已知腫瘤中的癌細胞異質性與腫瘤發展具有相關性，如轉移、抗藥性及復發。然而，腫瘤中異質性的癌細胞之間的訊息傳遞如何影響腫瘤發展尚未被了解。我們發現BMP-7在肺腫瘤細胞CL1-0中高度表達，其中BMP7表現量與淋巴結轉移呈負相關。在生長速度快的肺癌細胞CL1-0中，BMP7表現量降低會促進其細胞侵襲能力。降低CL1-0細胞的BMP7表現量並將其培養基加入CL1-5細胞中，發現CL1-5細胞生長會被抑制。細胞週期分析顯示CL1-0培養基加入CL1-5細胞會抑制CL1-5細胞進入S期。Q-PCR分析顯示CL1-0培養基在CL1-5中誘導p21CIP1表達。透過Q-PCR基因表達分析，我們確認BMP7在CL1-0和CL1-5細胞表達。Clonogenic分析顯示，CL1-0培養基中的BMP7表現量下降使得CL1-5細胞生長不會受到抑制。 Kaplan-Meier分析顯示，BMP7表現量與NSCLC患者的recurrence-free survival有正相關。我們的研究結果表明BMP7在肺癌細胞異質性和腫瘤發展中具有關鍵作用，具有作為肺癌復發和轉移的預測生物標誌的潛力。

The cancer cellular heterogeneity in tumors has been linked to tumor progression, such as metastasis, chemoresistance and recurrence. However, how the crosstalk between heterogeneous cancer cells in tumors affects tumor progression in unknown. In this study, we report that BMP-7 is highly expressed in a subgroup of lung tumors, in which BMP-7 expression is inversely associated with lymph node metastasis. In high proliferative CL1-0 lung cancer cells, BMP7 silencing promoted invasion. The conditioned medium from CL1-0 cells suppressed the growth of its high invasive descendant CL1-5 cells. Cell cycle analysis revealed that CL1-0 conditioned medium inhibits the S phase thus attenuating the cell cycle progression of CL1-5. Quantitative-PCR (Q-PCR) assays showed that CL1-0 conditioned medium induced p21CIP1 expression in CL1-5. Through gene expression profiling analysis followed by Q-PCR, we identified BMP-7 as one of genes differently expressed between CL1-0 and CL1-5 cells. Clonogenic analysis revealed that BMP-7-silencing in CL1-0 conditioned medium unleashed the growth inhibitory effect on CL1-5. Kaplan-Meier analysis showed that the BMP-7 expression correlated with a better recurrence-free survival in NSCLC patients. Our findings suggest a critical role of BMP-7 in lung cancer cellular heterogeneity and tumor progression with a potential to serve as a predictive biomarker for recurrence and metastasis of lung cancer.

摘要 4
Abstract 5
Introduction 7
Lung cancer 7
Cancer cellular heterogeneity 7
BMP-7 (Bone morphogenetic protein 7) 8
BMP-7 signaling 9
Study Aim: 10
Materials and Methods 10
Cell Culture 10
Lentiviral Infection 11
Cell Proliferation Assay 11
Cell-Cycle Analysis 12
RNA Extraction 12
Quantitative Real-time PCR (Q-PCR) 12
Immunoblotting 13
Clonogenic Assay 13
Live Cell Migration Assay 13
Transwell Invasion Assay 14
Immunohistochemistry Staining and Tissue array 14
Statistical Analysis 14
Table 1: Q-PCR primers 15
Results 16
Discussion 19

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