ABSTRACT
Objective The shRNA library was used to screen the tumor suppressor genes related to lung epithelial cells,and its function of inhibiting malignant transformation in lung cells was preliminarily verified,which provided a theoretical basis and a new therapeutic target for tumor prevention and treatment. Methods The shRNA retrovirus library was constructed by GP2 -293 virus packaging cell line to infect the immortalized lung bronchial epithelial BEAS-2B cells. The transfected epithelial cell clones were screened by soft agar colony formation,and a single transformed cell clone with a diameter greater than 1. 0 mm was selected. The in-serted shRNA fragment was amplified by PCR,and the target candidate gene corresponding to shRNA was determined by the conven-tional DNA sequencing and blast alignment. A candidate tumor suppressor gene INPP4B was verified by soft agar cloning and tumor formation in nude mice. MTT assay was used to detect the cell proliferation. Results Six lung epithelial malignant transformation in-hibitory factors were screened by soft agar colony formation. The candidate INPP4B gene was selected for functional experiments. Si-lencing INPP4B gene in BEAS-2B cells promoted the formation of clones in the soft agar plates,and the cell proliferation rate was accelerated. The silencing cell line showed the enhanced tumorigenicity in nude mice,indicating that INPP4B was involved in tumor formation. Conclusion shRNA library and soft agar colony formation assays are a powerful tool for screening tumor suppressor genes, and INPP4B is a malignant transformation inhibitor of lung epithelial cells.
ABSTRACT
Translationally controlled tumor protein (TCTP)is a small protein highly conserved in a variety of eukaryotic species.TCTP is overexpressed in various tumor cells and has been implicated in the regu-lation of cellular processes including apoptosis,DNA repair and drug resistance.By reviewing the recent pro-gress of TCTP research,TCTP is becoming an important regulator of DNA repair and a new molecular target for tumor therapy.
ABSTRACT
AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future.