下调S100A6基因对裸鼠肺腺癌移植瘤生长和凋亡的影响

doi:10.3877/cma.j.issn.1674-6902.2017.03.002

目的

探讨下调靶向基因S100A6对在体肺腺癌移植瘤生长和凋亡的影响。

方法

18只健康Balb/c雄性裸鼠随机分为空载体对照组、阴性对照组及S100A6基因RNA干扰组三组，每组6只动物；分别应用不携带目的基因的空载质粒、未转染任何质粒以及含有S100A6基因RNA干扰慢病毒质粒的A549肺腺癌细胞接种于裸鼠皮下，构建移植瘤动物模型；观察不同组移植瘤组织生长情况；采用Real-Time PCR、免疫组织化学及Western-blot等技术检测S100A6 mRNA和蛋白的表达，TUNEL法检测细胞凋亡。

结果

成功构建了裸鼠肺腺癌皮下移植瘤模型；病理学特征显示，阴性对照组和空载体对照组可见到成巢的肿瘤细胞，癌细胞核大，而干扰组肿瘤细胞较稀疏，间质组织明显；接种细胞2周时，阴性对照组、空载体对照组和RNA干扰组肿瘤组织体积和质量差异具有统计学意义(P<0.05)；S100A6基因和蛋白表达在三组之间差异具有统计学意义(P<0.05)；各组裸鼠移植瘤凋亡细胞呈现不同程度的棕褐色肿瘤细胞核，三组肿瘤细胞凋亡率的差异具有统计学意义(P<0.05)。

结论

下调肿瘤瘤组织S100A6蛋白表达，可抑制肿瘤生长，诱导细胞凋亡，为进一步开发肺腺癌分子靶点提供了新途径。

关键词: S100A6, 肺腺癌, 生长移植病, 细胞凋亡

Objective

S100A6 can regulate cell proliferation, differentiation, cell cycle and apoptosis by interacting with target proteins under the condition of calcium ion existence. S100A6 gene is highly expressed in multiple tumor tissues and is closely related to the occurrence and development of tumor. The aim of this study is to explore the influence of S100A6 gene down-regulation on growth and apoptosis of transplanted lung adenocarcinoma in nude rats.

Methods

Eighteen healthy male Balb/c nude mice were randomly divided into three groups: empty vector control group (n=6), negative control group (n=6) and S100A6 gene RNA interference (RNAi) group (n=6). The transplanted lung adenocarcinoma models were established by subcutaneous injection of A549 lung adenocarcinoma cells line with empty vector, non-carrier vector and pLenR-GPH-shRNAi-S100A6, respectively. The growth of transplanted tumor in different groups was observed. The expression of S100A6 gene was detected using real-time PCR, immunohistochemistry and western-blot methods. Apoptosis cells were analyzed by Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL).

Results

The transplanted lung adenocarcinoma models in nude rat were successfully established. The pathological features showed that typical tumor cells formed solid hest-like mass. The tumor cell nucleus in empty vector control group and negative control group got bigger. The tumor cells in S100A6 RNAi group were relatively looser and their interstitial components became obvious. The tumor volume and mass among three groups had significant difference(P<0.05). The expression of S100A6 gene of RNAi group was significantly lower than that of empty vector control group and negative control group(P<0.05). The apoptosis rate had significantly difference among three groups(P<0.05). The nucleus of apoptosis cell in transplanted lung adenocarcinoma showed brown.

Conclusions

Down-regulation expression of S100A6 in lung adenocarcinoma could inhibit tumor growth and induce cell apoptosis, which could provide new pathway for further study of molecular target spot of lung adenocarcinoma.

Key words: S100A6, Lung adenocarcinoma, Growth transplant disease, Apoptosis

图1 慢病毒感染A549肺腺癌细胞72hGFP表达；注：A：阴性对照组；B：空载体对照组；C：S100A6RNA干扰组(×200)

图2 三组裸鼠接种A549肺腺癌细胞第14天成瘤情况观察；注：A：阴性对照组；B：空载体对照组；C：S100A6RNA干扰组

图3 三组裸鼠接种A549肺腺癌细胞14天成瘤肿瘤组织体积及质量比较；注：A：体积比较；B：质量比较；1：阴性对照组；2：空载体对照组；3：S100A6 RNA干扰组；*与阴性对照组相比，P<0.05；#与空载体对照组相比，P<0.05

图4 裸鼠成瘤HE染色组织标本；注：A：阴性对照组；B：空载体对照组；C：S100A6RNA干扰组（×200）

图5 Real-time PCR检测移植瘤组织S100A6mRNA表达；注：三组mRNA相对表达比较；*与阴性对照组相比，P<0.05；#与空载体对照组相比，P<0.05

图6 裸鼠成瘤S100A6免疫组织化学；注：A：阴性对照组；B：空载体对照组；C：S100A6RNA干扰组（×200）

表1 应用免疫组织化学法检测S100A6在三组移植瘤肿瘤组织差异表达

组　别	例数	S100A6表达			P值
组　别	例数	＋	＋＋	χ²值	P值
阴性对照组	6	2(33.3%)	4(66.7%)	?	?
空载体对照组	6	1(16.7%)	5(83.3%)	5.850	0.054
RNA干扰组	6	5(83.3%)	1(16.7%)	?	?

表1 应用免疫组织化学法检测S100A6在三组移植瘤肿瘤组织差异表达

组　别	例数	S100A6表达			P值
组　别	例数	＋	＋＋	χ²值	P值
阴性对照组	6	2(33.3%)	4(66.7%)	?	?
空载体对照组	6	1(16.7%)	5(83.3%)	5.850	0.054
RNA干扰组	6	5(83.3%)	1(16.7%)	?	?

图7 Western-blot检测裸鼠成瘤S100A6蛋白表达；注：A: western-blot电泳条带，1－1阴性对照组，2－1空载体对照组，3－1 RNA干扰组；B：S100A6蛋白相对表达量比较，*与阴性对照组相比，P<0.05，#与空载体对照组相比，P<0.05

图8 TUNEL法检测移植瘤凋亡细胞及各组凋亡细胞数比较；注：A：阴性对照组，B：空载体对照组，C：RNA干扰组，D：各组凋亡细胞数比较，*与阴性对照组相比，P<0.05，#与空载体对照组相比，P<0.05(×400)

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Influence of S100A6 gene down-regulation on growth and apoptosis of transplanted lung adenocarcinoma in nude rats

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Influence of S100A6 gene down-regulation on growth and apoptosis of transplanted lung adenocarcinoma in nude rats