[STK15基因沉默诱导胃癌细胞有丝分裂停滞]
[Mitotic arrest of gastric cancer cells induced by silencing of STK15 gene].
作者信息
Lan Bin, Chen Xue-hua, Liu Bing-ya, Qu Ying, Zhang Xiao-qing, Cai Qu, Dai Qi-bao, Zhang Jian, Zhu Zheng-gang
机构信息
Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Affiliated Hospital of Shanghai Second Medical University, Shanghai 200025, China.
出版信息
Zhonghua Bing Li Xue Za Zhi. 2006 Feb;35(2):106-9.
OBJECTIVE
To investigate the role of STK15 in regulating mitosis of gastric cancer cells (MKN45) by gene silencing through RNA interference mechanism.
METHODS
RNA interference technique was used to inhibit STK15 expression in MKN45 cells. The expression levels of STK15 mRNA and protein were measured by real-time quantitative RT-PCR and Western blot respectively and cell morphological changes were investigated by reverse microscopy. In addition, cell cycle distribution and cellular proliferation were determined by flow-cytometry and MTT assay respectively. Finally, the mitotic phenotype of MKN45 cells was studied by immunofluorescence staining and confocal microscopy.
RESULTS
Silencing of STK15 gene by RNA interference was confirmed by marked decrease of STK15 mRNA and protein levels in the treated MKN45 cells. This silencing correlated with rounding of the cells, decreasing of DNA content in G(2) phase (P < 0.05) and a lowered proliferation index (P < 0.05), along with alterations of mitotic phenotype of MKN45 (P < 0.05).
CONCLUSION
STK15 gene may play a key role in regulating cellular mitosis and its inhibition by RNA interference leading to mitosis arrest in MKN45 cells.
目的
通过RNA干扰机制使基因沉默，研究丝氨酸/苏氨酸蛋白激酶15（STK15）在调控胃癌细胞（MKN45）有丝分裂中的作用。
方法
采用RNA干扰技术抑制MKN45细胞中STK15的表达。分别通过实时定量逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测STK15 mRNA和蛋白的表达水平，并用倒置显微镜观察细胞形态变化。此外，分别通过流式细胞术和MTT法测定细胞周期分布和细胞增殖情况。最后，通过免疫荧光染色和共聚焦显微镜研究MKN45细胞的有丝分裂表型。
结果
经RNA干扰后，处理的MKN45细胞中STK15 mRNA和蛋白水平显著降低，证实了STK15基因的沉默。这种沉默与细胞变圆、G2期DNA含量降低（P < 0.05）、增殖指数降低（P < 0.05）以及MKN45有丝分裂表型改变（P < 0.05）相关。
结论
STK15基因可能在调控细胞有丝分裂中起关键作用，RNA干扰对其抑制导致MKN45细胞有丝分裂停滞。
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