Transformational genes in rat hepatoma cells induced by irradiated nonparenchymal cells

Abstract

Objective To investigate the influence of irradiated liver microenvironment on irradiated hepatoma metastatic potentials and its possible molecular mechanism. Methods Primary rats’ nonparenchymal cells (NPCs) were separated and cultured, then they were divided into irradiated (6-Gy X-ray) and non-irradiated groups. After irradiation, conditioned media from irradiated (SR) or non-irradiated (SnonR) cultures were collected and added to the hepatoma McA-RH7777 cell line cells which were given single exposure of 6 Gy X-ray. Then, hepatoma cells were continuously passaged (RH 6 Gy and RH 6 Gy-R). The expression of mRNA of RH 6 Gy and RH 6 Gy-R cells was compared by gene microarray. Subsequent bioinformatic analysis of these target genes was performed by DAVID software (GO-analysis, Kyoto gene and genome encyclopedia (KEGG)-analysis and BIOCARTA-analysis). In vitro wound scratch assay was conducted to compare the metastatic potentials of RH 6 Gy and RH 6 Gy-R cells. Results The gene microarray test showed that the expression of genes related to metastasis was up-regulated in RH 6 Gy-R cells, such as cyclin dependent kinase 2 (CDK2), mitogen-activated protein kinase 9 (MAPK9), Cyclin A, Toll/interleukin-1R domain-containing adaptor protein (TIRAP), 3-phosphoinositide-dependent protein kinase 1/2 (PDK1/2) and phosphatidylinositol 3 kinase (PI3K). These mRNAs were enriched in pathways involved in the development and metastasis of cancer, such as adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, phosphatidylinositol signaling system, Ras signaling pathway.In vitro wound scratch assay revealed that the healing rate of RH 6 Gy and RH 6 Gy-R cells was (9.00±1.35)% and (41.30±6.32)% respectively. Conclusion Radiation can increase invasiveness and metastatic potentials of sublethally irradiated hepatoma cells, and co-culture with conditioned media from irradiated NPCs promotes these potentials. We speculated that some cytokines released by NPCs after irradiation promoted the expression of metastasis associated mRNA in HCC after irradiation, and thus activated the metastatic pathway. Key words: Hepatic nonparenchymal cell; Hepatoma cells; Metastasis; GO analysis; Kyoto gene and genome encyclopedia analysis