[Construction of recombinant lentiviral vectors containing Rheb gene and its mutant Rheb'D60K gene and their expression in human liver cancer cells]

Nan Fang Yi Ke Da Xue Xue Bao. 2012 Mar;32(3):341-4.
[Article in Chinese]

Abstract

Objective: To construct recombinant lentiviral vectors carrying Rheb gene and its mutant Rheb'D60K gene, and examine their expression in human liver cancer cells.

Methods: Rheb gene was amplified by PCR to construct the recombinant plasmid LV31-Rheb-WT and LV31-Rheb-D60K. HEK-293 FT cells were contransfected with the recombinant lentiviral vector together with a lentiviral package plasmid to produce the lentiviral particles. The expression of PS6 protein was detected in the lentivirus-infected MCF-7 cells. The apoptosis of SK-HEP-1 cells transfected with LV31-Rheb-WT or LV31-Rheb-D60K was observed.

Results: The recombinant LV31-Rheb-WT and LV31-Rheb-D60K vectors were confirmed by PCR and DNA sequencing. Western blotting showed that PS6 protein expression was increased in LV31-Rheb-WT-transfected cells while decreased in LV31-Rheb-D60K-transfected cells. LV31-Rheb-D60K-transfected SK-HEP-1 cells showed more obvious apoptosis after starvation than LV31-Rheb-WT-transfected cells.

Conclusion: Lentiviral vectors carrying Rheb gene and its mutant has been successfully constructed, which can be useful in further investigation of the role of Rheb gene in cancer cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / genetics
  • Carcinoma, Hepatocellular / metabolism
  • Carcinoma, Hepatocellular / pathology*
  • Genetic Vectors / genetics
  • HEK293 Cells
  • Humans
  • Lentivirus / genetics
  • Lentivirus / metabolism
  • Liver Neoplasms / metabolism
  • Liver Neoplasms / pathology*
  • MCF-7 Cells
  • Monomeric GTP-Binding Proteins / biosynthesis*
  • Monomeric GTP-Binding Proteins / genetics*
  • Mutant Proteins / genetics*
  • Neuropeptides / biosynthesis*
  • Neuropeptides / genetics*
  • Ras Homolog Enriched in Brain Protein
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Transfection

Substances

  • Mutant Proteins
  • Neuropeptides
  • RHEB protein, human
  • Ras Homolog Enriched in Brain Protein
  • Recombinant Proteins
  • Monomeric GTP-Binding Proteins