Nanashima Naoki, Horie Kayo, Yamada Toshiyuki, Shimizu Takeshi, Tsuchida Shigeki
Department of Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, Hirosaki 036-8564, Japan.
Department of Biochemistry and Genome Biology, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan.
Oncol Rep. 2017 May;37(5):2964-2970. doi: 10.3892/or.2017.5564. Epub 2017 Apr 11.
Keratins are fibrous proteins. Hair keratins constitute hard structures such as the hair and nails, and cytokeratins have been used as markers of breast carcinoma. However, the expression and function of full-size hair keratin genes have not been previously demonstrated in breast cancer. We investigated the expression of the hair keratin, KRT81, and its function in human breast cancer and normal mammary epithelial cells. Western blotting showed full size 55-kDa KRT81 expression in the human breast cancer cell lines, MCF7, SKBR3 and MDA-MB-231, normal human mammary epithelial cells (HMEC), and non-neoplastic cells (MCF10A). Reverse transcription-polymerase chain reaction revealed that the full size KRT81, including its 5' region is expressed in breast cells. Immunohistochemical and immunofluorescence analyses showed that KRT81 was located in the cytoplasm. To investigate the function of KRT81, we knocked down KRT81 by siRNA in MCF10A cells. Microarray analysis revealed that the expression of genes related to invasion such as matrix metallopeptidase (MMP)9 was decreased. In KRT81-knockdown MDA-MB231 cells, zymography revealed a decrease in MMP9 activity, while scratch and invasion assays revealed that KRT81-knockdown decreased cell migration and invasion abilities. This is the first study showing that full size KRT81 is expressed in normal breast epithelial cells and breast cancer cells. Moreover, our results indicate that KRT81 contributes to the migration and invasion of breast cancer cells.
角蛋白是纤维状蛋白质。毛发角蛋白构成毛发和指甲等坚硬结构，细胞角蛋白已被用作乳腺癌的标志物。然而，全长毛发角蛋白基因在乳腺癌中的表达和功能此前尚未得到证实。我们研究了毛发角蛋白KRT81在人乳腺癌细胞和正常乳腺上皮细胞中的表达及其功能。蛋白质免疫印迹法显示，在人乳腺癌细胞系MCF7、SKBR3和MDA-MB-231、正常人乳腺上皮细胞（HMEC）和非肿瘤细胞（MCF10A）中存在全长55 kDa的KRT81表达。逆转录-聚合酶链反应显示，包括其5'区域的全长KRT81在乳腺细胞中表达。免疫组织化学和免疫荧光分析表明，KRT81位于细胞质中。为了研究KRT81的功能，我们在MCF10A细胞中通过小干扰RNA敲低KRT81。基因芯片分析显示，与侵袭相关的基因如基质金属肽酶（MMP）9的表达降低。在KRT81敲低的MDA-MB231细胞中，酶谱分析显示MMP9活性降低，而划痕和侵袭试验显示KRT81敲低降低了细胞迁移和侵袭能力。这是第一项表明全长KRT81在正常乳腺上皮细胞和乳腺癌细胞中表达的研究。此外，我们的结果表明，KRT81促进乳腺癌细胞的迁移和侵袭。
