热休克蛋白70在乳腺癌中的表达及其对体外乳腺癌细胞株生物学功能的影响

Expression of heat shock protein 70 in breast cancer and its effect on the biological function of breast cancer cells in vitro

目的观察热休克蛋白70（HSP70）在乳腺癌中的表达，评估其对体外乳腺癌细胞株生物学功能的影响。方法应用免疫组织化学方法检测200例手术切除的乳腺癌组织中HSP70mRNA的表达，探讨其与临床病理特征的关系及对预后的影响。构建HSP70-小干扰RNA（siRNA）慢病毒载体，应用基因转录方法检测HSP70基因表达对体外乳腺癌MDA-MB-231细胞株的抗增殖作用和生物学功能的影响；通过Westernblot和实时定量聚合酶链反应（Real-timePCR）技术检测HSP＇／0-siRNA在MDA-MB-231细胞中的表达水平，并通过噻唑蓝（MTY）法、Transwell小室法、显微镜等观察比较转染多亮氨酸重复区免疫球蛋白样蛋白1（LRIGl）基因的RC-4B／C细胞的的迁移能力、黏附能力和形态学改变。结果（1）HSP70mRNA在单纯乳腺增生组织、不典型增生组织、乳腺癌组织中的表达分别为0．263±0．034、0．627±0．071、0．579±0．062，其在乳腺癌和不典型增生中的表达明显高于单纯乳腺增生（P〈0．05），但从不典型增生至乳腺癌的过程中HSP70的表达无明显变化（P〉0．05）。（2）HSP70mRNA的表达与肿瘤大小、病理类型、组织学分级、孕激素受体（PR）状态、人类表皮生长因子受体2（Her-2）状态，腋窝淋巴结转移状况无关，与雌激素受体（ER）表达呈正相关（rg=0．792，P〈0．01）。（3）HSP70-siRNA慢病毒转染的MDA．MB-231细胞中HSP70的表达在mRNA和蛋白水平均明显增强。其HSP70蛋白的水平是未转染组的2．8和3．7倍。（4）HSP70-siRNA转染组在1、2、4h的黏附能力分别为0．14±0．02、0．27±0．03和0．45±0．06。空载体组为0．05±0．01、0．15±0．02和0．32±0．03。未转染组为0．07±0．01、0．16±0．01和0．34±0．02。转染组MDA-MB-231细胞的增殖活性，黏附和侵袭能力明显增强（P〈0．05）。（5）显微镜下观察，体外培养2周后，空载体组和未转染组的MDA-MB-231细胞全部死亡，癌细胞明显漂浮，培养液中出现大量碎片。而转染HSP70．siRNA的MDA-MB-231细胞，生长状况良好，并可见阳性克隆。结论HSP70在乳腺癌组织中有-定程度的表达，HSP70阳性表达与乳腺癌患者生存期的缩短有关。HSP70基因转染对体外乳腺癌MDA-MB-231细胞株具有促进增殖侵袭和抗凋亡的作用。

Objective To investigate the expression of heat shock protein 70 （HSPT0） in breast cancer, and its effect on the biological function of breast cancer cells in vitro. Methods The expression of HSP70 mRNA in 200 cases of breast cancer was detected by immunohistochemical method, and its relation- ship with clinicopathological features and prognosis was studied. The lentiviral vectors of HSP70 - small in- terfering RNA （siRNA） were constructed. The gene transcription method was used to detect the effects of HSP70 gene expression on the antiproliferation and biological function ofin vitro breast cancer cell line MDA - MB - 231. By Western blotting and real - time quantitative polymerase chain reaction （ Real - time PCR）, the expression of HSP70 - siRNA was detected in MDA - MB - 231 ceils. The migration ability was measured by methyl thiazol tetrazolium （MTr） assay. Transwell chamber method and microscopy were used to observe and compare the transfection of leucine - rich repeats and immunoglobulin - like domains 1 （ LRIG1 ） gene RC - 4B/C cells and adhesion ability and morphological change. Results （ 1 ） The expres- sion of HSP70 mRNA in breast cancer and dysplasia was significantly higher than that in simple mammary gland hyperplasia （P 〈 0. 05 ）, but the expression of HSP70 had no significant change during the process of atypical hyperplasia to breast cancer （ P 〉 0. 05 ）. （2） The mRNA expression had a positive correlation with tumor size, pathological type, histological grade, progesterone receptor （ PR）, human epidermal growth factor receptor - 2 （ Her - 2） status, axillary lymph node metastasis, and estrogen receptor （ER） ex- pression （correlation coefficient rs = 0. 792 ,P 〈 0. 01 ）. （3） The expression of HSP70 mRNA and protein in MDA - MB -231 cells transfected with HSP70 - siRNA was significantly enhanced. （4） The adhesion ability of HSP70 - siRNA in 1, 2 and 4 h was 0. 14 ± 0. 02, 0. 27 ± 0. 03 and 0. 45 ± 0. 06, that in empty vector group was 0. 05 ±0. 01,0. 15 ±0. 02 and 0. 32 ±0. 03, and that in non - transfection group was 0. 07 ±0. 01, 0. 16±0. 01 and 0. 34±0.02 respectively. The proliferation activity of MDA - MB - 231 cells in HSPTO - siRNA transfection group was significantly enhanced by the adhesion and invasion ability. （5） Under the microscope, the MDA -MB -231 cells in the empty vector group and the non -transfection group after in vitro culture for 2 weeks were all dead, the cancer cells were floating, and there was debris in the culture fluid. The MDA -MB -231 cells transfected with HSP70 -siRNA grew well, and the positive clones were observed. Conclusion HSP70 was expressed in breast cancer tissues, and the expression of HSP70 was related to the survival of patients with breast cancer. HSP70 gene transfection can promote the proliferation and inhibit apoptosis of the MDA - MB -231 cell line in vitro.