Effect of shRNA targeting mouse CD99L2 gene in a murine B cell lymphoma in vitro and in vivo
Effect of shRNA targeting mouse CD99L2 gene in a murine B cell lymphoma in vitro and in vivo
复制标题
靶向小鼠 CD99L2 基因的 shRNA 对小鼠 B 细胞淋巴瘤体内外的影响
DOI：
10.3892/or.2013.2244
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发表时间：
2013-04-01
期刊：
影响因子：
4.2
通讯作者：
Zhao, Tong
中科院分区：
文献类型：
--
作者：
Liu, Fang;Zhang, Gong;Zhao, Tong
小鼠CD99抗原样2 （mCD99L2）先前已被本研究组证实在小鼠B淋巴瘤（A20）细胞中表达。本研究旨在建立mCD99L2下调的A20细胞系，在体外和体内研究靶向mCD99L2的shRNA在A20细胞中的作用。构建4个含有mCD99L2 shrna表达盒的pLenti6/mCD99L2表达载体，转染A20细胞，建立稳定的mCD99L2下调亚克隆A20-mCD99L2（-）细胞，并通过定量PCR和western blot分析鉴定。采用光镜、透射电镜、MTT法、流式细胞术和免疫荧光标记法观察其体外形态、生物学和表型特征。部分A20-mCD99L2（-）细胞表现为H/ rs细胞样形态，增殖能力下降，G2期延长，CD30和CD15表达增加。将细胞注射到裸鼠或免疫正常的BALB/c小鼠体内，观察肿瘤的发生、生长、形态和表型。A20-mCD99L2（-）细胞在裸小鼠和BALB/c小鼠中诱导肿瘤，但后者的效力低于对照组。在A20-mCD99L2（-）细胞诱导的肿瘤中观察到与体外相似的形态学、生物学和表型特征。当使用抗体阵列检测mCD99L2下调后，CD30T、IL-12p40/p70、IL-3、ifn - γ、CXCL16、MIP-1 α和CD40等细胞因子上调。western blot分析结果表明mCD99L2表达的调控可能涉及活化的核因子- κ B通路。本研究为进一步研究肿瘤细胞中的mCD99L2基因提供了数据。
Mouse CD99 antigen-like 2 (mCD99L2) has previously been confirmed to be expressed in murine B lymphoma (A20) cells by our group. The present study aimed to establish a mCD99L2-downregulated A20 cell line and to investigate the effect of shRNA targeting mCD99L2 in A20 cells in vitro and in vivo. Four pLenti6/mCD99L2 expression vectors containing the mCD99L2 shRNA-expressing cassette were constructed, transfected into A20 cells and stable mCD99L2-downregulated A20 subclones, termed A20-mCD99L2(-) cells, were established and identified by quantitative PCR and western blot analysis. Light and transmission electron microscopy, MTT assay, flow cytometry and immunofluorenscence labeling were used to observe the morphological, biological and phenotypic characteristics in vitro. Some of the A20-mCD99L2(-) cells exhibited H/RS-cell like morphology, a decreased proliferative ability, a prolonged G2 phase and increased CD30 and CD15 expression. Upon injecting cells into nude or immunocompetent BALB/c mice, tumorigenesis, tumor growth, morphology and phenotypes in vivo were observed. A20-mCD99L2(-) cells induced tumors in nude and BALB/c mice, but with less potency in the latter compared with the controls. Similar morphological, biological and phenotypic characteristics were observed in the A20-mCD99L2(-) cell-induced tumors as those in vitro. Several cytokines including CD30T, IL-12p40/p70, IL-3, IFN-gamma, CXCL16, MIP-1 alpha and CD40 were upregulated following mCD99L2 downregulation when detected using antibody arrays. The results from western blot analysis indicated that the regulation of mCD99L2 expression may involve the activated nuclear factor-kappa B pathway in the murine B lymphoma cells. The present study provides data for further investigation into the mCD99L2 gene in tumor cells.