SHIP基因突变对K562细胞中细胞周期相关蛋白及磷酸化Akt的影响

[Effects of SHIP gene mutation on cell cycle related proteins and phosphorylated Akt in K562 cells].

作者信息

Yang Lin, Luo Jian-min, Liu Xiao-jun, Wen Shu-peng, Yang Jing-ci, Zhang Jing-yu

机构信息

Department of Hematology, the 2nd Hospital of Hebei Medical University, Shijiazhuang 050000, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2009 Aug;30(8):548-52.

PMID:19954644

Abstract

OBJECTIVE

To investigate the effect of SHIP gene mutation on the cell cycle and its related gene expression in K562 cells.

METHODS

The recombined green fluorescent protein (GFP) containing FIV-SHIP gene was transfected into K562 cells. The transfection efficiency and cell cycle of K562/SHIP were assessed by flow cytometry (FCM). The proliferation of K562 cells was detected by MTT assay, the mRNA levels of SHIP by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR), and the protein levels of SHIP, Cyclin D1, p21(WAF1/CIPI) and p27(KIP1) by Western blot.

RESULTS

Wild type SHIP inhibited K562 cell proliferation and caused a G(0)/G(1) arrest [(34.2 +/- 7.8)% vs (0.7 +/- 8.3)% (P < 0.01)]; while the point mutation of SHIP gene did not show such effect. Western blot results showed that the Akt phosphorylation and cyclin D1 expression was significantly decreased (P < 0.01), and the expression of p27(KIP1) and p21(WAF1/CIPI) increased. Site-directed mutation of SHIP gene SH2 domain (TTC-->CTC, Phe-->Leu) did not influence the Akt phosphorylation and cyclins (P > 0.05).

CONCLUSION

(1) wtSHIP gene can down-regulate Akt phosphorylation and result in inhibition of cyclin D1 expression, up-regulating p27(KIP1) and p21(WAF1/CIPI) expression, finally leading to the reduction of K562 cell proliferation, and inducing G(0)/G(1) phase arrest. (2) SHIP gene suppresses the proliferation of K562, being dependent on its intact structure and function.

摘要

目的

研究SHIP基因突变对K562细胞周期及其相关基因表达的影响。

方法

将含FIV-SHIP基因的重组绿色荧光蛋白（GFP）转染至K562细胞。采用流式细胞术（FCM）评估K562/SHIP的转染效率和细胞周期。采用MTT法检测K562细胞的增殖情况，采用实时荧光相对定量逆转录PCR（FQ-PCR）检测SHIP的mRNA水平，采用蛋白质印迹法检测SHIP、细胞周期蛋白D1、p21（WAF1/CIPI）和p27（KIP1）的蛋白质水平。

结果

野生型SHIP抑制K562细胞增殖并导致G(0)/G(1)期阻滞[（34.2±7.8）%对（0.7±8.3）%，P<0.01]；而SHIP基因的点突变未显示出这种作用。蛋白质印迹结果显示，Akt磷酸化和细胞周期蛋白D1表达显著降低（P<0.01），p27（KIP1）和p21（WAF1/CIPI）表达增加。SHIP基因SH2结构域的定点突变（TTC→CTC，苯丙氨酸→亮氨酸）不影响Akt磷酸化和细胞周期蛋白（P>0.05）。