Construction Of Lentiviral Vector-mediated RNA Interference Of APE1 Gene And Observation Of Its Silence Efficiency In Human MHCC97-H Cell Line

Objective To investigate the effects of APE1 gene expression on human high invasion hepatocellular carcinoma MHCC97-H cells, the lentiviral vector-mediated RNA interference targeting APE1 gene was constructed and transfected into MHCC97-H cells, the expression of APE1 protein and mRNA was detected by Western blot and RT-PCR, and analysis its functional.It provides theoretical basis and experimental basis for further discussion on the invasion and metastasis mechanism of APE1 gene in hepatocellular carcinoma.Methods According to the information of APE1 gene in PubMed Home,4 small RNA interference sequences targeting APE1 gene were designed. The sequences were separately cloned into the pGCSIL-GFP vector to construct restructuring plasmid (nam- ed KD1~KD4), which were subsequently confirmed by PCR and DNA sequencing analysis. The most effective targets was screened by Western blot, The titer of lentivirus was determined after 293T cells when they were cotransfected with the most effective reorganization plasmid, pHelper 1. 0 and pHelper 2. 0. After sueeessful construction of the recombinant 1entiviruses, they were injected into MHCC97-H cells, the mRNA and protein expression of the APE1 gene were examined by RT-PCR and Western blot, in order to observe the change of APE1 protein localization,the immunocytochemistry was used.The proliferation of the MHCC97-H cells was detected by MTT assays and the cell cycle of the MHCC97-H cells was detected by FCM, The effects of LV-APE1-shRNA on migration and invasion of the MHCC97-H cells was measured by reconstituted matrigel invasion and polycarbonate filters migration experiments.The adhesive capacity to matrigel and FN was measured by MTT assays.Results1. PCR and DNA sequencing demonstrated that the inserted sequences were correct. The KD3 silence efficiency of APE1 gene nearly 90% was inhabited by Western blot, The titer of lentivirus was 4x108TU/ mL.2. After transfection with LV-APE1-shRNA,APE1expression in MHCC97-H cells was significantly inhibited at both mRNA and protein levels compared with non-transfected and empty vector transfected with MHCC97-H cells, the cellular immune chemistry showed that the expression of APE1 protein from the cell nucleus, cell plasma transferred to cell nucleus.3. As compared with non-transfected and empty vector transfected MHCC97-H cells, MTT assays showed that the proliferation of MHCC97-H cells was inhibited. FCM test showed that the cells cycle of MHCC97-H cells was changed: the G1 stage was prolonged,and the G2 stage and S stage was unit shortening.4. The adhesion and reconstituted matrigel invasion and polycarbonate filters migration experiments in vitro showed that the adhesive, migration and invasion of MHCC97-H cells was inordinately inhibited when compared with non-transfected and empty vector transfected MHCC97-H cells.Conclusion1. The high effective lentiviral RNAi vector can significantly, stably inhibit the expr- ession of APE1, which will provide reliable foundation for the subsequent research.2. The migration and invasion ability of MHCC97-H cells was inordinately inhibited after transfecting with LV-APE1-shRNA. And it implies that APE1 gene can probably regulate invasion and metastasis ability of hepatocellular carcinoma, and it provides the theoretical basis for hepatocellular carcinoma patients with tumor gene therapy, and it also provides the invasion and metastasis mechanism of hepatocellular carcinoma with experimental foundation for subsequent research.