mice (P﹤0.05). There was no difference in tumor mass wet-weights from any four of the different SNCG shRNAs (P > 0.05) (Table 3). Table 3. SNCG shRNA inhibition of breast cancer cell xenograft formation and growth Xenograft Group (×10-3g)a % of inhibitionb Liposome control 12.23 ±0. 679 0 Nonsense shRNA 11.60 ± 0.52 0.54 ± 0.236 SNCG -shRNA1 2.01 ±0. 08 8.80 ± 0.32 SNCG -shRNA2 2.14 ±0. 14 8.69 ± 0.24 SNCG -shRNA3 2.03 ±0. 09 8.79 ± 0.27 SNCG -shRNA4 2.10 ± 0.14 8.73 ± 0.19 a F = 986.134, P < 0.001 b F = 1750.94, P < 0.001 (% of inhibition); *% of inhibition = [(Liposome control – shRNA)/ liposome control] SNCG mRNA expression in mouse xenografts The levels of SNCG mRNA in mouse xenografts of the SNCG shRNA-transfected MCF-7 cells were assessed by semi-quantitative RT-PCR analysis. The levels of SNCG mRNA were much lower in SNCG shRNA-transfected tumor xenografts than in either the vector or nonsense control tumors (Fig. 2 and Table 4) (P < 0.05). There was no significant difference among the four SNCG shRNA-transfected groups (P > 0.05) or between the nonsense control and the normal control tumors (P > 0.05). Table 4. Levels of SNCG mRNA expression in breast cancer cell xenografts Group Levels of SNCG mRNA (mean ± SD) Liposome control 2.62 ± 0.68 Nonsense shRNA 2.19 ± 0.37 SNCG-shRNA1 1.28 ± 0.36 SNCG-shRNA1 1.28 ± 0.36 SNCG-shRNA3 1.21 ± 0.47 SNCG-shRNA4 0.86 ± 0.17 F = 12.492, P < 0.001, SNCG shRNA vs. the control SNCG protein expression in mouse xenografts Immunohistochemical analysis was performed to assess the levels of SNCG protein expression in the xenografts. The xenografts from all four SNCG shRNA-transfected MCF-7 sublines presented expression of SNCG protein that was significantly decreased from the levels detected in the controls (Fig. 3). Furthermore, SNCG protein levels were significantly reduced by SNCG shRNA in all four of the SNCG shRNA xenografts, as compared to the control groups (P < 0.05) (Table 5). Table 5. SNCG protein expression in breast cancer cell mouse xenografts Gene transfection SNCG protein levels (mean ± SD) Nonsense shRNA 16.56 ± 0.86 Liposome control 17.90 ± 1.38 SNCG-shRNA1 8.06 ± 0.21 SNCG-shRNA2 6.98 ± 0.43 SNCG-shRNA3 6.98 ± 0.72 SNCG-shRNA4 7.70 ± 0.47 F=215.479, P < 0.001, SNCG shRNA vs. the control DISCUSSION In the current study, we have determined the anti-tumor growth effects of SNCG knockdown using SNCG shRNA in vivo. In particular, we found that SNCG shRNA significantly reduced expression of SNCG mRNA and protein in mouse xenografts, as compared to controls. Furthermore, tumor masses were dramatically decreased in response to knockdown of SNCG expression. The data from the current study indicates the potential benefit of SNCG shRNA as a clinical strategy to control breast cancer. Future studies will aim to determine the underlying molecular mechanism of this phenomenon and evaluate the feasibility of patient safety and effectiveness. SNCG is a human gene ocalized at 10q23.20-23.3. Its cDNA is approximately 5 kb in length and comprised of five exons that translate into a protein of 127 amino acids[5,6]. Under physiological conditions, SNCG protein expression is localized to nervous tissues[7-9]; however, an analysis of disease-specific cDNA library[10] found SNCG to be highly expressed in the cancerous states of the mammary glands, liver, stomach, and pancreas, especially at advanced tumor stages. Collectively, the data suggested that SNCG may play a role in tumor progression and metastasis, and implicated it as a tumor marker. Since then, many studies have demonstrated that those metastatic breast and ovarian cancers that overexpress SNCG are particularly insensitive to certain chemotherapeutic drugs, suggesting that SNCG might be exploited as a therapeutic target[14-17]. RNA interference is an established and effective means to specifically knockdown gene expression, and is a promising strategy by which to accomplish gene therapy. RNAi is a sequence-specific double-stranded (dsRNA) RNA that is capable of degrading the homologous mRNA[18], resulting in specific gene silencing at the translational level. RNAi is generally accepted as being more effective than antisense technology. Therefore, RNAi or shRNA may be novel therapeutic tools for treatment of many diseases[19-21]. Many previous studies, using shRNA to such common cancer-associated factors as VEGF, WT1, and KDR, have demonstrated the usefulness of RNAi; all of which effectively inhibited tumor formation and growth in nude mouse xenograft models of breast cancer MCF7 cells[22-25]. Likewise, our data indicates that SNCG shRNA is capable of inhibiting tumor formation and growth in nude mice. Reference 1. Zhu X. The general survey of 34299 women with breast disease. 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