SASH1基因通过p53信号通路调节乳腺癌细胞增殖

SASH1 regulates proliferation of breast cancer cells via p53 signaling pathway

目的探讨SASH1基因对乳腺癌细胞增殖的作用及其与p53基因的关系。方法购买134例乳腺癌组织芯片(上海芯超科技有限公司,芯片型号:HBre-Duc150Sur-01),用SASH1和p53抗体进行组织芯片免疫组织化学染色分析。构建重组质粒SASH1-PEGFP-C3(GFP-SASH1)、HA-p53-pcD NA3.0(HA-Tp53),定点突变SASH1基因上581、594、605、610位点(D581V-SASH1、W594L-SASH1、F605S-SASH1、V610E-SASH1)和p53基因上175、248、273位点(R175H-p53、R248W-p53、R273H-p53)。实验分组如下:(1)将0、0.5、1.0、2.0、4.0μg的HA-Tp53单独转染或分别与2.0μg的GFP-SASH1共同转染至HEK-293T细胞,用Western blot检测p53对GFP-SASH1和endo-SASH1表达的影响。(2)将0、0.5、1.0、2.0、4.0μg的GFP-SASH1单独转染或分别与2.0μg的HA-Tp53共同转染至HEK-293T细胞,用定量RT-PCR检测p53 mRNA及蛋白表达。(3)2.0μg的HA-pcD NA3.0(空载体),野生型p53(wt-p53),突变型R175H-p53、R248W-p53、R273H-p53单独转染或分别与2.0μg的GFP-SASH1共同转染至HEK-293T细胞,用Western blot检测GFP-SASH1和endo-SASH1表达。(4)将2.0μg的空载体,野生型-SASH1,突变型D581V-SASH1、W594L-SASH1、F605S-SASH1、V610E-SASH1单独转染或与2.0μg的HA-Tp53共同转染HEK-293T细胞,另设未经处理细胞为空白对照,Western blot检测HA-Tp53和endo-Tp53表达。用Spearman相关系数法对SASH1与p53免疫组织化学结果进行分析,用单因素方差分析方法比较SASH1与p53的表达量,两两比较采用LSD-t法。结果在134例人乳腺癌组织芯片中,SASH1和p53的表达呈正相关(r=0.319,P<0.001)。其中,SASH1(-)4例、(+)42例、(++)67例、(+++)20例、(++++)1例,p53(-)82例、(+)36例、(++)10例、(+++)6例。随着HA-Tp53转染剂量的增大,外源性SASH1(GFP-SASH1)和内源性SASH1(endo-SASH1)的表达水平都增高(F=205.369、178.238,P均<0.001)。随着GFP-SASH1转染剂量的逐渐增大,HA-Tp53 mRNA及蛋白表达量逐渐增加(F=67.481、506.538,P均<0.001)。突变型SASH1使内源性和外源性p53表达上调(F=165.767、1136.930,P均<0.001);突变型p53抑制内源性和外源性SASH1表达(F=57.142、354.517,P均<0.001)。结论 SASH1与p53正相关,对乳腺癌细胞增殖起调节作用。

Objective To investigate the role of SASH1 gene in the proliferation of breast cancer ceilsand its relationship with p53 gene. Methods Totally 134 cases of breast cancer tissue microarrays were purchased from Shanghai Xinchao Technology Co. , No. HBre-Duc150Sur-01 ）, conjugated with SASH1 or p53 antibody for immunohistochemistry. HA-p53-pcDNA3.0 （ HA-Tp53 ） and SASH1-PEGFP-C3 （ GFP-SASH1 ） recombined plasmid were constructed by gene cloning. D581V, W594L, F605S and V610E mutants of SASH1 gene and R175H, R248W, R273H mutants of p53 gene were generated by the associated mutagenesis primers and mutagenesis kit. Experimental grouping was as follows： （1） the different dosages （0,0. 5,1.0,2. 0,4. 0 μg） of HA-Tp53 were transfected alone or co-transfected with 2. 0 μg of GFP-SASH1 into HEK-293T cells. Western blot was used to detect the expressions of GFP-SASH1 and endo-SASH1. （2） The different dosages （0,0. 5, 1.0,2. 0,4. 0 μg） of GFP-SASH1 were transfected alone or co-transfected with 2. 0 μg HA-Tp53 into HEK- 293T cells. The quantitative RT-PCR was used to detect the p53 mRNA and protein expressions. （3） The 2. 0 μg of HA-pcDNA3.0 （empty vector）, wild-type p53 （wt-p53）, R175H-p53, R248W-p53 or R273H-p53 was transfected into HEK-293T cells alone, or together with 2. 0 μg of GFP-SASH1. Western blot was used to detect GFP-SASH1 and endo-SASH1 expressions. （4） The 2. 0 μg of empty vector, wt-SASH1, D581V- SASH1, W594L-SASH1, F605S-SASH1 or V610E-SASH1 was transfected into HEK-293T cells alone, or together with 2. 0 μg of HA-Tp53. Western blot was used to detect HA-Tp53 and endo-Tp53 expressions. Spearman correlation analysis was used to process immunohistoehemieal results of SASH1 and p53. Single- factor variance analysis was used to compare the expressions of p53 and SASH1. Pairwise comparison was performed using LSD-t method. Results In 134 cases of the human breast carcinoma tissue array, SASH1 expression was positively correlated with p53 expression （r=0. 319, P〈0. 001 ）. There were 4 cases of SASH1 （-）, 42 （＋）, 67（＋＋）,2 （＋＋＋）, 2 （＋＋＋＋） , and82 cases of p53 （-）, 36 （＋） , 10 （＋＋）, 6 （＋＋＋）. With the increasing dose of transfected HA-Tp53, the expression levels of exogenous SASH/ （ GFP-SASH1 ） and endogenous SASH1 （endo-SASH1） were increased （F = 205. 369, 178. 238, both P〈0. 001 ）. With the increasing dose of transfected GFP-SASH1, HA-Tp53 mRNA and protein expressions were gradually increased （F = 67.481, 506. 538, both P〈 0. 001 ）. Mutant SASH1 up-regulated endogenous and exogenous p53 expressions （ F= 165.767,1136. 930, P〈0. 001 ） ; mutant p53 inhibited the expressions of endogenous and exogenous SASH1 （F=57. 142, 354. 517, P〈0. 001） Condusion SASH1 is positively correlated with p53, with a regulatory role in proliferation of breast cancer cells.