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Inhibition of HOXB7 Gene Expression in Melanoma Cells by Small Interfering RNA GE Lin-hu(葛林虎)1**, YU Bao-dan(于宝丹)2,

PENG Si-da(彭思达)2, ZHEN Li-xia(郑丽霞)2,

TAN Huo(谭获)2*, YE Xu(叶絮)1

WANG Chun-yang(王春燕)2

1

Department of Thoracic Surgery, First Affiliated Hospital, 2Centre of Oncology & Hematology, The First Affiliated Hospital, 3Division of Hematology, Second Affiliated Hospital, Guangzhou Medical College, Guangzhou 510260 CLC number: R739.5

Document code: A

Article: 1000-9604(2008)02-0090-10

10.1007/S11670-008-0090-4 ABSTRACT Objective: HOXB7 gene is a kind of transcription regulator over-expressed in malignant melanoma (MM) cell lines. It can specifically up-regulate the expression of angiogenic factors and tumor growth factors such as bFGF, GROa, VEGF and induce angiogenesis in melanoma, resulting in the proliferation and metastasis of tumor cells. We designed and synthesized HOXB7 specific siRNA to study its interfering effect on the expressions of HOXB7 and bFGF genes in melanoma A375 cell line and the biologic characteristics of A375 cells. Methods: Three synthesized siRNA with different sequences were separately transfected into A375 cells by lipofecter 2000. The expression of HOXB7 and bFGF mRNA in transfected cells was detected by RT-PCR 24 and 48 hours after transduction. The expression of bFGF protein in the transfected cells were detected by flowcytometry 48 hours after transfection. MTT assay was used to analyze the cell proliferation rate of siRNA transfected cells. Based on the in vitro experiment results, one effective siRNA sequence was selected for the construction of in vivo siRNA expression vector. Then, a malignant melanoma animal model was established. The siRNA expression plasmid was injected into the tumor foci and its influence on the growth and angiogenesis of tumor was observed. Results: The mRNA expressions of both HOXB7 and bFGF genes in the A375 cells reduced significantly 24 and 48 hour after transfection of siRNA. Expression level of the protein of angiogenic factor bFGF induced by HOXB7 gene in siRNA transfected cells was significantly lower than that in control cells 48 hours after transduction. Cell proliferation was also suppressed in siRNA transfected cells. Two of the three siRNA strands showed prominent interference effect. The in vivo study indicated that the tumor size and the microvessel density in the tumor both reduced after injection of HOXB7siRNA plasmid. Conclusion: Down-regulation of HOXB7 gene expression can effectively reduce the expression of angiogenic factor bFGF and the proliferation of MM cells. Besides, the growth and angiogenesis of MM tumor were also inhibited. Key words: Small interference RNA; Malignant melanoma cell; HOXB7 gene bFGF gene; siRNA expression vector

⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Received: Dec. 19, 2007; Accepted: Mar 10, 2008. This work was supported by the grants from the Research Foundation of Science & Technology Bureau of Guangzhou (2004Z2-E0011) and the Guangdong Province Natural Science Foundation (5002318). * Author to whom correspondence should be addressed. E-mail: [email protected] ** E-mail: [email protected]

INTRODUCTION Malignant Melanoma(MM) is a kind of cancer with high malignancy, active metastatic capability, resistance to current methods of therapy and poor prognosis[1]. The two major cellular biological factors affecting prognosis of MM are the proliferating index of tumor cells and the angiogenetic intensity of the tumor, the latter is more important. Tumor growth and

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metastasis rely on blood supply through angiogenesis. Several experiments and clinical pathologic studies have shown that, without blood supply, the development of a solid tumor can only reach a mass size of 1 to 2 mm3 and additional blood supply by angiogenesis is needed for further growth of the tumor[2]. Therefore, exploration for medicine fighting angiogenesis is important for the treatment and prevention of metastasis of tumors. The highly conservative Homeobox7 (HOXB7) gene which has been mapped on chromosome 17 belongs to homeobox-containing genes, which are a family of regulatory genes encoding transcription factors that primarily play a crucial role in embryonic development, cell proliferation and differentiation[3,4]. Besides, HOXB7 gene is also involved in oncogenesis of several kinds of malignancies including MM, glioblastoma and breast cancer. It is also expressed in a variety of leukemia lymphoma cell lines[5]. According to reports, HOXB7 is not expressed in normal melanocytes, but is invariably over-expressed in malignant melanoma cells[6]. It is reported that basic fibroblast growth factor (bFGF) gene, which is the main downstream target gene of HOXB7 in MM cells, is a kind of growth factors that can induce angiogenesis and cell growth. Treatment of melanoma cell lines with antisense oligomers targeting HOXB7 mRNA markedly inhibited cell proliferation and specifically abolished expression of bFGF mRNA. Transduction of antisense oligos to bFGF inhibited the growth of malignant cells[7,8]. Further studies indicated that expressions of angiogenetic factors and oncogenic factors including vascular endothelial growth factor(VEGF), melanoma growth-stimulatory activity/growth-related oncogenene α(GROα), interleukin-8 (IL-8), and angiopoietin-2 were up-regulated by HOXB7 transduction. The proliferation, metastasis and angiogenesis of transducted MM cells were also accelerated[6]. We inferred that HOXB7 gene would be a target to aim at to down-regulate the expression of tumor-associated growth factors and angiogenic factors, so as to treat MM. The recently found RNA interference (RNAi) phenomenon is a kind of defensive mechanism developed during evolution. Now, siRNA has become a useful research tool for gene silencing and its potential application in gene therapy has aroused widespread attention[9]. In this study, we used the RNA interference technology to explore the effects of inhibition of

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HOXB7 expression in A375 cells. Our study was divided into cell experiment part (in vitro study) and animal experiment part (in vivo study). In the in vitro study, 3 different siRNA sequences designed against HOXB7 gene sequence were transfected into A375 cell lines via lipofectamine 2000. The expression levels of cytokine bFGF and cell biological features of transfected A375 cells were analyzed after reduction of HOXB7 gene expression. The best siRNA sequence was selected to construct siRNA expression vector for in vivo study. Then, the in vivo tumor growth and inhibition of angiogenesis were observed to provide experimental data for the application of siRNA to interfere with the expression of HOXB7 gene and the treatment of MM.

MATERIALS AND METHODS Synthesis of Double-strand siRNAs The double-strand siRNAs were synthesized and purified by Ambion Co. (the U.S.). The sequences of siRNAs are: siRNA-1 sense 5’-GGGCAGAGGAAGAGACAUGtt-3’ and antisense 5’-CAUGUCUCUUCCUCUGCCCtt-3’; siRNA-2 sense 5’-GGAAACUCAAAUCGAAUAGtt-3’ and antisense 5’-CUAUUCGAUUUGAGUUUCCtg-3’; siRNA-3 sense 5’-GGAGGAAGAGGAAGAGUGAtt-3’ and antisense 5’-UCACUC UUCCUCUUCCU CCtt-3’ .

Cell Culture The A375 malignant melanoma cell line was bought from the Cell Bank of Chinese Scientific Academy, Shanghai. The cells were cultured in RPMI 1640 medium supplemented with 5 or 10% fetal calf serum at 37℃ in a 5% CO2 atmosphere. siRNA Transduction A375 cells were seeded in 24-well and 96-well culture plate until 50%~60% confluent. 50nmol/L siRNA was used to infect A375 cells according to the protocols of lipofectamine 2000. The experiments were divided into 5 groups, namely, the siRNA-1 transduction group, the siRNA-2 transduction group, the siRNA-3 transduction group, the empty vector group and the control group. Cells in all groups were cultured at 37℃ in 5% CO2 for 6 h. After a medium

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change, cells were grown for 24 h or 48 h and then harvested for experiments.

USA). Parameters including the positivity of stained cells and the mean fluorescence density of the cells were recorded.

RT-PCR Study Cell Proliferation Assay Then, 24 or 48 h after the cells were transfected with siRNA, total RNA was extracted from cells cultured in 24-well plate with Trizol (Invitrogen Co., USA) and was reverse transcribed into cDNA with reverse transcriptase AMV (Takara, Japan) and oligo(dT) as the primer. PCR method was used to detect the expression levels of HOXB7 and bFGF genes at transcription level after siRNA interference. β-actin was used as housekeeping gene control. The primers were synthesized by Boya Co. (China). The sequences are: HOXB7 dir 5’-CGCGCCCTTTGAGCAGAACC-3’ and rev 5’-CGCGGTCTTGTTCTCCTTTT-3’; bFGF dir 5’ATCCAAAACAGGACCTGGGC-3’ and rev 5’-AACTCCCATCACCAGCAGAG-3’; β-actin dir 5’CATTGTGATGGACTCCGGAGACGG-3’ and rev 5’-CATCTCCTGCTCGAAGTCTAGAGC-3’.

PCR amplification conditions were: HOXB7 94℃30s, 56℃45s, 72℃30s; bFGF 94℃30s, 58℃45s, 72℃30s for 28 cycles and then extended at 72℃ for 10 min. The products were subjected to electrophoresis on 1.5% agarose gel. The RT-PCR data were analyzed by scanning densitometry of gel bands with the gel image analysis software and by normalization to β-actin signals obtained for the same time. Expression levels of HOXB7 and bFGF were expressed as ratios of HOXB7/β-actin and bFGF/β-actin respectively. Every experiment was repeated 3 times. Flow Cytometry Analysis Indirect immune fluorescent staining method was used to detect the expression level of bFGF protein. 48 h after the A375 cells were seeded into the 6-well culture plates, 5°105 cells were collected from each experiment group and stained with trypan blue to ensure that 98% of the cells were alive. The collected cells were fixed, sealed, and incubated with rabbitanti-human bFGF antibody (Santa Cruz Co., USA) for 30 min at room temperature. After PBS washing, the cells were incubated with FITC-conjugated goat-antirabbit antibody and incubated in dark for 30 min at room temperature. Then, the cells were fixed again and were analyzed with flow cytometry (Beckman,

The cellular growth activity in vitro was analyzed by MTT colorimetry[10]. Single cell suspension was prepared and seeded into 96-well culture plate with a density of 2°103 per well. 24 h later, the cells were adherent and were added with siRNA and liposome to be cultivated for 24 h. Then, 20μl MTT was added to each well with a final concentration of 1mg/ml and incubated at 37℃ for 4 h until purple crystals were formed. Two hundred μl DMSO was added to solve the crystals. Absorbance at 490nm (A490) was read with microplate reader. The results were expressed as cell viability as follows: Cell viability (%)=(Absorbance of experiment group/ Absorbance of the control group) ×100%

In vivo Tumor Inhibition Assay A375 cells in exponential growth phase were injected s.c. at a dose of 5×106 into 4~6 week old BALB/C nude mice (half male, half female) purchased from and maintained at the Experimental Animal Center of Sun-yat-sen University (Guangzhou, China) under specific pathogen free (SPF) condition. Cell suspension was injected in a volume of 0.2 ml. When the maximal diameter of the tumor increased to 3~5mm, the mice were randomly divided into three groups with 6 of each. The groups were: (1) pSilencer3.1-H1-hygro/HOXB7 plasmid injection group; (2) pSilencer3.1-H1hygro/neo negative plasmid injection group; (3) normal saline control injection group. The plasmids were directly injected into the tumor with a single injection on days 1, 4, 7, and 10 days after grouping. The maximal and minimal lengths of tumors were measured with vernier intermittently and the tumor volume was calculated as: tumor volume=π×maximal length×minimal length×2/6[3]. Tumor inhibition rate=(tumor volume of the control group-tumor volume of the treatment group)/tumor volume of the control group×100%. Thirteen days later, the nude mice were killed. The tumors were peeled off subcutaneously, fixed in 4% paraformaldehyde for 12 h and paraffin sections were

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prepared and preserved at room temperature for further experiment.

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transfected cells, agarose gel electrophoresis of RNA was performed and the results were shown in Figure 1, confirming that the RNA is integrate.

Tumor Microvessel Density (MVD) Detection The paraffin sections were placed in oven at 60℃ for 45 min, dewaxed with dimethylbenzene and dehydrated with gradient ethanol. Then, the sections were put into citric acid repairing fluid, microwaved for 15 min, rinsed and incubated with 50μl peroxidase stopping solution at room temperature for 10 min. After that, the sections were incubated with 50μl non-immunologic animal serum sealing fluid at room temperature for 10 min, incubated with diluted first antibody (rabbit anti-human factor VIII antibody) at 4℃ overnight, with 50μl biotin- conjugated second antibody at room temperature for 10 min and with 50μl streptavidin- biotin-peroxidase solution at room temperature for 10 min. Finally, the sections were stained with DAB fluid, counter-stained with hematoxylin, rinsed with tap water until the sample color became blue, dehydrated, dewaxed and sealed with resinene. Microvessel density is counted according to Weidner’s methods[11]. Briefly, the whole section was scanned under LP and 3 randomly selected fields were further examined for microvessel count under HP (200×). Presence of cytosolic brownish yellow particles was regarded as vascular endothelial positive. A single or a clump of endothelial cells inside tumor tissues, no matter they were lumens or not, was considered a microvessel. Blood vessels with a lumen diameter more than the size of 8 erythrocytes or with muscular layer were excluded. The mean values of microvessel count in each patch of tissue were recorded. Statistical Analysis Data was analyzed with SPSS 10.0 statistics software and expressed in⎯x±s. A Student’s t-test was used for comparison. Analysis of variance was used for comparison of tumor volumes among groups and P<0.05 was considered significant.

RESULTS The purity and integrity of total RNA After the total RNA was extracted from siRNA

Fig.1. The total RNA extracted from cells after siRN A transduction.

The Interference Effects of siRNA on the mRNA Expression Levels of HOXB7 and bFGF Twenty four and 48 hours after A375 cells were transfected with siRNA, the mRNA expression levels of HOXB7 gene and the induced angiogenetic factor bFGF gene were detected with semi-quantitive RT-PCR method. The sizes of amplified fragments were 331bp respectively and 663bp and the size of amplified β-actin fragment was 260bp. The ratios of HOXB7/ β-actin and bFGF/β-actin were listed in Table 1 and Table 2. As indicated in Figure 2A, 24 hours after treatment with siRNA, the mRNA expression levels of HOXB7 and bFGF genes in si-HOXB7-1 group were reduced (59.0±2.5)% and (42.0±2.3)% respectively (P<0.05); those of the si-HOXB7-2 group were reduced (66.1±1.7)% and (56.5±0.9)% (P<0.05); those of the si-HOXB7-3 group were reduced (69.1±2.1)% and (59.3±1.5)% (P<0.05). As indicated in Figure 2B, 48 hours after treatment with siRNA, the mRNA expression levels of HOXB7 and bFGF genes decreased (13.6±1.9)% and (10.5±1.6)% (P>0.05) in si-HOXB7-1 group; decreased (69.5±2.3)% and (60.1±1.0)% (P<0.05) in si-HOXB7-2 group; decreased (71.8±1.7)% and (69.9±2.0)% (P<0.05) in si-HOXB7-3 group.

The Interference Effect of siRNA Targeting HOXB7 Gene on the Protein Expression of bFGF in MM Cells The bFGF expression level in cells of each group was detected with flow cytometry 48 h after transduction (Figure 3). The bFGF expression level

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decreased from 92.9% before treatment to 81.4% in si-HOXB7-1 group (P>0.05), to 33.3% in si-HOXB72 group (P<0.05) and to 14.8% in si-HOXB7-3 group (P<0.05). The bFGF expression levels in the control group and empty vector group were 92.9% and 91.3% respectively after transduction. The differences were not significant comparing with the baseline data (P>0.05)(Table 3).

Fig. 2. The RT-PCR electrophoresis results 24 and 48 h after siRNA transduction. A:

RT-PCR electrophoresis results 24 h after siRNA

transduction. Samples of a6 and b11 were Takara 100bp DNA Marker and the brightest band was the band of 500bp. Bands of a1, a7 and a12 reflected the mRNA levels of HOXB7 gene, bFGF gene andβ-actin gene in si-HOXB7-1 group. Bands of a2, a8 and a13 show the mRNA expression levels of the three genes in si-HOXB7-2 group. Bands of a3, a9 and a14 were indicative of the mRNA expression levels of the three genes in si-HOXB7-3 group. Bands of a4, a10 and a15 showed the mRNA levels of the three genes in the empty vector group. Bands of a5, a11 and a16 reflected the mRNA levels in control group. B: RT-PCR electrophoresis results 48 h after siRNA transduction. Bands of b1, b6 and b12 reflected the mRNA levels of the three genes in si-HOXB7-1 group. Bands of b2, b7 and b13 show the mRNA expression levels of the three genes in si-HOXB7-2 group. Bands of b3, b8 and b14 were indicative of the mRNA expression levels of the three genes in si-HOXB7-3 group. Bands of b4, b9 and b15 showed the mRNA levels of the three genes in the blank vector group. Bands of b5, b11 and b16 reflected the mRNA levels in control group.

Table 1 Ratios of HOXB7/β-actin and bFGF/β-actin 24 hours after transduction(⎯x±s)

HOXB7 bFGF #

Control group

Empty vector group

Si-HOXB7-1 group

Si-HOXB7-2 group

Si-HOXB7-3 group

1.653±0.053 1.306±0.053

1.487±0.044# 1.210±0.029#

0.678±0.067* 0.758±0.036*

0.545±0.064* 0.568±0.071*

0.511±0.043* 0.532±0.082*

P>0.05 compared with control group, *P<0.05 compared with control group Table 2 Ratios of HOXB7/β-actin and bFGF/β-actin 48 hours after transduction(⎯x±s)

HOXB7 bFGF #

Control group

Empty vector group

si-HOXB7-1 group

si-HOXB7-2 group

si-HOXB7-3 group

1.488±0.044 1.164±0.026

1.482±0.062# 1.143±0.053#

1.280±0.036* 1.042±0.075*

0.446±0.078* 0.465±0.061*

0.417±0.045* 0.350±0.026*

P>0.05 compared with control group, *P<0.05 compared with control group Table 3. The bFGF protein expression levels in each group after siRNA transduction(⎯x±s)

bFGF #

Control group

Empty vector group

si-HOXB7-1 group

si-HOXB7-2 group

si-HOXB7-3 group

92.9%

91.3% #

81.4%*

33.3%*

14.8%*

P>0.05 compared with control group, *P<0.05 compared with control group

Influence of HOXB7 Gene Silencing on the Proliferative Activity of A375 Cells The proliferative activity of A375 cells in each group 24 h after siRNA transduction detected by MTT

method was shown in Table 4. 24 h after transduction, cell viability was 72.77% in si-HOXB7-1 group (P<0.05), 37.08% in si-HOXB7-2 group (P<0.05) and 32.06% in si-HOXB7-3 group (P<0.05)(Figure 4).

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Fig.3. The bFGF protein expression levels in each group detected with flow cytometry 48 h after siRNA transduction. A: negative control; B: control group; C: empty vector group; D: si-HOXB7-1 transduction group; E: si-HOXB7-2 transduction group; F: si-HOXB7-3 transduction group.

Changes in Interference

Tumor

Volumes

after

siRNA

Tumor volumes in animals injected with normal saline or pSilencer3.1-H1 hygro/neo negative plasmid gradually expanded as time went by and the nude mice became exhausted day by day. There was no significant difference (P>0.05) in tumor volume between the above two groups 13 days after plasmid injection, that is, 3 days after the fourth plasmid injection. In HOXB7 plasmid transduction group, necrotic wounds appeared on the surfaces of tumors after plasmid injections. The tumor volumes in these groups decreased significantly comparing with those in normal saline group or negative plasmid group. Thirteen days after the first injection of plasmids, the tumor volume in HOXB7 plasmid groups was (0.134±0.039) cm3, significantly smaller than that in control group (P<0.05), suggesting that the tumor growth in HOXB7 plasmid injection group was

inhibited (Table 5). The variation curve of tumor volume in nude mice before and after injection is shown in Figure 5.

Fig.4. Influence of siRNA interference on the proliferative activity of A375 cells 1 cntrol group; 2 empty vector group; 3 si-HOXB7-1 group; 4 si-HOXB7-2 group; 5 si-HOXB7-3 group

Table 4. Proliferative activity in each group 24 h after transduction(⎯x±s) Control group After transfect #

1.600±0.347 *

Empty vector group

si-HOXB7-1 group

si-HOXB7-2 group

si-HOXB7-3 group

1.573±0.268#

1.168±0.307*

0.592±0.319*

0.513±0.422*

P>0.05 compared with control group, P<0.05 compared with control group

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www.springerlink.com/Chin J Cancer Res 20(2): 90-99, 2008 Table 5. Tumor volume (cm3) in nude mice before and after injection and tumor inhibition rate (TIR) (⎯x±s)

Before injection A B C

0.060±0.014 0.050±0.013 0.043±0.020

Days after first injection 10

4

7

0.239±0.021* 0.279±0.002* 0.415±0.0130

0.125±0.038* 0.802±0.080# 0.866±0.143

0.167±0.081* 0.909±0.156# 0.966±0.213

13

TIR

0.134±0.039* 0.929±0.157# 1.006±0.235

86.71% 7.79%

A: HOXB7 plasmid group; B: negative plasmid group; C: normal saline control group P>0.05 compared with control group, *P<0.05 compared with control group

#

in other two groups (P<0.05). There was no significant difference in tumor tissue MVD between normal saline control group and negative plasmid injection group (P>0.05).

Fig.5. The variation curves of tumor volume cm3 in nude mice of each group before and after injection. 1: before injection; 2: three days after the first injection 3: seven days after the first injection; 4: ten days after the first injection; 5: thirteen days after the first injection.

Changes in Tumor Histology As indicated in histological slides shown in Figure 6, tumor cells under microscope were polygonal in shape with pale cytoplasm, large and dark nucleus. Abundant microvessels with brown-colored endothelial cells could be seen on histological slides from negative plasmid group or normal saline control group. Only few microvessels along with patches of dissolved tumor cells and tissue necrosis foci could be seen on slides from HOXB7 transduction group.

Fig.6. The microscopic images of tumor tissues from nude mice in different group. A: normal saline control group; B: negative plasmid injection group; C: HOXB7 plasmid transduction group. A1,B1,C1: Image of immuno- histochemistry slide with factor VIII relative antigen staining(15×); A2,B2,C2: Image of immunohistochemistry slide with factor VIII relative antigen staining(30×). Table 6 Tumor tissue MVD in each group after injection(⎯x±s)

The Interference effect of siRNA Expression Vector on Microvessel Angiogenesis in Tumor Tissues of Nude Mice Tumor tissue MVD in HOXB7 plasmid transduction group, negative plasmid injection group and normal saline injection group were 2.8±1.9, 18.1±5.5 and 19.9±5.6 respectively. MVD in HOXB7 transduction group was significantly lower than those

MVD #

Normal saline group

Negative plasmid group

HOXB7 plasmid group

19.9±5.6

18.1±5.5#

2.8±1.9*

P>0.05 compared with control group, *P<0.05 compared with control group.

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DISCUSSION HOXB7 gene is overexpressed in MM. Treatment of melanoma cells with antisense oligomers targeting HOXB7 mRNA could markedly inhibit proliferation activity of tumor cells and specifically reduce the expression of its downstream angiogenetic bFGF mRNA. In this study, we adopted RNA interference technology and designed 3 siRNA sequences targeting HOXB7 gene. After transfecting the siRNAs into A375 cells by liposome, the decreased expression of bFGF and cell growth inhibition resulting from down-regulation of HOXB7 by siRNA were observed. The siRNA sequence which caused the most significant inhibition effect was screened out for in vivo study in animal models. We studied mRNA expression levels of HOXB7 and bFGF genes in A375 cells 24 hours and 48 hours after siRNA transduction. Results of RT-PCR tests showed that HOXB7 gene expression in A375 cells was inhibited after transduction. So was the bFGF gene induced by HOXB7. Among the 3 siRNA sequences we designed, si-HOXB7-3 had the most significant interference effect. Twenty four hours after siRNA transduction, the mRNA expression levels of HOXB7 and bFGF genes decreased (59.0±2.5)% and (42.0±2.3)% (P<0.05) respectively in si-HOXB7-1 group, decreased (66.1±1.7)% and (56.5±0.9)% (P<0.05) in si-HOXB7-2 group, decreased (69.1±2.1)% and (59.3±1.5)% (P<0.05) in si-HOXB7-3 group. Forty eight hours after siRNA treatment, the mRNA expression levels of HOXB7 and bFGF genes reduced (13.6±1.9)% and (10.5±1.6)% (P>0.05) respectively in si-RNA-1 group, reduced (69.5±2.3)% and (60.1±1.0)% (P<0.05) in si-HOXB7-2 group, reduced (71.8±1.7)%, (69.9±2.0)% (P<0.05) in si-HOXB7-3 group. The study conducted by Care et al[7] showed that there was a direct and selective interaction of the HOXB7 protein with the sequence encompassing nucleotides 1130 to 1159 of the bFGF promoter. Our study results also confirmed that siRNA targeting HOXB7 gene could reduce the mRNA expression of bFGF gene which is regulated by HOXB7 and expression of both genes were down-regulated at similar levels. In our study, the expression level of bFGF protein was analyzed by flow cytometry 48 hours after siRNA transduction into A375 cells. The results showed that bFGF protein expression levels decreased from 92.9% before treatment to 81.4% after treatment in si-HOXB7-1 group, to 33.3% in si-HOXB7-2 group,

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to 14.8% in si-HOXB7-3 group, that is, the protein expression level of bFGF gene were down-regulated about 11.5%, 59.6% and 78.1% respectively. The fact that the mRNA expression level and protein expression level of bFGF gene tend to be down-regulated in parallel after HOXB7 gene was inhibited by siRNA suggests that inhibition of HOXB7 gene by siRNA can effectively reduce the expression of its downstream bFGF gene. Meanwhile, the protein expression levels of bFGF gene in control group and empty vector group were 92.9% and 91.3% with no significant difference between them. Transduction of HOXB7 gene can not only induce the expression of bFGF gene, but also up-regulate the expression of angiogenetic factors and oncogenic factors including VEGF, GROα, IL-8 and stimulate the growth and metastasis of MM[6]. In our study, MTT method was used to detect the growth of A375 cells after siRNA transduction. We found that, compared with control group, the cell proliferation rate of transduction groups 24 hours after treatment were 72.77% in si-HOXB7-1 group, 37.08% in si-HOXB7-2 group and 31.56% in si-HOXB7-3 group. The differences were significant in the latter two groups. Our results proved that siRNA specifically targeting HOXB7 gene could effectively inhibit the growth of A375 cells. The cell proliferation rate of empty vector group was 92.8% that of control with no significant difference. It is the target sequence that determines the interference effect of siRNA. In the in vitro study, we designed 3 siRNA sequences based on the sequence of HOXB7 gene so as to screen out the best one for in vivo study. The selection of the coding sequences for siRNA was empirically determined that they started with GG and were analyzed by blast research to ensure that they did not have significant sequence homology with other genes. Usually, the searching of targeting site of siRNA is started from the initiation code (AUG) of the target gene with 21 nt leaded by AA. As the combination of RISC complex and mRNA may be influenced by the interaction between activation protein and the target mRNA and reduce the interference effect, the mRNA region which may interact with protein should be avoided in the selection of interference site[12,13]. Our results showed that, comparing with control or empty vector group, the expressions of HOXB7 mRNA and protein were reduced and cell proliferation rate was also most inhibited in si-HOXB7-3 group among the three si-HOXB7 groups. As indicated by RT-PCR results,

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the interference effect of si-HOXB7-1 weakened gradually 48 h after transfection with the mRNA expression level of HOXB7 in the transfected cells close to that of control or empty vector group. Whereas, the interference effect of the si-HOXB7-2 and the si-HOXB7-3 groups lasted much longer and was more significant after 48 h than after 24 h. In our study, the si-HOXB7-3 sequence contained 52.3% of GC, while si-HOXB7-1 contained 58.2% of GC. Besides, the less effectiveness of si-HOXB7-1 may also be related with the localization of its targeting sequence. Thomas Tuschl has pointed out that the target region from the open reading frame of a given cDNA sequence is preferably 50 to 100 nt downstream of the start codon and should avoid 50 or 30 untranslated regions (UTRs) or regions close to the start codon as these sites may be richer in regulatory protein binding sites. It is conceivable that UTR-binding proteins and/or translation initiation complexes could interfere with binding of RISC to the target RNA[14]. We selected the best siRNA sequence based on our in vitro study results and inserted its corresponding sense and antisense sequences downstream of RNApol III promoter of the vector and constructed the siRNA expression vector for in vivo study of the interference effect of siRNA on angiogenesis and other biological features in MM tumor. The results showed that tumor cells in si-HOXB7 plasmid group grew slowly with the presence of vairous necrotic foci on tumor surface and the tumor volume was markedly smaller than those in normal saline control group and negative plasmid group. However, in the normal saline control group and the negative plasmid group, the volumes of the grafted tumors increased significantly and the mice weaned away as they survived. Three days after the last injection (the fourth injection), the mice were killed. The average volume of tumors in HOXB7 plasmid group (0.134±0.039 cm3) was significantly smaller than those in the normal saline control group (1.006±0.235 cm3) and the negative plasmid group (0.929±0.157 cm3). The tumor volume was barely affected by the injection of negative plasmid comparing with that of the normal saline group. As shown in the variation curve of tumor volume, there was no significant difference in tumor volume among the three groups within 3 days after the first injection. After the second injection on the fourth day, tumor in HOXB7 plasmid group began to grow slowly. On the seventh day, the average tumor volumes in HOXB7 plasmid group, the negative plasmid group and the

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normal saline control group were (0.125±0.038)cm3 and(0.802±0.080)cm3, (0.866±0.143)cm3 respectively. The tumor volume in the HOXB7 plasmid group was significantly smaller than those in the other two groups and smaller than that after the first injection, suggesting that the effect of down- regulation of the expression of the target gene resulting from transduction of siRNA targeting HOXB7 gene into tumor may not be seen until a certain period of time. The fact that tumor volume in the HOXB7 plasmid group after the fourth injection (0.134±0.039cm3) was even smaller than that after the first injection may be explained that the transfected siRNA expression vector can steadily suppress the expression of the target gene in the cell, so that the tumor growth was continuously inhibited. Besides, repeated injection of plasmid into tumors may produce an accumulative effect leading to reduction in tumor volume after more injections. To mark and count the tumor vessels, we used immunohistochemistry staining method. The commonly used antibodies for labeling endothelial cells include anti-factor VIII/von Willebrand factor related antigen antibody, anti-CD31 antibody and anti-CD34 antibody. Each has its own strong and weak points and they are variable in specificity and sensitivity[13]. Among them, anti-factor VIII/von Willebrand factor related antigen antibody is the most commonly used, which stains the endothelial cell membrane and the staining result may provide much more information on blood vessel distribution in tumors. Factor VIII related antigen staining can label microvessels in tumors with endothelial cells being stained into brownish yellow color. The stained microvessels may present as lumens or single endothelial cell or endothelial cell bundle[15]. Our results showed that tumor MVD in HOXB7 plasmid group (2.8±1.9) was significantly less than normal saline control group (19.9±5.6) and negative plasmid group (18.1±5.5) and many necrotic foci also presented as a result of deficiency in blood supply. The tumor microvessel generation in negative plasmid group was not significantly suppressed comparing with the control group. In conclusion, the results of our in vitro study demonstrated that HOXB7 specific siRNA is able to significantly reduce the expression of HOXB7 gene in A375 cells and further down-regulate the expression of its downstream target bFGF gene, together with the growth of A375 cells. The nude mice xenograft tumor model results proved that down-regulation of the expression of HOXB7 gene can result in remarkable inhibition of the growth of

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