Establishment of phosphatase of regenerating liver-1 stably-transfected HCC cell lines and the biological functions in Huh7 cells

Objective To construct phosphatase of regenerating liver-1(PRL-1)eukaryotic expression vector, establish stably-transfected Huh7 cell lines and investigate biological functions of PRL-1 in Huh7 cells. Methods The coding sequences(CDS) fragment of PRL-1 was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR).BgiⅡ/XhoⅠrestriction sites were introduced into the flank of the target fragment.Then, p MSCV-Hemagglutinin(HA)-PRL-1 vector was constructed by cloning the target fragment into p MSCV-PIG plasmid.PCR and DNA sequencing verified recombinant plasmid was successfully constructed.Huh7 cells were infected by retrovirus supernatant which was packaged using 293T cells.Following infection, cells were selected with 1 mg/L puromycin for 2 weeks.Then the expression of PRL-1 in Huh7 cells was detected by fluorescence microscope, flow cytomety, real-time fluorescent quantitative polymerase chain reaction(FQ-PCR)and Western blotting.Biological functions of PRL-1 in Huh7 cells were investigated by cell counting kit-8(CCK-8)assay, plate colony formation assay and Transwell invasion assay. Results DNA sequencing demonstrated target fragment inserted into p MSCV-PIG vector was exactly correct.Fluorescence microscope and flow cytometry showed stably-transfected efficiency was over 90%.FQ-PCRassay showed the mRNA level of PRL-1 in overexpression group was(4. 4±1. 5)times as compared with that in control group(P<0. 05).Western blotting assay indicated PRL-1 was exogenously overexpressed in Huh7 cells.Compared with control cells,the proliferation rate increased in the PRL-1 stable transfected cells(P<0. 01).Colony formation efficiency was higher in PRL-1 stably tranfected cells[(26. 7 ±1. 9)% vs.(7. 3±0. 8)%, P< 0. 01], and overexpression group had much stronger invasive capability (P<0. 01). Conclusion PRL-1 stably-transfected Huh7 cell line is successfully established and PRL-1 enhances proliferation, colony formation and invasion in Huh7 cells. Key words: Hepatocellular carcinoma; Phosphatase of regenerating liver-1; Retrovirus vector; Biological functions