Construction Of MiR-128Lentiviral Vector And Functional Studies In Glioma

Gliomas, accounting for approximately35~60%of intracranial tumors, arethe most common primary tumors of the central nervous system, with features ofhigh incidence, high recurrence rate, high mortality, and low cure rate. It isdifficult for surgical removal of glioma because of poor differentiation and stronginfiltration. Though the survival rate of glioma suffers is improved to a certainextent by the comprehensive treatment including surgery, radiotherapy andchemotherapy, the treatment effect is limited because the pathogenesis of gliomais not yet fully elucidated. Further research about the pathogenesis and newtreatment strategies for glioma is urgently required.With the discovery of miRNAs and the in-depth study about theirintracellular mechanisms, the regulatory role of miRNAs in glioma gains moreand more attention. miRNAs, play a role that similar with oncogenes and tumorsuppressor genes, can regulate the growth of tumor cells. miR-128, which is richin brain tissue and reduced expression in glioma, plays a role similar with thetumor suppressor gene in glioma pathogenesis. In the study, lentiviral-mediatedapproach was introduced to increase the expression of miR-128in glioma cellsand the effect of high expressed miR-128on human glioma U87cells was investigated to find new therapeutic approach for glioma gene therapy.Objective: To detect miR-128expression in a number of glioma samples. Toconstruct lentivirus vectors containing the precursor of miR-128-1andmiR-128-2and screen U87cells that stably express target genes. To study theeffect of miR-128on the proliferation of glioma.Methods: There are three sections in the study.1. Fresh glioma tissue andbrain tissue obtained by surgery in Xijing Hospital was used in the study. Theglioma grades were determined by pathological examination and immunologicalanalysis. Total RNA was extracted and the expression of miRNA in gliomasamples was detected by real-time quantitative PCR to investigate the overallexpression of miR-128.2. After BamH I and XhoI digestion of amplifiedhsa-miR-128precursor fragment and pENTR3C, T4DNA ligase the BamH I/Xho I fragment to pENTR3C plasmid and confirmed by sequencing. Thesequence was further confirmed in adapter plasmid Plenti6.3by LR clonase IIenzyme. The constructed plasmid and two helper plasmids, PMD2.G and psPAX2,were transducted into293T cells by liposome2000. The titers of the lentiviralvectors was determined.3. Human glioma U87cells were infected with packedlentivirus. U87cells with high miR-128expression were screened by Bsd. Theproliferation of U87cells with high miR-128expression was determined by MTTand flow cytometry and compared with uninfected U87cells.Results: The results showed that miR-128expression in gliomas was lowerthan that in normal brain tissue and the expression in high-grade gliomas waslower than that in low-grade gliomas. Two recombinant lentiviral vectorsPlenti6.3-miR-128-1and Plenti6.3-miR-128-2were successfully constructed.Packing of lentivirus was validated by the expression of mCherry fluorescenceprotein and real-time quantitative PCR after Blasticidin screening. Titer was1.1× 10~7~8.3×10~7TU/mL. Flow cytometry and MTT showed that there wasinhibited proliferation in U87cells with high expression of miR-128-1andmiR-128-2.Conclusion: miR-128expression was negatively correlated with grades ofhuman gliomas. lentiviral vectors with two miR-128precursors were successfullypacked and cell lines with stable high expression of miR-128were screened. BothmiR-128-1and miR-128-2inhibits the proliferation of U87cells.