ABSTRACT
Objectives: Ring finger protein 187 (RNF187) was recently demonstrated to be up-regulation and function as a promoter in multiple cancers. However, the roles of RNF187 in osteosarcoma (OS) are unclear. Here, we tried to reveal the clinicopathological and biological roles of RNF187 in OS. Materials and Methods: We employed the quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) to determine the expression of RNF187 in OS tissues and cells. Migration and invasion capacities were analyzed by wound healing and transwell assays, and colony formation and CCK8 assays were performed to investigate proliferative ability. The functional effects of RNF187 on OS drugs resistance were further determined by CCK8 and western blot assays. Then, the relationship between RNF187 expression and clinical implications was analyzed by tissue microarrays (TMAs) including 51 OS cases. Moreover, the prognostic value was also determined by Kaplan-Meier analysis. Results: We reported that RNF187 mRNA was significantly increased in OS tissues compared to matched nontumorous tissues (3.83 ±0.79 vs. 1.70 ± 0.63), which was in line with the IHC assay in TMAs. By RNA interference and cDNA transfection, we showed high level of RNF187 increased the migration, invasion and proliferation of OS cells. Moreover, we demonstrated that elevated RNF187 expression induced OS cell drugs resistance, activated the ERK1/2 molecular and markedly enhanced the BCL-2 expression. Clinically, OS patients with high level of RNF187 was associated with Histologic differentiation (p=0.001), an advanced Enneking stage (p=0.001), response to chemotherapy (p=0.004), and metastasis (p= 0.001). Clinically, our data displayed that the RNF187 overexpression in OS samples associated with shorten overall survival (p=0.001) and high tumor recurrence (p=0.001) in postoperative OS patients. Conclusions: Our results indicate that Elevated RNF187 expression is a new adverse outcomes marker for OS patients and may be used as a new therapeutic target of OS.
ABSTRACT
Cortex Eucommiae is used worldwide in traditional medicine, various constituents of Cortex Eucommiae, such as chlorogenic acid (CGA), has been reported to exert anti-osteoporosis activity in China, but the mechanism about their contribution to the overall activity is limited. The aims of this study were to determine whether chlorogenic acid can prevent estrogen deficiency-induced osteoporosis and to analyze the mechanism of CGA bioactivity. The effect of CGA on estrogen deficiency-induced osteoporosis was performed in vivo. Sixty female Sprague-Dawley rats were divided randomly among a sham-operated group and five ovariectomy (OVX) plus treatment subgroups: saline vehicle, 17α-ethinylestradiol (E2), or CGA at 9, 27, or 45 mg/kg/d. The rats' femoral metaphyses were evaluated by micro-computed tomography (µCT). The mechanism of CGA bioactivity was investigated in vitro. Bone mesenchymal stem cells (BMSCs) were treated with CGA, with or without phosphoinositide 3-kinase (PI3K) inhibitor LY294002. BMSCs proliferation and osteoblast differentiation were assessed with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and alkaline phosphatase, with or without Shp2 interfering RNA (RNAi). The results display that CGA at 27 and 45 mg/kg/day inhibited the decrease of bone mineral density (BMD) that induced by OVX in femur (p< 0.01), significantly promoted the levels of bone turnover markers, and prevented bone volume fraction (BV/TV), connectivity density (CoonD), trabecular number (Tb.N), trabecular thickness (Tb.Th) (all p< 0.01) to decrease and prevented the trabecular separation (Tb.Sp), structure model index (SMI)(both p< 0.01) to increase. CGA at 1 or 10 µM enhanced BMSC proliferation in a dose-dependent manner. CGA at 0.1 to 10 µM increased phosphorylated Akt (p-Akt) and cyclin D1. These effects were reversed by LY294002. CGA at 1 or 10 µM increased BMSC differentiation to osteoblasts (p< 0.01), Shp2 RNAi suppressed CGA-induced osteoblast differentiation by decreasing Shp2, p-Akt, and cyclin D1. This study found that CGA improved the BMD and trabecular micro-architecture for the OVX-induced osteoporosis. Therefore, CGA might be an effective alternative treatment for postmenopausal osteoporosis. CGA promoted proliferation of osteoblast precursors and osteoblastic differentiation of BMSCs via the Shp2/PI3K/Akt/cyclin D1 pathway.
