Original Article
Nm23-H1 was involved in regulation of KAI1 expression in high-metastatic lung cancer cells L9981
Abstract
Background: The tetraspanin KAI1/CD82 was identified as a tumor metastasis suppressor that down-regulated in malignant progression of lung cancer. However, the underlying mechanism of anti-metastasis role of KAI1 in lung cancer is hardly known. In this paper, we sought to study the function and regulatory mechanism of KAI1 in high metastasis lung cancer cell line.
Methods: KAI1 expression was detected in high/low metastatic large lung cancer cell line L9981/NL9980 by quantitative real-time polymerase chain reaction (qRT-PCR). The tumor suppressor function of KAI1 was determined by wound healing assay after over-expression or knockdown of KAI1 in L9981 or NL9980 cells. Invasion assay was performed to detect the invasion ability of L9981 by transfection of KAI1. The effect of tumor suppressor p53 on KAI1 expression was measured by western blot and luciferase assay. Then the regulation of KAI1 due to over-expression of metastasis suppressor nm23-H1 was monitored by qRT-PCR, western blot and reporter gene assay. The progression of L9981 cells after p53 and nm23-H1 expression was detected by invasion assay. Also, methylation status of KAI1 promoter in NL9980 and L9981 cells were examined by bisulfite sequencing and methylation-specific PCR.
Results: We found that KAI1 is down-regulated in high metastatic L9981 cells compare with NL9980 cells. The migration and invasion of L9981 cells were remarkably suppressed in vitro by KAI1 transfection. The migration ability of NL9980 was enhanced by inhibition of KAI1. Furthermore, KAI1 expression was induced after over-expression of p53 or nm23-H1, while cell invasion was inhibited in L9981 cells. The results of reporter analysis indicated that KAI1 promoter region between −922 to −846 could response to nm23-H1. In addition, we discovered only slight methylation of KAI1 promoter, which showed that loss expression of KAI1 in L9981 cells may not due to promoter methylation.
Conclusions: The results suggested that nm23-H1 was involved in the KAI1-regulated inhibition of metastasis in lung cancer cells. More insights into the relationship between KAI1 and other metastasis suppressors will pave the way for the elucidation of anti-metastasis mechanism in lung cancer.
Methods: KAI1 expression was detected in high/low metastatic large lung cancer cell line L9981/NL9980 by quantitative real-time polymerase chain reaction (qRT-PCR). The tumor suppressor function of KAI1 was determined by wound healing assay after over-expression or knockdown of KAI1 in L9981 or NL9980 cells. Invasion assay was performed to detect the invasion ability of L9981 by transfection of KAI1. The effect of tumor suppressor p53 on KAI1 expression was measured by western blot and luciferase assay. Then the regulation of KAI1 due to over-expression of metastasis suppressor nm23-H1 was monitored by qRT-PCR, western blot and reporter gene assay. The progression of L9981 cells after p53 and nm23-H1 expression was detected by invasion assay. Also, methylation status of KAI1 promoter in NL9980 and L9981 cells were examined by bisulfite sequencing and methylation-specific PCR.
Results: We found that KAI1 is down-regulated in high metastatic L9981 cells compare with NL9980 cells. The migration and invasion of L9981 cells were remarkably suppressed in vitro by KAI1 transfection. The migration ability of NL9980 was enhanced by inhibition of KAI1. Furthermore, KAI1 expression was induced after over-expression of p53 or nm23-H1, while cell invasion was inhibited in L9981 cells. The results of reporter analysis indicated that KAI1 promoter region between −922 to −846 could response to nm23-H1. In addition, we discovered only slight methylation of KAI1 promoter, which showed that loss expression of KAI1 in L9981 cells may not due to promoter methylation.
Conclusions: The results suggested that nm23-H1 was involved in the KAI1-regulated inhibition of metastasis in lung cancer cells. More insights into the relationship between KAI1 and other metastasis suppressors will pave the way for the elucidation of anti-metastasis mechanism in lung cancer.
