The ORF of CsGRN was cloned into the pEGFP-C1 eukaryotic expression vector, and the recombinant plasmids were confirmed by sequencing (Additional file 1: Figure S1). Viable cell images of most cells showed green fluorescence and grew well after the recombinant plasmid pEGFP-C1-CsGRN transduction (Fig. 5a). To exclude the possibility that transfection plasmids could affect cell survival, the cellular viability of the transfected cells was detected by Annexin V-APC/7AAD staining with FACS analysis. Neither the pEGFP-C1 plasmids nor the pEGFP-C1-CsGRN plasmids had any effect on cell viability (Fig. 5b). The qRT-PCR and Western blotting results showed the relative mRNA level and the protein expression of CsGRN were significantly raised in the PLC-GRN and RBE-GRN cells compared with that in PLC-GFP/RBE-GFP cells (Fig. 5c, d), indicating that overexpressed-CsGRN PLC/RBE cell line was successfully constructed.Fig. 5Successful construction of stable CsGRN-overexpressed hepatoma cells and cholangiocarcinoma cells. a Green fluorescence was observed under an inverted microscope and was emitted by PLC and RBE cells transformed with pGFP-C1 and pGFP-C1-CsGRN, respectively. The images were magnified at 100× (scale-bar: 200 μm).b Cellular viability of transfected PLC and RBE cells (24 h, 48 h and 72 h after transfection) was detected by Annexin V-APC/7AAD double staining with FACS analysis. c qRT-PCR was used to detect the expression of CsGRN mRNA between the experimental group and control group. d Western blot analysis was used to determine the CsGRN protein expression between the experimental group and control group. *P < 0.05, ***P < 0.001 compared with a matched group