The Investigation For Hepcidin Expression In Non-small Cell Lung Cancer And Promoting Growth Of Lung Cancer Cell

Objective:Lung cancer is the most common cancer all over the world, as well as the major cause of cancer death in men, there are nearly1.2million new cases per year. Among women, it is the fourth most common cancer and the second leading cause of cancer death. Histologically, lung cancer can be classified into non-small cell lung cancer (NSCLC) and SCLC. NSCLC accounts for>85%of primary lung cancers. Iron is essential for fundamental cellular processes in all living organisms. Many animal models and epidemiological surveys showed that excess iron is one of important reasons for promoting the generation of malignant tumors. Studies found that the regulation of iron metabolism was closely associated with Hepcidin in the body. Hepcidin is a small peptide which is synthesized and secreted by the liver, and is expressed in the heart, brain, lung and pancreas. Human Hepcidin was transcribed and translated by HAMP gene. Hepcidin can combine with ferroportin on the cell membrane, which leads to its phosphorylation, internalization and degradation, thereby inhibiting the output of intracellular iron. Recently, more attention was in studying the relationship between Hepcidin and tumors, studies have shown that Hepcidin expression is decreased in hepatocellular carcinoma and renal cell carcinoma, but increased in colon cancer and breast cancer. For now, there are no studies reported the relationship between Hepcidin and lung cancer, so this project focuses on the study of Hepcidin-25expression in blood circulation of NSCLC patients, analyzing the relationship between Hepcidin and its associated signaling pathway factor IL-6and BMP in patients with NSCLC; Detected the level of Hepcidin in lung cancer tissue samples, and analyzing its relationship with pathological features; Further analyze the relationship between Hepcidin and the growth of lung cancer cells by cell experiments, and study the mechanism of Hepcidin action in the growth of lung cancer cells. Method:Serum hepcidin-25, bone morphogenetic protein (BMP)2, and interleukin (IL)-6concentration in53patients and16normal individuals was measured by ELISA, which was analyzed their relationship with pathological features of lung cancer. Reverse transcriptase quantitative polymerase chain reaction was utilized to study the expression of hepcidin mRNA in paired tumor and non-tumor lung tissues in surgical specimens from65patients with NSCLC, as well as in six types of lung cancer cell line and human bronchial epithelial (HBE) cells. Hepcidin protein expression and cellular localization in NSCLC was determined by immunohistochemistry. Further construct the Hepcidin eukaryotic expression vector, due to Hepcidin over expression in HBE cell lines, and study the impact of increased Hepcidin expression for the proliferation and growth of cells by WST-8and colony formation experiments. Reduce the expression of Hepcidin in H520cell lines by RNA interference, and study the the impact of decreased Hepcidin expression for the proliferation and growth of cells by WST-8and colony formation experiments. Detect the impact of Hepcidin binding domain (HBD) for H520cells by WST-8experiment, and analyzed the action of HBD in the growth of lung cancer cells. In addition, detect the labile iron pool in HBE cells with increased Hepcidin expression and H520cells with decreased Hepcidin expression by fluorescence measurement, and further elucidate the mechanism of Hepcidin promoting the proliferation and growth of lung cancer cells.Result:The serum Hepcidin-25level of the patients with NSCLC was significantly higher than in healthy volunteers (P=0.004); The increased serum Hepcidin-25level of the patients with NSCLC was associated with the incidence of lymph node metastasis (P=0.044). Meanwhile, the serum Hepcidin-25level was positively related with TNM clinical stage, and increased with the improvement of clinical stage (P=0.017). We found that IL-6was no significant different between NSCLC patients and healthy volunteers by further measurement (P0.917), but the IL-6level was decreased with the incidence of lymph node metastasis (P=0.018) and improvement of TNM clinical stage (P=0.041). The level of serum BMP-2in patients with NSCLC was significantly higher than in healthy volunteers (P<0.01), and was positively related with incidence of lymph node metastasis (P=0.011) and improvement of TNM clinical stage (P=0.001). Through Spearman’s correlation coefficient analysis, we found that the level of Hepcidin-25was negatively related with IL-6(r=-0.242, P=0.049), and positively related with BMP-2(r=0.737, P<0.001).We found that there were67.7%cases of Hepcidin mRNA in lung cancer tissue samples of all65patients with NSCLC was higher than in normal lung tissues by Real-time qPCR detection, and30.8%cases was lower than in normal lung tissues, only1.5%cases was not different with normal lung tissues, it indicated that the Hepcidin mRNA expression in NSCLC tissues was increased (P<0.01). We standardizing the ratio of hepcidin mRNA relating to beta-actin (ie.2-ΔCT value) by log10, and founded that Hepcidin mRNA expression was increased in lung cancer tissue (P=0.001). In addition, we founded that Hepcidin mRNA in lung cancer tissue samples was positively related with incidence of lymph node metastasis (P=0.041), and was positively related with serum Hepcidin-25level (r=0.280, P=0.042). It showed that positive expression of Hepcidin in NSCLC was higher than in normal lung cancer (P<0.05).We found that Hepcidin mRNA expression in4cell lines of all6NSCLC cell lines was increased by Real-time qPCR, and only which in2cell lines was decreased. We chose H520cell lines which express highest level of Hepcidin mRNA for immunofluorescence experiment, and found that Hepcidin was significantly expressed in H520cell lines, the fluorescence signal were concentrated in the cytoplasm and nearly membrane fraction. We successfully constructed the Hepcidin eukaryotic expression vector, and transferred it to HBE cells which could stable overexpress Hepcidin; Using WST-8test and colony formation experiment, we found that Hepcidin overexpression was significant role in promoting the growth of HBE cell. Hepcidin gene was silenced by RNA interference in H520cells; Using WST-8test and colony formation experiment, we found that decreased expression of Hepcidin has a significant inhibitory effect on the growth of H520cells. The synthetic HBD can inhibit the proliferation and growth of H520cells after intervention. The result of measuring the intracellular liable iron pool by fluorescence measurement showed that, increased Hepcidin expression in HBE cells can improve the iron concentration, and decreased Hepcidin expression in H520cells can reduce the intracellular iron concentration.Conclusion:Hepcidin expression was significantly increased in the serum and lung cancer tissues of patients with non-small cell lung cancer, and it was positively correlated with lymph node metastasis and clinical stage of lung cancer. Meanwhile, the serum BMP-2level of NSCLC patients was significantly increased, and was positively correlated with lymph node metastasis, clinical stage and serum Hepcidin-25. It means that BMP-2may be a key factor in regulating Hepcidin. Vitro experiments confirmed that Hepcidin can promote the growth of lung cancer cells, and it was related with the change in the concentration of intracellular iron. It elaborated mechanism of action in proliferation and growth of lung cancer cells by the control shaft of Hepcidin-Ferroportin-iron. This study considered that Hepcidin can be used as a new target for cancer threatment, and it can suggest a new research basis and ideas for tumor treatment. In addition, HBD may be used as a new molecule drugs for the treatment of tumors.