DHRS7对人乳腺癌细胞MCF-7细胞周期的影响及其在乳腺癌组织中的表达

Effect of DHRS7 on cell cycle of human breast cancer MCF-7 cells and its expression in breast cancer tissue

目的:探究DHRS7基因过表达和DHRS7特异性短发夹RNA(shRNA)重组质粒对人乳腺癌细胞MCF-7细胞周期的影响及DHRS7与乳腺癌浸润癌的关系,阐明DHRS7在乳腺癌发生、发展中的作用。方法:利用RT-PCR技术将DHRS7基因全长克隆入真核表达载体pcDNA3.1(+)质粒中,构建pcDNA3.1-DHRS7重组真核表达质粒。实验分为pcDNA3.1-DHRS7组、阴性对照组(pcDNA3.1组)、空白对照组和DHRS7-shRNA-pGenesil组,用Fugene HD转染试剂将各种质粒转染MCF-7细胞,采用RT-PCR和Westernblotting法检测各组MCF-7细胞中DHRS7的表达情况,流式细胞术分析细胞周期变化,免疫组织化学SP法检测乳腺原位癌和乳腺浸润癌组织中DHRS7蛋白的表达情况。结果:成功构建pcDNA3.1-DHRS7重组表达质粒。PCR和Western blotting结果显示,pcRNA-DHRS7组蛋白的表达量高于空白对照组(P<0.05),DHRS7-shRNA-pGenesil组蛋白的表达量低于空白对照组,差异均有统计学意义(P<0.05)。流式细胞术显示,DHRS7表达上调使MCF-7细胞S期细胞增多(P<0.05),DHRS7的表达下调使处于G2期的细胞增多(P<0.05)。免疫组织化学染色显示,DHRS7蛋白在原位癌中高表达,阳性表达率为100%(37/37);在乳腺浸润癌的表达明显降低,阳性率为18.9%(7/37),两者比较差异有统计学意义(P<0.01)。结论:DHRS7基因参与了MCF-7细胞细胞周期的调控过程,可以抑制细胞增殖,DHRS7蛋白的表达丢失可能促进乳腺癌浸润。

Objective To investigate the effects of overexpression of DHRS7 gene and short hairpinRNA（shRNA） recombinant plasmid on cell cycles of the human breast cancer MCF-7 cells and the relationship between DHRS7 and breast cancer invasion,and to clarify the effect of DHRS7 on the development of breast cancer.Methods The cDNA fragment encoding human DHRS7 was obtained from mRNA of breast cancer cells MCF-7 by RT-PCR.Then it was cloned into vector pcDNA3.1（＋） plasmid to conduct DHRS7-shRNA-pGenesil.There were four groups in the experiment： pcDNA3.1-DHRS7 group,negative control group（pcDNA3.1 group）,blank control group and DHRS7-shRNA-pGenesil group.Then the plasmids were transfected into MCF-7 cells using Fugene HD transfection agent,and the expressions of DHRS7 were determined by RT-PCR and Western blotting.The cell cycles were analyzed by flow cytometry.The expression of DHRS7 protein in carcinoma in stiu and infiltrating carcinoma were detected by immunohistochemical staining.Results The recombinant plasmid pcDNA3.1-DHRS7 was constructed correctly.RT-PCR analysis and Western blotting showed that the expression of DHRS7 protien in pcDNA-DHRS7 group was higher than that in blank control group（P〈0.05）,and the expression of DHRS7 protien in DHRS7-shRNA-pGenesil group was lower than that in blank control group（P〈0.05）.Flow cytometry showed the overexpression of DHRS7 increased the cells in S phase（P〈0.05） and the low expression of DHRS7 increased the cells in G2 period（P〈0.05）.Immunohistochemical staining showed that DHRS7 protein expression was higher in the breast cancer in situ,and the positive rate was 100%（37/37）;the DHRS7 protein expression was lower in the breast infiltrating carcinoma tissues,and the positive rate was 18.9%（7/37）,there was significant difference between them（P〈0.01）.Conclusion DHRS7 gene may be involved in the regulation of cell cycle in MCF-7 cells,which can inhibit cell proliferation and the expression missing of DHRS7 protein may promote invasion of breast cancer.