Studies On The Expression Of NLK As Well As The Correlation Between NLK And The Progression Of Non-small Cell Lung Cancer

ObjectiveTo investigate the expression of Nemo-like kinase(NLK) in non-small cell lung cancer(NSCLC), and analyze the expression variation and the role of NLK during the proliferation process of NSCLC. To investigate the correlation between NLK and the progression of NSCLC, and clarify the correlation of NLK as well as the occurrence and development of NSCLC.Methods1. The expression of NLK, PCNA, and GAPDH was evaluated by Western blot in 8 paired fresh NSCLC tissues. And analyzed the relations between NLK, Ki-67 and the clinical feactures about NSCLC patients’ age, sex,tumor grade, clinical stage, lymph node metastasis by immunohistochemistry(IHC) on 83 paraffin-embedded slices. With COX regression analysis, we analyzed the relationship of NLK expression as well as the clinical characteristics and patients’ prognosis.2. In this study, serum starvation and release synchronization process cell lung adenocarcinoma cell line A549 was used to detect changes in the cell cycle by flow cytometry. Western blot technique was peformed to detect NLK, PCNA and cyclin A expression in this process. After interfere with NLK by lentivirus infection in A549 and SK-MES-1 cells, Western blot was used to detect NLK,PCNA and cyclin A expression. Meanwhile, the proliferation of A549 cells was analyzed by flow cytometry cell cycle and CCK-8 cell activity. Furthermore,clone formation ananlysis was used in A549 cells. At last, luciferase reporter gene assay and Western blot were performed in A549 cells to find the detailmechanism of NLK in Wnt/β-catenin signaling pathway for NSCLC progression.Rests1. Western blot and IHC analysis showed that the expression of NLK in NSCLC tissues was significantly lower than the corresponding adjacent tissues,while the expression of PCNA in NSCLC was significantly higher than that in adjacent tissues. The relationship between NLK expression and NSCLC histological grade(P=0.002), clinical stage(P=0.001), lymph node metastasis(P=0.015), and Ki-67(P<0.001) were statistically significant. COX regression analysis showed that clinical stage(P<0.001), histological grade(P<0.001),NLK expression(P=0.023), and Ki-67 expression(P=0.025) could determine the non-small cell lung cancer prognosis.2. Serum starvation caused A549 cell growth cycle of stagnation. After serum stimulation of the release of A549 cells, NLK protein expression decreased, while cyclin A and PCNA expression increased. Infected with lentivirus, we found that NLK could inhibit the proliferation of A549 and SK-MES-1 cells. Luciferase reporter gene assay found that NLK could inhibit the transcriptional activity of β-catenin, as well as the expression of Wnt/β-catenin signaling pathway target genes c-myc and cyclin D1 in A549 cells.Conclusions1. NLK was lowly expressed in NSCLC compared with adjacent tissues.Patients with high expression of NLK had better prognosis than those with low expression of NLK, suggesting that NLK might be associated with the development of NSCLC.2. The cell cycle of A549 and SK-MES-1 cells would accelarate when infected with NLK-shRNA lenti-viruses. NLK might inhibit the proliferation of A549 and SK-MES-1 cells. In A549 cells, NLK could negtively regulate the transcription activity of β-catenin. And NLK could reduce the expression of c-myc and cyclin D1, thus affect the progression of NSCLC.