Construction And Identification Of Lentiviral Vector Carrying MYCT-1Gene

Gastric cancer is one of the malignant tumors with highest incidence. Theexact molecular mechanism of the occurrence of gastric cancer is unclear andthere is no effective approach for the early clinical diagnosis and treatment ofgastric cancer. Therefore, it is of importance to study the genes which play keyroles during the carcinogenesis and development of gastric cancer. The gene ofMYCT-1(myc target), once named as MTLC (myc target from laryngealcarcinoma cells), locates on6q25. MYCT-1protein mainly locates in thenucleus of most kinds of normal tissue cells. In many kinds of cancer cells, suchas gastric cancer, laryngeal cancer, breast cancer, liver cancer, the expression ofMYCT-1decreased significantly, which indicates that MYCT-1may serve as anovel tumor suppressor gene. Studies showed that MYCT-1is an importantmember within c-myc cell signaling pathway and is involved in the regulation ofnormal cellular physiological processes through its activity as transcriptionfactor. The expression of MYCT-1in76%of gastric cancer tissues isdown-regulated, and its expression level in normal tissues, cancer tissues andmetastatic cancer tissues decreased successively. Results from gene transfectionexperiments showed that MYCT-1over expression in BGC823cell lineinhibited cell growth and promoted cell apoptosis, which also indicates that thedown regulation of MYCT-1gene expression plays important roles in thedevelopment of gastric cancer. When MYCT-1gene was silenced in gastric mucosal cells GES-1by RNA interference, the cellular proliferation sloweddown, the cellular apoptosis increased and S phase became much longer.Although the silence of MYCT-1gene does not directly lead to malignanttransformation of normal gastric mucosal cells, the results indicated thatMYCT-1may serve to inhibit DNA synthesis.Lentiviral vector is one kind of gene therapy vector based on humanimmunodeficiency virus (HIV). Lentivirus can infect both dividing andnon-dividing cells, can exist and express target protein within cells for a longtime and is with good bio-safety.In present study, we constructed recombinant lentiviral expression vectorthrough incorporation of MYCT-1-encoding DNA fragment into enzymedigested lentiviral vector. Lentivirus were packaged in293T cells throughplasmids transfection, and then its titer was determined for further experiments.Our experiments created good condition for the subsequent animal experimentsto study the functions of MYCT-1in caner development.ObjectiveTo construct recombinant lentiviral vector carrying MYCT-1geneGV218-MYCT-1and lay the foundation for further studies and animalexperiments.MethodsMYCT-1gene fragment was amplified by PCR, and then ligated withenzyme digested lentiviral vector to construct recombinant lentivirus vector GV218-MYCT-1. The positive clones were identified by PCR and DNAsequencing. The recombinant vector was then transfected into293T cells and theexpression of GFP and MYCT-1were observed by fluorescence microscope andWestern Blot, respectively. Lentivirus carrying MYCT-1were packaged within293T cells by cotransfection of recombinant vector and lentivirus packagingsystem. The titer of lentivirus was determined by real-time PCR.Results1.Construction and identification of GV218-MYCT-1lentiviral vector overexpressing MYCT-1The target gene MYCT-1was prepared by PCR amplification and thenincorporated into vector GV218. All of the clones were identified by PCR. ThePCR product from positive clones were912bp and that from negative cloneswere198bp. The DNA sequencing results showed that GV218-MYCT-1lentiviral vector was constructed successfully.2. Protein expression in293T cells transfected with recombinantGV218-MYCT-1vectorAfter recombinant GV218-MYCT-1vector was transfected into293T cells,GFP could be observed with high intensity, which indicated that plasmidtransfection and the protein marker expression were correct. Through WesternBlot, the band with around52kD molecular weight was observed, which furthershowed that the plasmid was constructed successfully.3. GV218-MYCT-1lentivirus packaging and titer determination The recombinant lentiviral vector and other two kinds of helper plasmids(pHelper1.0and pHelp-er2.0) were co-transfected into293T cells. Cellularsupernatant was harvested48h later and the virus was concentrated. The finalvirus titer was2×10~8TU/ml determined by real-time PCR.ConclusionsMYCT-1gene was liagted with enzyme-digested lentiviral vector GV218to construct recombinant vector GV218-MYCT-1. The recombinant vector wastransfected into293T cells and the protein expression was determined byWestern Blot. The results showed that the recombinant vector GV218-MYCT-1was constructed successfully. The lentivirus carrying MYCT-1was packagedwith293T cell by co-trasfection of three kinds of plasmids. After collection andconcentration, the lentivirus with titer2×10~8TU/ml was prepared successfully.These results laid good foundation for the further study to research the roles ofMYCT-1in cancer development and treatment.