Cloning Of Human Soluble Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (sTRAIL) And The Antitumor Effects Of The STRAIL Vector On Mice Glioma In Vitro And In Vivo

Glioma is the most common and malignant tumor in brain with unfavorable outcome. Despite of the progress of neuroscience, gliomas, glioblastoma (GBM) tumors in particular, are refractory to all current therapeutic approaches. Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been reported to specifically kill malignant cells with programmed cell death after combination with death receptor (DR) on tumor celluar membrane, but to be relatively nontoxic to normal cells. However, TRAIL has been showed some hepacellar side effects in some studies, while sTRAIL didn't show such toxicity. In order to investigate the antitumor effects of sTRAIL in glioma, we cloned the gene of human sTRAIL from spleen and constructed eukaryotic expression vector. C6 cells were transfected by Lipofectin in vitro and glioma mice model was established in vivo, and the antitumor effects were presented. This is a foundation for us to find a new way to kill glioma cells and advance the therapy of this malignant brain tumor in future.Materials and methods1. Main reagent: E.coli TG1, TaqDNA polymerase, T4DNA ligase, Reverse transcriptase, RNA enzyme inhibitor, DMSO, trypsinase, Trizol, restriction endonuclease, Access RT-PCR System, PUCm-T vector, PcDNA3.1, Gel retrieval kit, plasmid extraction kit, DMED, Lipofectin, G418 et al. The Primer constuction and sequence identification was executed by Shanghai Sangon biological engineering & technology and service Co.Ltd.2. Primer design: Based on GeneBank data, asisted by Primer design software DNA star, we design the Primer as follow:Up stream: 5-GCGAATTCAGTATGGTGAGAGAAAGAGGTC-3; Down stream: 5-ACAGTAGGATCCTCCAGGTCAGTTAGCCA-3.3. Abstraction of total RNA and cloning of target gene: After obtaining traumatic spleen tissue, the scrap tissue was ground and suspended. Total RNA was sbstracted with Trizol by one-step way. Based on the protocol of Access RT-PCT system, cDNA was obtained from reverse transcription of total RNA. We amplified target gene through PCR technique, led by the Primer that designed before, with the RT product as template. The strap at 860bp was reclaimed and purified afterwards. The target gene was linked to PUCm-T vector, and transformed into the competent E.coli TG1. The purified plasmid was identitied by restriction endonuclease and the sequence of the target DNA was identified by dideoxy chain termination method.4. Construction of eukaryotic expression vector: Identified sTRAIL gene was digested by restriction endonuclease EcoR-I and BamH-I, and was inserted into eukaryotic expression vector PcDNA3.1. The Product named as PcDNA-sTRAIL.5. Effects of PcDNA-sTRAIL on glioma: In vitro, the antitumor effects of PcDNA-sTRAIL on C6 glioma cell were assessed by such methods as MTT. In vivo, the effects were also investigated in mude glioma model.Results1. The extraction of product was examed by gel electrophoresis with EB dying, and the strip of 18S and 28S was evident under ultraviolet lamp, suggesting the complete RNA.2. The product after PCR was examed by gel electrophoresis, there was specific strip around 540bp DNA marker strip, which match to anticipated molecular weight of sTRAIL. The product was successfully retrievalled.3. Identification of the sequence executed by shanghai Sangon biological engineering& technology and service Co.Ltd., confirmed the product was the sTRAIL gene.4. Construction and identification of PcDNA-sTRAIL: PcDNA-sTRAIL was digested by restriction endonuclease EcoR-I and Bamh-I respectively, and the specific strip around 5.9kb and 540bp DNA marker strip can be identified, ascertained that eukaryotic expression vectors were successfully constructed.5. The antitumor effects of PcDNA-sTRAIL on glioma cells: In vitro, the antitumor effects of PcDNA-sTRAIL on C6 glioma cell were assessed by such methods as MTT. The the growth and proliferation of C6 cells treated by PcDNA-sTRAIL were significantly inhibited compared with control group.6. The C6 glioma nude mice were established.7. The effects of PcDNA-sTRAIL on glioma nude model: In vivo, subcutaneous glioma, PcDNA-sTRAIL could inhibit tumor growth. Histological examination showed that the obvious tumor necrosis and infiltration of inflammatory cells were present around the C6 glioma of the tumor-bearing mice with PcDNA-sTRAIL.Conclusions1. The cloning vector of T-sTRAIL is constructed with the correct sequence.2. The eukaryotic expression vector of PcDNA-sTRAIL is constructed successfully confirmed by such methods as double restriction endonuclease.3. The constructed PcDNA-sTRAIL has the stable and sufficient ability to express sTRAIL.4. The C6 glioma nude mice are successfully established.5. PcDNA-sTRAIL can inhibit tumor growth, with obvious tumor necrosis and infiltration of inflammatory cells in glioma. The sTRAIL expression vector has an application value in treatment of gliomas in the future.