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目的:检测造血祖细胞激酶1(HPK1)在人非特异型浸润性乳腺癌组织及乳腺癌细胞中的表达,并构建HPK1慢病毒载体以探究HPK1过表达对人乳腺癌细胞增殖、凋亡的影响。方法:采用Western blot检测48例乳腺癌组织和配对癌旁组织中HPK1蛋白的表达,采用Western blot、RT-PCR法检测正常乳腺上皮细胞(MCF10A)和乳腺癌MCF-7细胞中HPK1的表达水平。PCR扩增HPK1序列,构建慢病毒p CDH-HPK1-puro重组载体,包装病毒、转染MCF-7细胞(过表达组),以感染空载体病毒的MCF-7细胞作为对照组,分别采用Western blot、RT-PCR检测2组细胞HPK1的表达水平,MTT法检测细胞增殖能力的变化,流式细胞术检测细胞凋亡及细胞周期。结果:与癌旁组织相比,HPK1蛋白在乳腺癌组织中低表达(P=0.036)。与MCF10A细胞相比,HPK1蛋白及mRNA在MCF-7细胞中低表达;与对照组相比,过表达组中HPK1蛋白及mRNA的表达水平上调,细胞增殖能力下降,细胞凋亡率升高,G0/G1期细胞比例升高(P均<0.05)。结论:HPK1在乳腺癌组织及细胞中低表达,HPK1过表达可抑制MCF-7细胞增殖和诱导凋亡,并使细胞周期阻断在G0/G1期。
OBJECTIVE: To detect the expression of hematopoietic progenitor kinase 1 (HPK1) in human non-specific invasive breast cancer and breast cancer cells and to construct HPK1 lentiviral vector to investigate the effect of HPK1 overexpression on proliferation and apoptosis of human breast cancer cells influences. Methods: Western blot was used to detect the expression of HPK1 protein in 48 cases of breast cancer tissues and matched paracancerous tissues. Western blot and RT-PCR were used to detect the expression of HPK1 in normal breast epithelial cells (MCF10A) and breast cancer MCF-7 cells . The HPK1 sequence was amplified by PCR. The recombinant lentiviral vector pCDH-HPK1-puro was constructed and packaged into MCF-7 cells (overexpression group). MCF-7 cells infected with empty vector were used as control group, The expression of HPK1 was detected by Western blot and RT-PCR. The proliferation of cells was detected by MTT assay. The apoptosis and cell cycle were detected by flow cytometry. Results: HPK1 protein was overexpressed in breast cancer tissues compared with adjacent tissues (P = 0.036). Compared with MCF10A cells, HPK1 protein and mRNA were down-regulated in MCF-7 cells. Compared with the control group, the expression of HPK1 protein and mRNA was up-regulated, the cell proliferation ability was decreased and the apoptosis rate was increased, G0 / G1 phase cells increased (P all <0.05). Conclusion: HPK1 is low expressed in breast cancer tissues and cells. HPK1 overexpression can inhibit the proliferation and induce apoptosis of MCF-7 cells, and block the cell cycle in G0 / G1 phase.