| About 1.2 million women suffer from breast cancer every year in the world. In Western Europe, North America and other developed countries, breast cancer is the most common malignant disease in women. It is noteworthy that the incidence of breast cancer in China grows rapidly at 3% per year in recent years. Breast cancer has become one of the leading causes of death in Chinese in the city. In these patients, it is not the primary tumor, but its metastasis at distant sites that are the main cause of death. So it is very significative to research the molecular mechanism of tumor metastasis.Previous studies have revealed Twist, a basic helix-loop-helix transcription factor, plays an important role in breast cancer metastasis. To clarify the molecular mechanism of its involvement in cancer metastasis, Twist was silenced by RNAi in highly metastatic murine breast cancer 4T1 cells. Real-time RT-PCR assay indicated that Twist was markedly silenced in 4T1 cells. An experimental animal model for breast cancer metastasis was used to assay the effect of Twist on cancer metastasis. The results demonstrated that Twist knockdown in 4T1 cells significantly suppressed the lung metastasis in vivo. Then microarray chips were used to investigate the gene-expression profiles of the Twist-knockdown 4T1 cells and the mock transfected 4T1 cells. Direct comparison of gene-expression profiles showed that 167 genes in Twist-knockdown cells differed dramatically in expression levels from those in control cells. Among the 167 genes, 26 genes are well-known tumor-associated, including 15 up-regulated and 11 down-regulated genes. These genes appear to be regulated by Twist during breast tumorigenesis.Lipocalin 24p3, one of the 26 tumor-associated genes, is abnormally expressed in some malignant human cancers and may play an important role in tumor metastasis. The result of the microarray showed that 24p3 was up-regulated in the Twist-knockdown 4T1 cells. Based on these results, we proposed that Twist might promote tumor metastasis by regulating 24p3.In order to determine whether 24p3 played a causal role in tumor metastasis mediated by Twist, 24p3 gene was stably overexpressed in 4T1 cells by retroviral vector mediated gene transfection. The proliferation, migration and anchorage-independent growth assays were used to detect the roles of 24p3 in breast cancer malignance. The results indicated that increasing the expression level of 24p3 in 4T1 cells couldn't affect the ability of proliferation and anchorage-independent growth but significantly stimulate cell migration in vitro. The role of 24p3 in cancer metastasis was then directly detected in vivo, and the results demonstrated that the overexpression of 24p3 in 4T1 cells didn't affect the tumor growth but significantly enhanced the lung metastasis in vivo. Twist knockdown in 4T1 cells up-regulated the expression of 24p3 and suppressed the metastasis. However, the overexpression of 24p3 couldn't inhibit but promote the tumor metastasis. Taken together, the results suggested that Twist couldn't affect the tumor metastasis by regulating 24p3.In conclusion, Twist and 24p3 played crucial roles in breast cancer metastasis. 24p3 was up-regulated in the Twist-silencing 4T1 cells, however, it was not involved in breast tumor metastasis mediated by Twist. These findings will provide new insights into the mechanism by which Twist and 24p3 are involved in tumor progress, suggesting the usefulness of Twist and 24p3 as targets for therapy of breast cancer.
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