The Human Synuclein-γ Eukaryotic Expression Vector Was Transfected Into Glioma Cells And Its Overexpression In Paclitaxel Resistance

Objective: WHO reported that each year about7.6million people died ofcancer and the disability and mortality rates of malignant gliomas wassignificantly higher than other tumors. Despite treated with the optimaltreatment, the median survival time of glioblastoma patients is only12-15months, and the patients with advanced tumors always died fromchemotherapy drug resistance. Even use of Temozolomide, which was the"star drug" for treating Malignant glioma, alone in recurrent and progressiveglioblastoma may failed or show only moderate efficacy. Apoptosis machinerydid exist in glioblastomas. But recurrent glioblastomas may lack or harbormolecular markers of cell survival even when they were challenged with apowerful chemotherapeutic drug such as Taxol. Identification of suchmolecular markers that modulate cell survival in recurrent glioblastomas mayhelp design more rational therapeutics for patients. According to the study onglioma etiology and pathogenesis, synuclein-γ (SNCG) was found to havesignificant high expression levels in malignant glioblastoma and anaplasticependymoma, and a close relationship with recurrent and progressive gliomas.SNCG even lost its original tissue-specific expression and showed highexpression in a variety of malignant tumors. It also promoted tumorproliferation, invasion, metastasis, and reduced the effecet of chemotherapydrugs, which made SNCG expected to become the new tumor marker andtherapeutic target. Our study was designed to transfect SNCG eukaryoticexpression vector into human malignant glioma cell lines of U87and U251,then established a new glioma cell line which is stably overexpressed SNCG.So we can study how overexpression of SNCG influences the glioma cell linesfrom gene, cell cycle and drug intervention three aspects. Methods: First, Pre-synthesised pIRES2-EGFP-SNCG plasmid vectorfragment were connected DH5α E. coli with the transformation of the bacteriainto uniform coating containing kanamycin50mg/L of the LB agar plate,37℃inverted culture12h, resistant colonies to be grown single colony pickedafter transfer to liquid medium in shake culture. By plasmid extraction kitinstructions mention the small plasmid, recombinant plasmid as a template forPCR amplification of plasmid identification. SNCG positive expressionplasmid was transfected into human glioma cells U87and U251by FuGENEHD Transfection Reagent. The stable transfection of SNCG U87cell line wasselected by G418. The expression of SNCG was detected by real-time RT-PCRand Immunofluorescence. CCK-8was used to determine Inhibition rate of cellproliferation of transfected SNCG group and the experimental control group.Cell cycle distribution was detected by FCM.Result: Before Plasmid pIRES2-EGFP-SNCG was transfected intoU87MG and U251MG, it had been converted, amplified, extract and identified.And then the87/SNCG cell line model was established which stablytransfected SNCG. After the recombinant plasmid pIRES2-EGFP-SNCGstably transfected U373cells, used fluorescent microscope observing them inthe same field, all the cells showed a visible view in the green fluorescence.The RT-qPCR results showed that U87/SNCG cells SNCG mRNA expressionlevel was significantly increased, and U87cells without transfection has nodifference between U87/neo and U87cells. The relative fold change of SNCGin U87/SNCG cells was7.217compared with U373/neo cells, and it comparedwith U87cells was9.05. And the relative fold change of SNCG inU251/SNCG cells was4.523compared with U373/neo cells, and it comparedwith U87cells was6.296. Without SNCG plasmid transfection inU87,U87/neo cells and U251U251/neo, there were no significant differenceamong them at the SNCG mRNA expression level. Meanwhile,Over-expressing SNCG decreased the blockage of cell mitosis which wasinduced by anti-microtubule drugs, with decrease in the G2/M phase andenhance the anti-apoptotic ability of tumor cells, reducing the U87/SNCG apoptosis/necrosis ratio.Conclusion: Successed to build U87/SNCG cell lines which was stablytransfected by vector pIRES2-EGFP-SNCG. CCK-8tested that SNCGoverexpression reduced drug sensitivity of glioma cells to paclitaxel. Cellcycle analysed by flow cytometry (FCM), tentatively confirmed theexpression of SNCG may reduce the anti-microtubule drug-induced U87cellmitosis arrest by enchanced tumor cell proliferation and anti-apoptoticcapacity, that lays a base for further study about the role of SNCG in glialtumor’s invasion and metastasis in vitro.