The Role Of EMMPRIN In The Lung Cancer Cells-mediated Osteoblastic Differentiation

Background and Objectives:Lung cancer is the leading cause of cancer death in the world and can be classified into two major classes based on its biology, therapy and prognosis: non-small cell lung cancer(NSCLC) and small cell lung cancer(SCLC). NSCLC is the most common type that accounts for 85% and approximately 30%-40% of patients with NSCLC develop bone metastases during the process of their disease. The mechanism involved in lung cancer bone metastasis is not fully understood, however the further study is a “vicious cycle” of bone metastases. Metastatic cancer cells produce many cytokines and other factors to affect bone microenvironment, which disturbes the normal bone homeostasis maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Meanwhile, many cytokines released in the process of bone destruction are capable of promotion of cancer cells growth or invasion. Thus, a series of serious bone related complications such as bone pain, pathological bone fractures, spinal cord compression, and nerve compression syndromes leading to a poor quality life are induced.Extracellular matrix metalloproteinase inducer(EMMPRIN), also known as CD147, is a highly glycosylated transmembrane protein belonging to the immunoglobulin superfamily. EMMPRIN induces tumor cell invasion and metastasis by stimulating production of matrix metalloproteinases(MMPs) and enhances in vivo tumor angiogenesis by increasing the synthesis of vascular endothelial growth factor(VEGF). It has been widely recognized that high levels of EMMPRIN expression is closely associated with poor prognosis of breast cancer, prostate cancer, NSLC. Recent studies have shown that increasing EMMPRIN expression can stimulate the “vicious cycle” of bone metastases and enhance osteoclastogenesis, which enhances breast cancer-induced osteolytic lesions. However, the role of EMMPRIN in lung cancer cells-induced osteoblastic differentiation is not fully understood.The aim of this study is to explore the role of EMMPRIN in lung cancer cells-induced osteoblastic differentiation and its possible mechanism, which opens up a novel way for prevention and treatment of lung cancer bone metastasis.Methods:EMMPRIN overexpression, short hairpin RNA(sh RNA) or negative control(NC) lentivirus were infected into A549 cells and stable clones of A549 cells were obtained respectively. To begain with, we observe the effects of EMMPRIN on the proliferation, migration, and invasion of lung cancer A549 cells, and explore its possible mechanism. What is more, collecting corresponding lung cancer conditioned medium(CM), co-culturing with osteoprogenitor MC3T3-E1 cells, and observing the effect of EMMPRIN on lung cancer cells-induced osteoblastic differentiation. Finally, detecting the effect of EMMPRIN on the expression of DKK1 in lung cancer A549 cells and the activation of β-catenin, the key mediator of the Wnt/β-catenin signaling pathway, in osteoprogenitor MC3T3-E1 cells.The effects of EMMPRIN on the proliferation, migration, and invasion of lung cancer A549 cells.1. EMMPRN overexpression, short hairpin RNA(sh RNA), negative control(NC) lentivirus were infected into lung cancer A549 cells. Stable clones of A549 EMP cells with EMMPRN overexpression, A549 sh EMP cells with EMMPRIN silencing and A549 NC cells were obtained respectively. Effects of EMMPRIN on the proliferation, migration, and invasion of lung cancer A549 cells were measured by using CCK-8(cell counting kit-8), wound scratch assay and transwell test respectively.2. The nuclear expression level of β-catenin in lung cancer A549 cells was detected by Western-blot analysis.Mechanism of EMMPRIN in lung cancer cells-induced osteoblastic differentiationThe CM was collected from each A549 EMP, A549 sh EMP, A549 NC or A549 cells, and osteogenic medium( OM) were formulated based on 20% diverse CM, then the osteoprogenitor MC3T3-E1 cells were co-cultured with diverse OM.1. The activity of alkaline phosphatase of MC3T3-E1 in each group was determined by BCIP/NBT Alkaline Phosphatase stain Kit in the third day.2. The bone nodule formation of MC3T3-E1 in each group in fourteenth day was detected by Alizarin Red S staining.3. The relative m RNA expression levels of Col1α1, RUNX2/Cbfa1 and OCN of MC3T3-E1 in each group were measured by Realtime PCR.4. The m RNA and protein expressional levels of DKK1 in lung cancer A549 cells and CM were determined by Realtime PCR, Immunofluorescence analysis and Western-blot analysis respectively.5. The nuclear expression level of β-catenin of MC3T3-E1 in each group was determined by Western-blot analysis.Results:The effects of EMMPRIN on the proliferation, migration, and invasion of lung cancer A549 cells1. The m RNA and protein expression levels of EMMPRIN in diverse lung cancer A549 cells were detected by Realtime PCR and western blot. The results showed that: compared with blank group A549 cells, the m RNA and protein expression levels of EMMPRIN in A549 EMP cells were greatly increased(P < 0.05). On the contrary, the m RNA and protein expression levels of EMMPRIN in A549 sh EMP cells were obviously decreased(P < 0.05).2. EMMPRIN could enhance lung cancer A549 cells proliferation, migration, and invasion.1) Compared with blank group A549 cells, the abilities of proliferation, migration and invasion of A549 EMP cells were greatly improved(P < 0.05). On the contrary, the proliferation, migration, and invasion of A549 sh EMP cells were obviously inhibited.(P < 0.05).2) Increasing CD147 expression level in lung cancer A549 cells upregulated significantly the nuclear expression of β-catenin, whereas, silencing CD147 inhibited the nuclear expression of β-cateninMechanism of EMMPRIN in lung cancer cells-induced osteoblastic differentiation1. Down-regulation of EMMPRIN rescued A549 cells conditioned medium-induced osteoblastic differentiation1) Activity of alkaline phosphatase: The activity of alkaline phosphatase of MC3T3-E1 in A549 sh EMPCM and A549 CM group were lower than those of Control group, and the activity of alkaline phosphatase of MC3T3-E1 in A549 sh EMPCM group was higher than those of A549 CM group.2) Mineralization: The quantity and area of bone nodule formation of MC3T3-E1 in A549 sh EMPCM and A549 CM group were less than those of Control group, and the quantity and area of bone nodule formation of MC3T3-E1 in A549 sh EMPCM group was more than those of A549 CM group.3) Expression of osteoblastic gene: The m RNA expression levels of Col1α1, RUNX2/Cbfa1 and OCN of MC3T3-E1 in A549 sh EMPCM and A549 CM group were lower than those of Control group, and the m RNA expression levels of Col1α1, RUNX2/Cbfa1 and OCN of MC3T3-E1 in A549 sh EMPCM group were higher than those of A549 CM group.(P< 0.05).2. EMMPRIN regulates the activity of β-catenin through regulation of DKK1 expression, which involves in the progression of lung cancer cells-mediated osteoblastic differentiation.1) DKK1: Down-regulation of EMMPRIN could obviously inhibit the m RNA and protein expression levels of DKK1 in lung cancer A549 cells or A549 CM. On the contrary, up-regulation of EMMPRIN could greatly improve the m RNA and protein expression levels of DKK1 in lung cancer A549 cells or A549 CM.2) The nuclear protein expression of β-catenin in MC3T3-E1: The nuclear protein expression levels of β-catenin of MC3T3-E1 in A549 sh EMPCM and A549 CM group were lower than those of Control group(as positive control), and the nuclear protein expression levels of β-catenin of MC3T3-E1 in A549 sh EMPCM group were higher than those of A549 CM group.(P< 0.05). Meanwhile, the nuclear protein expression levels of β-catenin of MC3T3-E1 in A549 EMPCM group were opposite(P < 0.05).Conclusion:1. EMMPRIN could enhance lung cancer A549 cells proliferation, migration, and invasion, it may be regulated by the β-catenin activity.2.EMMPRIN regulates the activity ofβ-catenin through regulation of DKK1expression,which involves in the progression of lung cancer cells-mediated osteoblastic differentiation.Mill-Q Biocel超纯水仪Millipore美国PB-20 p H计Sartorious德国电磁炉POVOS中国...