<i>The prognostic value of</i> ADAMTS8 and its role as a tumor suppressor in breast cancer

A disintegrin-like and metalloprotease with therombospondin type1 motif 8 (ADAMTS8) plays an important role in many malignancies. However, the clinical and biological significance of ADAMTS8 in breast cancer remain unknown. In this study, the clinical data from 1066 breast cancer patients were analyzed by The Cancer Genome Atlas (TCGA) database, and were analyzed using the correlation between ADAMTS8 expression and the clinicopathological features and prognoses. The CCK-8 assay, clone formation assay, flow cytometry and Transwell assay were used to characterize the effects of ADAMTS8 on proliferation, migration and invasion of breast cancer cells. Gene set enrichment analysis (GSEA) and western blotting were used to identify the potential molecular mechanism on how ADAMTS8 exert its biological function. ADAMTS8 overexpression correlated longer overall survival (OS) and progression-free survival (PFS). ADAMTS8 was considered as an independent prognostic factor for OS. ADAMTS8 overexpression inhibited breast cancer cell proliferation, migration and invasion in vitro, and induced G2/M cell cycle arrest. ADAMTS8 was also involved in cell cycle regulation and was associated with the EGFR/Akt signaling pathway. ADAMTS8 knockdown showed the reverse effect. Together, the results showed that ADAMTS8 functioned as a tumor suppressor gene (TGS) and could be a prognostic biomarker for breast cancer.</p

Zeb1 Is a Potential Regulator of Six2 in the Proliferation, Apoptosis and Migration of Metanephric Mesenchyme Cells

Nephron progenitor cells surround around the ureteric bud tips (UB) and inductively interact with the UB to originate nephrons, the basic units of renal function. This process is determined by the internal balance between self-renewal and consumption of the nephron progenitor cells, which is depending on the complicated regulation networks. It has been reported that Zeb1 regulates the proliferation of mesenchymal cells in mouse embryos. However, the role of Zeb1 in nephrons generation is not clear, especially in metanephric mesenchyme (MM). Here, we detected cell proliferation, apoptosis and migration in MM cells by EdU assay, flow cytometry assay and wound healing assay, respectively. Meanwhile, Western and RT-PCR were used to measure the expression level of Zeb1 and Six2 in MM cells and developing kidney. Besides, the dual-luciferase assay was conducted to study the molecular relationship between Zeb1 and Six2. We found that knock-down of Zeb1 decreased cell proliferation, migration and promoted cell apoptosis in MM cells and Zeb1 overexpression leaded to the opposite data. Western-blot and RT-PCR results showed that knock-down of Zeb1 decreased the expression of Six2 in MM cells and Zeb1 overexpression contributed to the opposite results. Similarly, Zeb1 promoted Six2 promoter reporter activity in luciferase assays. However, double knock-down of Zeb1 and Six2 did not enhance the apoptosis of MM cells compared with control cells. Nevertheless, double silence of Zeb1 and Six2 repressed cell proliferation. In addition, we also found that Zeb1 and Six2 had an identical pattern in distinct developing phases of embryonic kidney. These results indicated that there may exist a complicated regulation network between Six2 and Zeb1. Together, we demonstrate Zeb1 promotes proliferation and apoptosis and inhibits the migration of MM cells, in association with Six2

Six2 Is a Coordinator of LiCl-Induced Cell Proliferation and Apoptosis

The metanephric mesenchyme (MM) cells are a subset of kidney progenitor cells and play an essential role in mesenchymal-epithelial transition (MET), the key step of nephron generation. Six2, a biological marker related to Wnt signaling pathway, promotes the proliferation, inhibits the apoptosis and maintains the un-differentiation of MM cells. Besides, LiCl is an activator of Wnt signaling pathway. However, the role of LiCl in cellular regulation of MM cells remains unclear, and the relationship between LiCl and Six2 in this process is also little known. Here, we performed EdU assay and flow cytometry assay to, respectively, detect the proliferation and apoptosis of MM cells treated with LiCl of increasing dosages. In addition, reverse transcription-PCR (RT-PCR) and Western-blot were conducted to measure the expression of Six2 and some maker genes of Wnt and bone-morphogenetic-protein (BMP) signaling pathway. Furthermore, luciferase assay was also carried out to detect the transcriptional regulation of Six2. Then we found LiCl promoted MM cell proliferation at low-concentration (10, 20, 30, and 40 mM). The expression of Six2 was dose-dependently increased in low-concentration (10, 20, 30, and 40 mM) at both mRNA and protein level. In addition, both of cell proliferation and Six2 expression in MM cells declined when dosage reached high-concentration (50 mM). However, Six2 knock-down converted the proliferation reduction at 50 mM. Furthermore, Six2 deficiency increased the apoptosis of MM cells, compared with negative control cells at relative LiCl concentration. However, the abnormal rise of apoptosis at 30 mM of LiCl concentration implies that it might be the reduction of GSK3β that increased cell apoptosis. Together, these demonstrate that LiCl can induce the proliferation and apoptosis of MM cells coordinating with Six2