关键词: 黑色素瘤, 中枢神经系统转移, 软脑膜转移, 脑转移, HMGA2

Abstract:

Background and purpose: Leptomeningeal metastasis is a form of central nervous system metastasis of melanoma. High mobility group A2 (HMGA2) has been proven to play an important role in the occurrence and development of various tumors, but its biological functions in leptomeningeal metastatic melanoma cells remain unclear. On the basis of building mouse models of central nervous system metastasis of melanoma, this study investigated the differences in cell migration and cell proliferation among leptomeningeal metastatic melanoma cells, primary site melanoma cells and brain parenchymal metastatic melanoma cells, and further clarified the effects of differentially expressed gene HMGA2 on cell migration and proliferation of leptomeningeal metastatic melanoma cells. Methods: B16 mouse melanoma cells (B16-parental cells, B16-Par) stably expressing tdTomato and luciferase were generated by lentiviral infection. Subsequently, B16 specific brain parenchymal metastatic cells (B16-brain metastatic cells, B16-BrM) and B16 specific leptomeningeal metastatic cells (B16-leptomeningeal metastatic cells, B16-LM) were collected after adaptive screening of metastatic sites in vivo. The differences in migration and proliferation among B16-Par, B16-BrM and B16-LM were assessed by wound healing assay and cell counting kit-8 (CCK-8). RNA sequencing (RNA-seq) was used to analyze differential gene expression in B16-Par, B16-BrM and B16-LM, and HMGA2 gene specifically upregulated in B16-LM was screened out. The results were verified by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot. Gene ontology (GO) analysis was performed for genes which were upregulated in B16-LM specifically. siRNA was used to interfere with the expression of HMGA2 gene in B16-LM, and the knock-down effect was verified by RTFQ-PCR and Western blot. The effects of knocking down HMGA2 on cell migration and proliferation were detected by wound healing assay and CCK-8 assay. Using GSE174401 data in Gene Expression Omnibus (GEO), the specificity of HMGA2 gene expression in leptomeningeal metastatic melanoma cells from patients was verified. Results: Compared with Par cells, tumor cells screened by the brain environment were more likely to colonize the central nervous system. B16-LM had stronger migration and proliferation abilities, and upregulated the expression of HMGA2 gene. GO analysis revealed that HMGA2 was associated with many biological processes such as angiogenesis and cell proliferation. When the expression of HMGA2 gene was knocked down, the migration and proliferation of B16-LM could be inhibited. HMGA2 was upregulated in leptomeningeal metastatic melanoma cells from patients. Conclusion: Leptomeningeal metastatic melanoma cells had relatively unique cellular characteristics, which promoted cell migration and proliferation by upregulating HMGA2 gene expression.

Key words: Melanoma, Central nervous system metastasis, Leptomeningeal metastasis, Brain metastasis, HMGA2