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| Construction of Lentiviral Expression Vector Expressing Human RAB27A and Investigation Its Effect on the Proliferation of HepG2 Cell Lines |
| LIU Xue-jie1,2, LIN Wei-ran2, TANG Li-chun2, SUN Wei2, WEI Han-dong2, JIANG Ying1,2 |
1. Graduate School, Anhui Medical University, Hefei 230032, China;
2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China |
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Abstract Objective:To over express RAB27A by lentiviral system in human hepatocellular carcinoma (HCC) cell line HepG2 and investigate the effect.Methods:GFP-RAB27A protein coding gene sequences were amplified from plasmid pEGFP-C1-RAB27A, the PCR products were digested by double restriction enzymes and cloned into shuttle plasmid pENTR/U6, then pHAGE-GFP-RAB27A vector were constructed by Gateway technology. Recombinant plasmid pHAGE-GFP-RAB27A was mixed with the packaging plasmid(psPAX2)and the envelope plasmid (pMD2.G)and then co-transfected into HEK-293T cells. HepG2 cells were transfected with concentrated recombinant lentivirus particle. Western blot analysis were used to detect RAB27A expression in HepG2 cell lines. The proliferative activity was determined by CCK-8 and colony forming assay and the cell cycle analysis by flow cytometry. Results:The recombinant lentiviral expression vector pHAGE-GFP-RAB27A was constructed correctly. After transfected to HEK-293T, the lentivirus was successfully prepared. Strong green fluorescence was observed in transfected HepG2 cells under fluorescent microscope which showed the recombinant lentivirus expressing. The RAB27A protein was over expressed showed by Western-blot. The proliferation of infected HepG2 showed inhibited, and the percentage of S-phase cells was obviously decreased (P<0.01). Conclusion: The exogenous over expressed RAB27A was found inhibited the proliferation of transfected HepG2 cell lines. RAB27A is a key protein in the hepatocellular carcinogenesis and proliferation.
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Received: 27 April 2014
Published: 25 September 2014
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