WIF-1 or 5-aza-2'-deoxycytidine demethylation suppresses tumor growth in a mouse model of osteosarcoma

doi:10.3969/j.issn.2095-4344.2016.27.005

Abstract

Abstract:

BACKGROUND: WIF-1 is a tumor suppressor gene. Promoter hypermethylation causes WIF-1 down- regulation in most tumors. DNA methylation inhibitor can lead to gene demethylation and restore its expression.
OBJECTIVE: To observe the differences of tumor pathology and, WIF-1 mRNA and protein changes using WIF-1 or 5-aza-2'-deoxycytidine demethylation in animal models of osteosarcoma.
METHODS: Murine osteosarcoma models were established and divided into three groups. In the control group, no treatment was given. In the 5-aza-2'-deoxycytidine group, an appropriate amount of 5-aza-2'-deoxycytidine was injected in each mouse daily. In the WIF-1 group, an appropriate amount of Wnt/β-catenin signal transduction pathway inhibitor WIF-1 was injected in each mouse daily. Seven days after medication, the weight of nude mouse was weighed every 7 days. Short tumor diameter (a) and the long diameter (b) were measured. The relative tumor volume was calculated. The relative growth rate of tumor was calculated at 7, 14, 21, 28 and 56 days. Four nude mice from ach group were sacrificed by pulling the neck at 7, 14, 21, 28 and 56 days after medication. Tumor tissues were stripped and the weight of them was weighed. Pathological analysis of the tumor was conducted. The expression of WIF-1 protein and WIF-1 mRNA was detected in osteosarcoma at 56 days after medication in the three groups.
RESULTS AND CONCLUSION: (1) Compared with the medication and control groups, the weight of nude mice was increased at 7, 14, 21, 28 and 56 days in the treatment group. No significant difference was found between the medication and control groups. (2) The tumor size was significantly smaller in the medication group than in the control group. WIF-1 mRNA and WIF-1 protein expression was increased in the medication group compared with the control group to different degrees. (3) Results suggested that WIF-1 gene promoter methylation is one of the mechanisms of the development of osteosarcoma. Use of WIF-1 or 5-aza-2'-deoxycytidine demethylation can inhibit tumor growth in animal models of osteosarcoma.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Osteosarcoma, Deoxycytidine, Methylation, Tissue Engineering

CLC Number:

R318

Duan Fei, Li Shu-zhong, Zhu Wan-ping, Kang Xue-hua, Zhang Heng-jia, Dai Sheng-jie, Tian Yan-peng. WIF-1 or 5-aza-2'-deoxycytidine demethylation suppresses tumor growth in a mouse model of osteosarcoma[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(27): 3984-3991.

Figures/Tables 3

2.1 实验动物数量分析参加实验动物60只，每组20只，接种后未发现感染，各组裸鼠存活良好，实验中未出现死亡。 2.2 各组裸鼠体质量比较见表1。实验前3组裸鼠体质量为(22.8±0.15) g，各组裸鼠体质量差异无显著性意义。用药后5-氮杂-2’-脱氧胞苷组与WIF-1组裸鼠体质量较对照组有所增加，但用药后第7，14，21，28，56天测量的裸鼠体质量5-氮杂-2’-脱氧胞苷组与对照组之间差异无显著性意义：第7天(t=1.667，P > 0.05)，第14天(t=1.866，P > 0.05)，第21天(t=1.910，P > 0.05)，第28天(t=0.643，P > 0.05)，第56天(t=3.184，P > 0.05)。用药后第7，14，21，28，56天测量的裸鼠体质量WIF-1组与对照组之间差异无显著性意义：第7天(t=2.828，P > 0.05)，第14天(t=4.899，P > 0.05)，第21天(t=9.245，P > 0.05)，第28天(t=7.000，P > 0.05)，第56天(t=4.700，P > 0.05)。 2.3 各组裸鼠瘤质量比较见表2。5-氮杂-2’ -脱氧胞苷组与WIF-1组瘤质量较对照组明显减小。5-氮杂-2’-脱氧胞苷组用药后第7，14，21，28，56天测量的瘤质量较对照组明显减小，差异有显著性意义(P < 0.05)。WIF-1组用药后第7，14，21，28，56天测量的瘤质量较对照组明显减小，差异有显著性意义(P < 0.05)。 2.4 各组裸鼠骨肉瘤生长曲线和相对增长率用药后第7天开始每7 d用游标卡尺测量肿瘤短径(a)和长径(b)，根据公式V=0.5×a2×b计算相对肿瘤体积并绘制生长曲线(图1)，对照组较5-氮杂-2’-脱氧胞苷组、WIF-1组增长明显较快。根据公式RTVn=Vn/V8计算第7，14，21，28，56天的肿瘤相对增长率(图2)，5-氮杂-2’-脱氧胞苷组、WIF-1组较对照组肿瘤增长速率明显减慢，WIF-1组较5-氮杂-2’ -脱氧胞苷组肿瘤增长速率有所减慢。 2.5 5-氮杂-2’-脱氧胞苷对裸鼠骨肉瘤中WIF-1 mRNA表达的影响 RT-PCR 结果显示对照组肿瘤组织中未见目的条带，5-氮杂-2’-脱氧胞苷组、WIF-1组肿瘤组织可见不同程度的 WIF-1 mRNA表达，WIF-1组肿瘤组织中WIF-1 mRNA的表达量比5-氮杂-2’-脱氧胞苷组略微减少，见图3。 2.6 5-氮杂-2’-脱氧胞苷对裸鼠骨肉瘤中WIF-1蛋白表达的影响采用Western Blot技术检测3组肿瘤组织中WIF-1蛋白的表达情况。结果显示WIF-1蛋白在对照组无表达，在5-氮杂-2’-脱氧胞苷组、WIF-1组有不同程度的表达，见图4。2.7 各组肿瘤组织的病理分级及对比用药第7，14，21，28，56天的瘤体进行苏木精-伊红染色做病理分析，对照组较5-氮杂-2’-脱氧胞苷组、WIF-1组分化较差，核大深染，病理性核分裂像较多，见图5-9。"

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